The document provides instructions for compiling a complete lab report from four biology labs on genomic databases, primer design, PCR, and molecular cloning. It outlines the required sections of the report, including an introduction, materials and methods, results with data, and a discussion/conclusion section. It also provides discussion questions on building a reporter gene or transgene, defining key terms and outlining the necessary gene elements and ideas for using the transgene. The report should integrate results and instructions from all four labs.
viriology1) Describe and explain the structure , genomic org.docxdickonsondorris
viriology
1) Describe and explain the structure , genomic organization , and infection cycle bacteriophages .
2) Compare and contrast ssRNA , dsRNA , and DNA phages.
3) Discuss the theories of putative virus evolution
4) Explain how viruses can evolve
5) Compare and contrast emerging and re-emerging viruses
6) Discuss 3 (re-)emerging viruses, discuss their transmission , host , epidemiology , and elaborate on the reasons for their (re-) emergence amongst other aspects
7) Explain how viruses can result in the development of cancer , and elaborate on how onco-viruses can be countered/treated
8) Discuss the purpose of virus vaccinations, , the different types of viruses , and how vaccines are developed
9) Discuss the different classes of anti-viral drugs, their use and efficacy , and how they are developed
10) Discuss the structure , function , emergency , and disease conditions of prions .
11) Describe the morphology , genomic organization , life-cycle , and pathogenesis of viruses in the family Coronaviridae , and hallmark virus from the family as a representative case
12) Describe the morphology , genomic organization , life-cycle , and pathogenesis of viruses in the family Arenaviridae ,and use a hallmark virus from the family as a representative case
13) Describe the morphology , genomic , organization , life-cycle , and pathogenesis of viruses in the family Bunyaviridae , and use a hallmark virus from the family as a representative case
14) Describe the morphology , genomic organization , life –cycle , and pathogenesis of viruses in the family Flaviviridae , and use a hallmark virus from the family as a representative cas
15) Describe the morphology , genomic organization , life –cycle , and pathogenesis of viruses in the family Filoviridae , and use a hallmark virus from the family as a representative
Name:
Date:
Instructor’s Name:
Assignment: SCIE211 Phase 5 Lab Report
Title: Identifying Environmental Hazards
Instructions: You will write a 1-page lab report using the scientific method to answer the following questions:
· Why do you see increases and decreases in the invasive species population?
· What are the implications associated with these alterations to the ecosystem as a whole?
When your lab report is complete, post it in Submitted Assignment files.
Part I: Using the lab animation, fill in the data table below to help you generate your hypothesis, outcomes, and analysis.
Years
Zebra and Quagga Mussel (density/m2)
Phytoplankton (µg/ml)
Zooplankton (µg/ml)
Cladophora Biomass (g/m2)
Foraging Fish (kilotons)
Lake Trout (kilotons)
0
3
7
10
13
15
20
Part II: Write a 1-page lab report using the following scientific method sections:
· Purpose
· State the purpose of the lab.
· Introduction
· This is an investigation of what is currently known about the question being asked. Use background information from credible references to write a short summary about concepts in the ...
1
Phylogenetic Analysis Homework assignment
This assignment will be completed on your own and turned in the week of 11/8-11/10.
Introduction
Molecular evolution is the study of how proteins and nucleic acids evolve. Included in this
field are studies of mutations and chromosomal rearrangements, the evolutionary process,
the identification of sequence patterns conferring function in proteins and nucleic acids,
and the reconstruction of the evolutionary history of organisms and the molecules that
they make. All of these studies rely on comparisons of nucleotide or amino acid sequences.
In this tutorial, you will be introduced to some of the fundamental principles of molecular
evolution and the types of bioinformatics tools that are used in evolutionary studies. We
will begin by carrying out a manual sequence comparison, so that the basic concepts can
be introduced, and the remainder of the project will be carried out at The Biology
Workbench, a set of bioinformatics analysis programs managed by The San Diego
Supercomputing Center at the University of California, San Diego.
Objectives
• To introduce the principles of molecular evolution
• To acquaint you with the tools that are available to compare nucleotide and
amino acid sequences
• To learn about the use of protein sequences in reconstructions of evolutionary history
Project
Branching evolution occurs when one ancestral species gives rise to two or more progeny
species. However, speciation events don't involve the vast majority of the genes in a
genome. That is, for most genes, both of the progeny species inherit identical genes from
the ancestor. Following speciation, these genes evolve independently in the separate
lineages. Studies of molecular evolution therefore rely heavily on comparisons of related
sequences from different organisms.
Shown below is an alignment of two homologous sequences that we will use as a starting
place. Homologous sequences are sequences that have descended from a common
ancestral sequence. You can't meaningfully compare sequences unless they are
homologous. This alignment uses the single letter amino acid code, in which G represents
glycine, Q represents glutamine, etc. The aligned proteins have been shown to be involved
in the metabolism of similar, but different, toxic compounds. As you can see, these amino
acid sequences are very similar and it is easy to recognize that they are related by common
descent.
2
dntAc: KMGVDDEVIVSRQNDGSVR
nahAc: KMGIDDEVIVSRQSDGSIR
An expanded version of this alignment is shown below. In this expanded alignment, both
the amino acids and the corresponding DNA nucleotides are shown. For ease of analysis,
the codons have been broken into separate entries in a table.
Alignment of nahAc and dntAc sequences.
K M G V D E V I V
dntAc AAA ATG GGC GTC GAT GAA GTC ATC GTC
nahAc ...
LamiaFinal data ( results).docx1- label all lanes, label ma.docxDIPESH30
Lamia/Final data ( results).docx
1- label all lanes, label marker sizes, and indicate which three lanes, containing at least one BSA sample and one E. coli sample, you are writing about.
2- lanes 2, 5, 6, 9, and 11 are BSA, lanes 14 and 15 are empty, and lanes 3, 4, 7, 8, 10, 12, and 13 are E. coli.
Lamia/Graphing page.pdf
Lamia/Guidelines.doc
Biology 105 Laboratory Fall 2013
Instructor: Ayça Akal-Strader
Guidelines for Lab Report
Lab 2: Quantification of Protein (Bradford Assay)
Your report for Lab 2: Quantification of Protein (Bradford Assay) is due the week of October 7/8/9/10. Please include the following information in your report:
Hypothesis: as usual
Introduction:
• Background/theory of Bradford Assay
• Purpose of the experiment
Results:
In addition to the specific data discussed below, your Results section should always include one or more paragraphs of text that provide:
• A brief description of the procedure
• Explanations of any charts, graphs, figures, or calculations that are included
• Statements about the most interesting/noteworthy data
Data:
1. Table of measured absorbances (like Table 2 on p. 31).
2. Table showing protein concentrations of unknowns (like Table 3 on p. 31). Say which unknowns—1, 2, or both—you used.
**Please re-make the tables for your report. DO NOT simply tear out p. 31 from your lab manual and staple it to your report.
3. Standard Curve:
• Label with title and caption
• Label axes: x-axis = Concentration (μg/ml); y-axis = Absorbance at 595 nm. Be sure to include units on Concentration. Remember that absorbance (optical density; OD) has no units.
• Plot points, leaving room to plug in your unknown absorbances to find their concentrations
• Connect the dots
(Note: Do NOT draw a straight line—unless your data really looks like a straight line. The samples we measured did not fall into the “linear range” of the spectrophotometer, and everyone’s data that I saw flattened out a lot at the high concentration end of the range. Connect your data points with a curve.)
• Indicate by drawing horizontal and vertical lines how you found the concentration of your unknowns.
Discussion:
• Did your results match your expectations? If not, why not?
• Did you have any difficulty finding the concentration of any of your unknowns?
• Do you think your measurement of protein concentration was accurate? Did your duplicates agree well? For your standards, did your absorbances increase as your protein concentrations increased?
Conclusion: as usual
Lab Report Rewrites
You may rewrite TWO of your first FIVE lab reports in an effort to improve your grade.
You do not need to rewrite the entire report; just fix the problems that caused you to lose points the first time around.
You MUST hand in the original version of your report along with your corrected version. If you do not have the original attached, we will not accept your rewrite.
Your final grade on the rewritten report will be ...
This document provides instructions for a machine learning lab assignment. Students are asked to use the Weka machine learning tool to classify RNA-binding proteins using various algorithms, including Naive Bayes, J48 decision tree, SVM with linear and RBF kernels. Performance is measured using 5-fold cross-validation on the training set and classification of a separate test protein. Results for accuracy and other metrics are recorded in tables.
The document provides instructions for a machine learning lab experiment using the Weka machine learning software. Students are asked to run several classifiers on a dataset containing RNA-binding protein sequences to predict whether amino acids bind to RNA or not. Classifiers include Naive Bayes, J48 decision tree, support vector machine (SVM) with linear and RBF kernels. Students record performance metrics from 5-fold cross validation and testing on a separate protein sequence, and analyze which classifier worked best.
BioAssay Express: Creating and exploiting assay metadataPhilip Cheung
The challenge of accurately characterizing bioassays is a real pain point for many drug discovery organizations. Research has shown that some organizations have legacy assay collections exceeding 20,000 protocols, the great majority of which are not accurately characterized. This problem is compounded by the fact that many new protocol registrations are still not following FAIR (Findability, Accessibility, Interoperability, and Reusability) Data principles.
BioAssay Express is a tool focused on transforming the traditional protocol description from an unstructured free form text into a well-curated data store based upon FAIR Data principles. By using well-defined annotations for assays, the tool enables precise ontology based searches without having to resort to imprecise keyword searches.
This talk explores a number of new important features designed to help scientists accelerate the drug discovery process. Some example use-cases include: enabling drug repositioning projects; improving SAR models; identifying appropriate machine learning data sets; fine-tuning integrative-omic pathways;
An aspirational goal for our team is to build a metadata schema based on semantic web vocabularies that is comprehensive to the extent that the text description becomes optional. One of the many possibilities is to take the initial prospective ELN entry for a bioassay protocol and feed it directly to an automated instrument. While there are many challenges involved in creating the ELN-to-robot loop, we will provide some insights into our collaborations with UCSF automation experts.
In summary, the ability to quickly and accurately search or analyze bioassay data (public or internal) is a rate limiting problem in drug discovery. We will present the latest developments toward removing this bottleneck.
https://plan.core-apps.com/acs_sd2019/abstract/6f58993d-a716-49ad-9b09-609edde5a3f4
Apollo annotation guidelines for i5k projects Diaphorina citriMonica Munoz-Torres
Apollo is a web-based application that supports and enables collaborative genome curation in real time, allowing teams of curators to improve on existing automated gene models through an intuitive interface. Apollo allows researchers to break down large amounts of data into manageable portions to mobilize groups of researchers with shared interests.
S.N.Sivanandam & S.N. Deepa - Introduction to Genetic Algorithms 2008 ISBN 35...edwinray3
This document provides an introduction to genetic algorithms. It discusses the historical development of evolutionary computation, including genetic algorithms, genetic programming, evolutionary strategies, and evolutionary programming. The key features of evolutionary computation are described, such as particulate genes and population genetics, the adaptive code book, and the genotype/phenotype dichotomy. Advantages of evolutionary computation are highlighted, including conceptual simplicity, broad applicability, hybridization with other methods, parallelism, robustness to dynamic changes, and solving problems with no known solutions. The document concludes with a discussion of applications of evolutionary computation.
viriology1) Describe and explain the structure , genomic org.docxdickonsondorris
viriology
1) Describe and explain the structure , genomic organization , and infection cycle bacteriophages .
2) Compare and contrast ssRNA , dsRNA , and DNA phages.
3) Discuss the theories of putative virus evolution
4) Explain how viruses can evolve
5) Compare and contrast emerging and re-emerging viruses
6) Discuss 3 (re-)emerging viruses, discuss their transmission , host , epidemiology , and elaborate on the reasons for their (re-) emergence amongst other aspects
7) Explain how viruses can result in the development of cancer , and elaborate on how onco-viruses can be countered/treated
8) Discuss the purpose of virus vaccinations, , the different types of viruses , and how vaccines are developed
9) Discuss the different classes of anti-viral drugs, their use and efficacy , and how they are developed
10) Discuss the structure , function , emergency , and disease conditions of prions .
11) Describe the morphology , genomic organization , life-cycle , and pathogenesis of viruses in the family Coronaviridae , and hallmark virus from the family as a representative case
12) Describe the morphology , genomic organization , life-cycle , and pathogenesis of viruses in the family Arenaviridae ,and use a hallmark virus from the family as a representative case
13) Describe the morphology , genomic , organization , life-cycle , and pathogenesis of viruses in the family Bunyaviridae , and use a hallmark virus from the family as a representative case
14) Describe the morphology , genomic organization , life –cycle , and pathogenesis of viruses in the family Flaviviridae , and use a hallmark virus from the family as a representative cas
15) Describe the morphology , genomic organization , life –cycle , and pathogenesis of viruses in the family Filoviridae , and use a hallmark virus from the family as a representative
Name:
Date:
Instructor’s Name:
Assignment: SCIE211 Phase 5 Lab Report
Title: Identifying Environmental Hazards
Instructions: You will write a 1-page lab report using the scientific method to answer the following questions:
· Why do you see increases and decreases in the invasive species population?
· What are the implications associated with these alterations to the ecosystem as a whole?
When your lab report is complete, post it in Submitted Assignment files.
Part I: Using the lab animation, fill in the data table below to help you generate your hypothesis, outcomes, and analysis.
Years
Zebra and Quagga Mussel (density/m2)
Phytoplankton (µg/ml)
Zooplankton (µg/ml)
Cladophora Biomass (g/m2)
Foraging Fish (kilotons)
Lake Trout (kilotons)
0
3
7
10
13
15
20
Part II: Write a 1-page lab report using the following scientific method sections:
· Purpose
· State the purpose of the lab.
· Introduction
· This is an investigation of what is currently known about the question being asked. Use background information from credible references to write a short summary about concepts in the ...
1
Phylogenetic Analysis Homework assignment
This assignment will be completed on your own and turned in the week of 11/8-11/10.
Introduction
Molecular evolution is the study of how proteins and nucleic acids evolve. Included in this
field are studies of mutations and chromosomal rearrangements, the evolutionary process,
the identification of sequence patterns conferring function in proteins and nucleic acids,
and the reconstruction of the evolutionary history of organisms and the molecules that
they make. All of these studies rely on comparisons of nucleotide or amino acid sequences.
In this tutorial, you will be introduced to some of the fundamental principles of molecular
evolution and the types of bioinformatics tools that are used in evolutionary studies. We
will begin by carrying out a manual sequence comparison, so that the basic concepts can
be introduced, and the remainder of the project will be carried out at The Biology
Workbench, a set of bioinformatics analysis programs managed by The San Diego
Supercomputing Center at the University of California, San Diego.
Objectives
• To introduce the principles of molecular evolution
• To acquaint you with the tools that are available to compare nucleotide and
amino acid sequences
• To learn about the use of protein sequences in reconstructions of evolutionary history
Project
Branching evolution occurs when one ancestral species gives rise to two or more progeny
species. However, speciation events don't involve the vast majority of the genes in a
genome. That is, for most genes, both of the progeny species inherit identical genes from
the ancestor. Following speciation, these genes evolve independently in the separate
lineages. Studies of molecular evolution therefore rely heavily on comparisons of related
sequences from different organisms.
Shown below is an alignment of two homologous sequences that we will use as a starting
place. Homologous sequences are sequences that have descended from a common
ancestral sequence. You can't meaningfully compare sequences unless they are
homologous. This alignment uses the single letter amino acid code, in which G represents
glycine, Q represents glutamine, etc. The aligned proteins have been shown to be involved
in the metabolism of similar, but different, toxic compounds. As you can see, these amino
acid sequences are very similar and it is easy to recognize that they are related by common
descent.
2
dntAc: KMGVDDEVIVSRQNDGSVR
nahAc: KMGIDDEVIVSRQSDGSIR
An expanded version of this alignment is shown below. In this expanded alignment, both
the amino acids and the corresponding DNA nucleotides are shown. For ease of analysis,
the codons have been broken into separate entries in a table.
Alignment of nahAc and dntAc sequences.
K M G V D E V I V
dntAc AAA ATG GGC GTC GAT GAA GTC ATC GTC
nahAc ...
LamiaFinal data ( results).docx1- label all lanes, label ma.docxDIPESH30
Lamia/Final data ( results).docx
1- label all lanes, label marker sizes, and indicate which three lanes, containing at least one BSA sample and one E. coli sample, you are writing about.
2- lanes 2, 5, 6, 9, and 11 are BSA, lanes 14 and 15 are empty, and lanes 3, 4, 7, 8, 10, 12, and 13 are E. coli.
Lamia/Graphing page.pdf
Lamia/Guidelines.doc
Biology 105 Laboratory Fall 2013
Instructor: Ayça Akal-Strader
Guidelines for Lab Report
Lab 2: Quantification of Protein (Bradford Assay)
Your report for Lab 2: Quantification of Protein (Bradford Assay) is due the week of October 7/8/9/10. Please include the following information in your report:
Hypothesis: as usual
Introduction:
• Background/theory of Bradford Assay
• Purpose of the experiment
Results:
In addition to the specific data discussed below, your Results section should always include one or more paragraphs of text that provide:
• A brief description of the procedure
• Explanations of any charts, graphs, figures, or calculations that are included
• Statements about the most interesting/noteworthy data
Data:
1. Table of measured absorbances (like Table 2 on p. 31).
2. Table showing protein concentrations of unknowns (like Table 3 on p. 31). Say which unknowns—1, 2, or both—you used.
**Please re-make the tables for your report. DO NOT simply tear out p. 31 from your lab manual and staple it to your report.
3. Standard Curve:
• Label with title and caption
• Label axes: x-axis = Concentration (μg/ml); y-axis = Absorbance at 595 nm. Be sure to include units on Concentration. Remember that absorbance (optical density; OD) has no units.
• Plot points, leaving room to plug in your unknown absorbances to find their concentrations
• Connect the dots
(Note: Do NOT draw a straight line—unless your data really looks like a straight line. The samples we measured did not fall into the “linear range” of the spectrophotometer, and everyone’s data that I saw flattened out a lot at the high concentration end of the range. Connect your data points with a curve.)
• Indicate by drawing horizontal and vertical lines how you found the concentration of your unknowns.
Discussion:
• Did your results match your expectations? If not, why not?
• Did you have any difficulty finding the concentration of any of your unknowns?
• Do you think your measurement of protein concentration was accurate? Did your duplicates agree well? For your standards, did your absorbances increase as your protein concentrations increased?
Conclusion: as usual
Lab Report Rewrites
You may rewrite TWO of your first FIVE lab reports in an effort to improve your grade.
You do not need to rewrite the entire report; just fix the problems that caused you to lose points the first time around.
You MUST hand in the original version of your report along with your corrected version. If you do not have the original attached, we will not accept your rewrite.
Your final grade on the rewritten report will be ...
This document provides instructions for a machine learning lab assignment. Students are asked to use the Weka machine learning tool to classify RNA-binding proteins using various algorithms, including Naive Bayes, J48 decision tree, SVM with linear and RBF kernels. Performance is measured using 5-fold cross-validation on the training set and classification of a separate test protein. Results for accuracy and other metrics are recorded in tables.
The document provides instructions for a machine learning lab experiment using the Weka machine learning software. Students are asked to run several classifiers on a dataset containing RNA-binding protein sequences to predict whether amino acids bind to RNA or not. Classifiers include Naive Bayes, J48 decision tree, support vector machine (SVM) with linear and RBF kernels. Students record performance metrics from 5-fold cross validation and testing on a separate protein sequence, and analyze which classifier worked best.
BioAssay Express: Creating and exploiting assay metadataPhilip Cheung
The challenge of accurately characterizing bioassays is a real pain point for many drug discovery organizations. Research has shown that some organizations have legacy assay collections exceeding 20,000 protocols, the great majority of which are not accurately characterized. This problem is compounded by the fact that many new protocol registrations are still not following FAIR (Findability, Accessibility, Interoperability, and Reusability) Data principles.
BioAssay Express is a tool focused on transforming the traditional protocol description from an unstructured free form text into a well-curated data store based upon FAIR Data principles. By using well-defined annotations for assays, the tool enables precise ontology based searches without having to resort to imprecise keyword searches.
This talk explores a number of new important features designed to help scientists accelerate the drug discovery process. Some example use-cases include: enabling drug repositioning projects; improving SAR models; identifying appropriate machine learning data sets; fine-tuning integrative-omic pathways;
An aspirational goal for our team is to build a metadata schema based on semantic web vocabularies that is comprehensive to the extent that the text description becomes optional. One of the many possibilities is to take the initial prospective ELN entry for a bioassay protocol and feed it directly to an automated instrument. While there are many challenges involved in creating the ELN-to-robot loop, we will provide some insights into our collaborations with UCSF automation experts.
In summary, the ability to quickly and accurately search or analyze bioassay data (public or internal) is a rate limiting problem in drug discovery. We will present the latest developments toward removing this bottleneck.
https://plan.core-apps.com/acs_sd2019/abstract/6f58993d-a716-49ad-9b09-609edde5a3f4
Apollo annotation guidelines for i5k projects Diaphorina citriMonica Munoz-Torres
Apollo is a web-based application that supports and enables collaborative genome curation in real time, allowing teams of curators to improve on existing automated gene models through an intuitive interface. Apollo allows researchers to break down large amounts of data into manageable portions to mobilize groups of researchers with shared interests.
S.N.Sivanandam & S.N. Deepa - Introduction to Genetic Algorithms 2008 ISBN 35...edwinray3
This document provides an introduction to genetic algorithms. It discusses the historical development of evolutionary computation, including genetic algorithms, genetic programming, evolutionary strategies, and evolutionary programming. The key features of evolutionary computation are described, such as particulate genes and population genetics, the adaptive code book, and the genotype/phenotype dichotomy. Advantages of evolutionary computation are highlighted, including conceptual simplicity, broad applicability, hybridization with other methods, parallelism, robustness to dynamic changes, and solving problems with no known solutions. The document concludes with a discussion of applications of evolutionary computation.
This document provides an overview of a webinar introducing the Web Apollo genome annotation tool. The webinar aims to help researchers in the Ceratitis capitata research community learn to identify homologous genes, become familiar with the Web Apollo interface and annotation process, and access resources for the Ceratitis capitata genome. The webinar covers what Web Apollo is, the manual annotation process, and a demonstration of Web Apollo's functionality.
Drosophila Three-Point Test Cross Lab Write-Up Instructions.docxharold7fisher61282
This document provides instructions for writing a lab report on a three-point testcross experiment in Drosophila. The report should include an abstract, introduction, methods, results, discussion, and conclusions section. The introduction should provide background on genetic mapping and crossover frequency. The methods should describe the experimental design, scoring, and calculations. The results should present phenotypic counts, genetic maps, and chi-square tests comparing expected and observed values. The discussion should interpret results in light of hypotheses, published data, and difficulties encountered. The conclusions should summarize key findings and ways to improve the experiment.
This document provides instructions for a student project to build a 3D model of DNA. It explains that DNA contains four nitrogen bases (adenine, guanine, cytosine, thymine) that make up its code. Students will work individually or in groups with roles like research scientist, artistic consultant, or building supervisor to construct a model matching a given amino acid sequence. They will turn in individual reports and be evaluated based on their model and report. The goal is for students to understand how DNA codes for amino acids and proteins.
GIAB Integrating multiple technologies to form benchmark SVs 180517GenomeInABottle
Genome in a Bottle aims to provide well-characterized human genomes as benchmarks to validate genome sequencing and variant calling. The summary characterizes five genomes that have been analyzed to provide benchmark calls for simple and some complex variants, though many challenges remain, particularly for structural variants and difficult genomic regions. Integration of multiple data types and analyses from diverse technologies is key to improving benchmark calls over time in an open and transparent manner.
This document provides instructions for students to conduct basic BLAST searches using unknown DNA sequences as queries to identify homologous sequences and determine the identity of the unknown sequences. It describes how to analyze the BLAST results, including the descriptions, alignments and significance values. Students are guided to find homologs of a known sequence on GenBank and explore the BLAST results, alignments and formatting options.
Bioinformatics is the use of computer science and statistical techniques to analyze and interpret biological data. It involves developing tools to access and manage biological data, analyzing sequences like DNA and proteins, and developing algorithms to understand relationships within large data sets. The main areas of bioinformatics are molecular, cellular, and organismal/community levels. It is used for tasks like gene finding, predicting protein structure and function, understanding evolutionary relationships, and aiding drug discovery.
Web Apollo Tutorial for the i5K copepod research community.Monica Munoz-Torres
Introduction to Web Apollo for the i5K i5K copepod research community. WebApollo is genome annotation editor; it provides a web-based environment that allows multiple distributed users to review, edit, and share manual annotations. This presentation includes information specific to the projects of the Global Initiative to sequence the genomes of 5,000 species of arthropods, i5K. Let's get started!
An introduction to Web Apollo for the Biomphalaria glabatra research community.Monica Munoz-Torres
Web Apollo is a web-based, collaborative genomic annotation editing platform. We need annotation editing tools to modify and refine precise location and structure of the genome elements that predictive algorithms cannot yet resolve automatically.
This presentation is an introduction to how the manual annotation process takes place using Web Apollo. It is addressed to the members of the Biomphalaria glabatra research community.
This presentation explains the meaning of curation and includes an introduction to the Apollo genome annotation editing tool and its curation environment.
An introduction to Web Apollo for i5K Pilot Species Projects - HemipteraMonica Munoz-Torres
Introduction to Web Apollo for the i5K Pilot species project. WebApollo is genome annotation editor; it provides a web-based environment that allows multiple distributed users to review, edit, and share manual annotations. This presentation includes information specific to the projects of the Global Initiative to sequence the genomes of 5,000 species of arthropods, i5K. Let's get started!
The document discusses using comparative gene neighborhood analysis and visualization to help understand bacterial gene function from large genome sequence datasets. It describes how genes involved in similar biological processes are often located near each other in bacterial genomes. By comparing gene neighborhoods across different genomes, functions can be predicted for unknown genes. However, this requires analyzing many gene neighborhoods to identify statistically significant patterns. The author's thesis examines designing a new visualization called BactoGeNIE that can scale to "big data" sizes and large displays to enable experts to explore and analyze comparative gene neighborhood data in an interactive way.
Chapter 13 Activity Sport Imagery QuestionnaireYouve now had a .docxcravennichole326
Chapter 13 Activity: Sport Imagery Questionnaire
You've now had a chance to experiment with imagery vividness and control, the two keys to good images. This activity will help you evaluate your images to determine what aspects of imagery you need to focus on to develop your imagery skills. This evaluation is similar to what you might experience before an imagery training program.
Reprinted, by permission, from American Coaching Effectiveness Program, 1987, Sport psychology, level two (Champaign, IL: Human Kinetics), 69-71.
Instructions:
1. Read the four imagery situations.
2. Create an image for each situation. Provide as much detail from your imagination as possible to make the image seem real. Think of specific examples of the skill, the people involved, the place, and the time. There are, of course, no right or wrong images.
3. Rate your imagery in the tables provided, using a scale where 1 = very poor and 5 = very well.
Top of Form
Imagery Situations
Situation 1: Select a specific skill or situation in your sport. Imagine yourself performing the activity in the place where you would normally practice, without anyone else present. Now close your eyes for about a minute. Try to see yourself at this place: hear the sounds, feel the body movements, and be aware of your mood.
Very poor
Very well
Rate how well you...
1
2
3
4
5
Saw yourself performing the activity
Heard the sounds
Felt yourself performing the activity
Were aware of your mood
Controlled your image
Situation 2: You are performing the same activity as in situation 1, but this time the coach and your teammates are present. You make a mistake that everyone notices. Now close your eyes for about a minute and imagine making the error and what occurs immediately afterward.
Very poor
Very well
Rate how well you...
1
2
3
4
5
Saw yourself
Hear the sounds
Felt yourself performing the movements
Controlled your image
Situation 3: Think of a teammate performing a specific activity unsuccessfully in a contest (e.g., missing a 20-foot shot, being passed by other runners, falling from the balance beam, missing a field goal.) Now close your eyes for about a minute to imagine watching your teammate performing this activity unsuccessfully in a critical part of the contest as vividly and realistically as possible.
Very poor
Very well
Rate how well you...
1
2
3
4
5
Saw your teammate
Heard the sounds
Felt your own physical presence or movement
Felt your own emotions
Controlled your image
Situation 4: Imagine yourself performing the same activity that you imagined your teammate performing in situation 3. Imagine yourself performing the activity very skillfully. Spectators and teammates show their appreciation. Now close your eyes for about a minute and imagine the situation as vividly as possible.
Very poor
Very well
Rate how well you...
1
2
3
4
5
Saw yourself
Heard the sounds
Felt yourself ma ...
Running Head Title1Title3TitleNameSCI 207 De.docxagnesdcarey33086
Running Head: Title
1
Title
3
Title
Name
SCI 207: Dependence of man on the environment
Instructor
Date
*This template will provide you with the details necessary to finalize a quality Final Lab Report. Utilize this template to complete the Week 5 Final Lab Report and ensure that you are providing all of the necessary information and proper format for the assignment. Before you begin, please note the following important information:
1. Carefully review the Final Lab Report instructions before you begin this assignment.
2. The Final Lab Report should cover all 3 experiments from your Week Two Lab.
3. Review instructor feedback from the Week Three outline of the Final Lab Report and make changes as necessary.
4. Review the Sample Final Lab Report for an example of a final product on a different topic. Your format should look like this sample report before submission.
5. Run your Final Lab Report through Turnitin using the student folder to ensure protection from accidental plagiarism
Title
Abstract
The abstract should provide a brief summary of the methods, results, and conclusions. It should very briefly allow the reader to see what was done, how it was done, and the results. It should not exceed 200 words and should be the last part written (although it should still appear right after the title page).
Introduction
The introduction should describe the background of water quality and related issues using cited examples. You should include scholarly sources in this section to help explain why water quality research is important to society. When writing this section, make sure to cite all resources in APA format.
The introduction should also contain the objective for your study. This objective is the reason why the experiment is being done. Your final report should provide an objective that describes why we want to know the answer to the questions we are asking.
Finally, the introduction should end with your hypotheses. This section should include a hypothesis for each one of the three experiments. These hypotheses should be the same ones posed before you began your experiments. You may reword them following feedback from your instructor to illustrate a proper hypothesis, however, you should not adjust them to reflect the “right” answer. You do not lose points for an inaccurate hypothesis; scientists often revise their hypotheses based on scientific evidence following an experiment.
Materials and Methods
The materials and methods section should provide a brief description of the specialized materials used in your experiment and how they were used. This section needs to summarize the instructions with enough detail so that an outsider who does not have a copy of the lab instructions knows what you did. However, this does not mean writing every little step like “dip the pH test strip in the water, then shake the test strips,” these steps can be simplified to read “we used pH test strips to measure water pH”, etc. Additionally, this se.
The document describes a lab experiment analyzing gene expression data from human fibroblasts in response to serum using microarray analysis. The aims are to analyze the gene expression data using Excel and the ArrayTrack workbench. Key steps include importing microarray data into Excel and pre-treating the data by centering and scaling. ArrayTrack is then used to analyze the data through descriptive statistics, exploring gene expression profiles of gene lists, and using the significance analysis of microarrays (SAM) tool. Additional online databases like Gene Atlas and ArrayExpress are queried to find expression profiles and experimental data for a specific gene, APT13A2, under different conditions.
This document provides an overview and introduction to bioinformatics. It discusses the large amounts of biological sequence data that have been generated and how bioinformatics is needed to analyze this data computationally. The document outlines topics that will be covered, including databases, sequence alignment tools like BLAST, gene finding, and protein analysis. Practical workshops are described that will involve database searching, multiple sequence alignments, and interpreting results to understand molecular biology and solve biomedical problems. Questions are welcomed throughout the workshops.
Here are a few things not to include in a cover letter when submitting a revised manuscript:
- Details about previous rejections from other journals
- Criticism of previous reviewers/editors' assessments
- Apologies for lack of impact or interest
- Excessive focus on the manuscript's weaknesses or limitations
- Requests for special treatment or exceptions to normal policies
The cover letter should focus on addressing issues raised in the previous review, changes made to strengthen the work, and why the revised manuscript is a good fit for the journal. It's best to maintain a positive tone that emphasizes the manuscript's strengths and significance within the journal's scope.
The document discusses two programs - BLASTing AmiGOs and "33" - that were designed to automatically generate Gene Ontology (GO) terms from gene/protein sequences. BLASTing AmiGOs takes FASTA sequences as input and outputs the associated GO terms without manual input. "33" queries a GO database using gene products from another group to retrieve GO terms and evidence codes. Manually collecting the same GO term data for 32 genes took 4-5 hours, while the programs could generate the terms automatically. The document compares the manual and automated methods and discusses using computational tools to help biologists more efficiently organize and access expanding genomic data.
MYSTERY MOLECULE PROJECT, PART II(20 points total)BIO1001.docxdohertyjoetta
MYSTERY MOLECULE PROJECT, PART II
(20 points total)
BIO1001
Fall 2020
To complete the remainder of this project and prepare for your presentation, follow the instructions below. Your presentation should include the answers to all questions indicated below. For your final
presentation, you are expected to
organize your research and
present a rehearsed, 10-12
minute presentation using the rubric at the end of this document as a guideline for preparation.
Learning Objectives
At the conclusion of this phase of the mystery molecule project, students will be able to
1. Effectively navigate sequence databases of the NCBI website
a. BLAST DNA sequence against the human genome
b. Analyze alignment data from genomic
databases
c. Identify an unknown gene based on DNA sequence
2. Use
the scientific literature (primary and secondary resources)
to analyze gene products, and research the cellular and molecular basis of a disease gene.
3. Organize scientific content into a coherent presentation.
4. Communicate research results to a group of peers.
Step 1: BLAST your DNA sequence
1. Go to:
http://www.ncbi.nlm.nih.gov/
2. Click on BLAST (under “popular resources” at the right side of the screen)
3. Choose the nucleotide blast program (under “Web BLAST”)
4. In the “blastn” tab (on left) enter your DNA sequence into the “enter query sequence” box (obtain your mystery DNA sequences in the Mystery Molecule content area in D2L).
a. In “choose search set” click the “genomic + transcript databases”
b. From the drop down menu below, select “Human genomic + transcript (Human G + T)”
c. Under “program selection” in the next section down, optimize for highly similar sequences (megablast)
5. Submit query (click on “BLAST” at the bottom of the screen) and wait– this may take up to a few minutes.
6. A colorized map of sequence alignment scores will appear. Red indicates very good sequence alignment.
7. Scroll down to descriptions of sequences that produced statistically significant alignments. You should see options for genomic alignments and transcripts. An e-value close to zero, and maximum sequence identity close to 100% are optimal.
8. Identify your mystery gene/transcript from the description, e-value, and % sequence identity.
Step 2: Analysis of gene product
Click on the reference number (left, in the column labeled, “Accession”) for the mRNA transcript or genomic sequence of your mystery gene. Scroll down; you will see many references (titles, authors, years of publication) describing your gene. Clicking on any one of the PubMed reference numbers will link you directly to the original publication. You will need these publications to characterize your gene for your presentation. Bookmark this page—all your references must be from primary or secondary sources (no google, wikipedia, textbooks, blogs, etc.).
You must use a minimum of 7 references that can be found by searching NCBI.
A workshop is intended for those who are interested in and are in the planning stages of conducting an RNA-Seq experiment. Topics to be discussed will include:
* Experimental Design of RNA-Seq experiment
* Sample preparation, best practices
* High throughput sequencing basics and choices
* Cost estimation
* Differential Gene Expression Analysis
* Data cleanup and quality assurance
* Mapping your data
* Assigning reads to genes and counting
* Analysis of differentially expressed genes
* Downstream analysis/visualizations and tables
To get PhDs, Masters and Bachelors??
To provide solutions to complex problems
To investigate laws of nature
To make new discoveries
To develop new products
To save costs
To improve our life
Human desires
The document provides instructions for an ESL student to submit a response in two parts. For the first part, the student must find an online reading or video about making the world better for a specific group and copy/paste the URL. For the second part, the student must answer three questions about the source material: identifying its genre or type, intended audience, and overall purpose. The student has one day to complete and submit both parts of the response.
This document provides an outline for a project on hazardous waste in Kuwait. The project should include: 1) basic concepts of hazardous waste, 2) historical, current and projected data on hazardous waste in Kuwait through data analysis, 3) approaches, opportunities and barriers to handling hazardous waste along with raising local and global awareness, and 4) how hazardous waste relates to or impacts ethical, cultural and religious practices in Kuwait from an ethical perspective. The document requests the outline for this project on hazardous waste in Kuwait.
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Chapter 13 Activity: Sport Imagery Questionnaire
You've now had a chance to experiment with imagery vividness and control, the two keys to good images. This activity will help you evaluate your images to determine what aspects of imagery you need to focus on to develop your imagery skills. This evaluation is similar to what you might experience before an imagery training program.
Reprinted, by permission, from American Coaching Effectiveness Program, 1987, Sport psychology, level two (Champaign, IL: Human Kinetics), 69-71.
Instructions:
1. Read the four imagery situations.
2. Create an image for each situation. Provide as much detail from your imagination as possible to make the image seem real. Think of specific examples of the skill, the people involved, the place, and the time. There are, of course, no right or wrong images.
3. Rate your imagery in the tables provided, using a scale where 1 = very poor and 5 = very well.
Top of Form
Imagery Situations
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Very poor
Very well
Rate how well you...
1
2
3
4
5
Saw yourself performing the activity
Heard the sounds
Felt yourself performing the activity
Were aware of your mood
Controlled your image
Situation 2: You are performing the same activity as in situation 1, but this time the coach and your teammates are present. You make a mistake that everyone notices. Now close your eyes for about a minute and imagine making the error and what occurs immediately afterward.
Very poor
Very well
Rate how well you...
1
2
3
4
5
Saw yourself
Hear the sounds
Felt yourself performing the movements
Controlled your image
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Very poor
Very well
Rate how well you...
1
2
3
4
5
Saw your teammate
Heard the sounds
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Controlled your image
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Very poor
Very well
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1
2
3
4
5
Saw yourself
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*This template will provide you with the details necessary to finalize a quality Final Lab Report. Utilize this template to complete the Week 5 Final Lab Report and ensure that you are providing all of the necessary information and proper format for the assignment. Before you begin, please note the following important information:
1. Carefully review the Final Lab Report instructions before you begin this assignment.
2. The Final Lab Report should cover all 3 experiments from your Week Two Lab.
3. Review instructor feedback from the Week Three outline of the Final Lab Report and make changes as necessary.
4. Review the Sample Final Lab Report for an example of a final product on a different topic. Your format should look like this sample report before submission.
5. Run your Final Lab Report through Turnitin using the student folder to ensure protection from accidental plagiarism
Title
Abstract
The abstract should provide a brief summary of the methods, results, and conclusions. It should very briefly allow the reader to see what was done, how it was done, and the results. It should not exceed 200 words and should be the last part written (although it should still appear right after the title page).
Introduction
The introduction should describe the background of water quality and related issues using cited examples. You should include scholarly sources in this section to help explain why water quality research is important to society. When writing this section, make sure to cite all resources in APA format.
The introduction should also contain the objective for your study. This objective is the reason why the experiment is being done. Your final report should provide an objective that describes why we want to know the answer to the questions we are asking.
Finally, the introduction should end with your hypotheses. This section should include a hypothesis for each one of the three experiments. These hypotheses should be the same ones posed before you began your experiments. You may reword them following feedback from your instructor to illustrate a proper hypothesis, however, you should not adjust them to reflect the “right” answer. You do not lose points for an inaccurate hypothesis; scientists often revise their hypotheses based on scientific evidence following an experiment.
Materials and Methods
The materials and methods section should provide a brief description of the specialized materials used in your experiment and how they were used. This section needs to summarize the instructions with enough detail so that an outsider who does not have a copy of the lab instructions knows what you did. However, this does not mean writing every little step like “dip the pH test strip in the water, then shake the test strips,” these steps can be simplified to read “we used pH test strips to measure water pH”, etc. Additionally, this se.
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Here are a few things not to include in a cover letter when submitting a revised manuscript:
- Details about previous rejections from other journals
- Criticism of previous reviewers/editors' assessments
- Apologies for lack of impact or interest
- Excessive focus on the manuscript's weaknesses or limitations
- Requests for special treatment or exceptions to normal policies
The cover letter should focus on addressing issues raised in the previous review, changes made to strengthen the work, and why the revised manuscript is a good fit for the journal. It's best to maintain a positive tone that emphasizes the manuscript's strengths and significance within the journal's scope.
The document discusses two programs - BLASTing AmiGOs and "33" - that were designed to automatically generate Gene Ontology (GO) terms from gene/protein sequences. BLASTing AmiGOs takes FASTA sequences as input and outputs the associated GO terms without manual input. "33" queries a GO database using gene products from another group to retrieve GO terms and evidence codes. Manually collecting the same GO term data for 32 genes took 4-5 hours, while the programs could generate the terms automatically. The document compares the manual and automated methods and discusses using computational tools to help biologists more efficiently organize and access expanding genomic data.
MYSTERY MOLECULE PROJECT, PART II(20 points total)BIO1001.docxdohertyjoetta
MYSTERY MOLECULE PROJECT, PART II
(20 points total)
BIO1001
Fall 2020
To complete the remainder of this project and prepare for your presentation, follow the instructions below. Your presentation should include the answers to all questions indicated below. For your final
presentation, you are expected to
organize your research and
present a rehearsed, 10-12
minute presentation using the rubric at the end of this document as a guideline for preparation.
Learning Objectives
At the conclusion of this phase of the mystery molecule project, students will be able to
1. Effectively navigate sequence databases of the NCBI website
a. BLAST DNA sequence against the human genome
b. Analyze alignment data from genomic
databases
c. Identify an unknown gene based on DNA sequence
2. Use
the scientific literature (primary and secondary resources)
to analyze gene products, and research the cellular and molecular basis of a disease gene.
3. Organize scientific content into a coherent presentation.
4. Communicate research results to a group of peers.
Step 1: BLAST your DNA sequence
1. Go to:
http://www.ncbi.nlm.nih.gov/
2. Click on BLAST (under “popular resources” at the right side of the screen)
3. Choose the nucleotide blast program (under “Web BLAST”)
4. In the “blastn” tab (on left) enter your DNA sequence into the “enter query sequence” box (obtain your mystery DNA sequences in the Mystery Molecule content area in D2L).
a. In “choose search set” click the “genomic + transcript databases”
b. From the drop down menu below, select “Human genomic + transcript (Human G + T)”
c. Under “program selection” in the next section down, optimize for highly similar sequences (megablast)
5. Submit query (click on “BLAST” at the bottom of the screen) and wait– this may take up to a few minutes.
6. A colorized map of sequence alignment scores will appear. Red indicates very good sequence alignment.
7. Scroll down to descriptions of sequences that produced statistically significant alignments. You should see options for genomic alignments and transcripts. An e-value close to zero, and maximum sequence identity close to 100% are optimal.
8. Identify your mystery gene/transcript from the description, e-value, and % sequence identity.
Step 2: Analysis of gene product
Click on the reference number (left, in the column labeled, “Accession”) for the mRNA transcript or genomic sequence of your mystery gene. Scroll down; you will see many references (titles, authors, years of publication) describing your gene. Clicking on any one of the PubMed reference numbers will link you directly to the original publication. You will need these publications to characterize your gene for your presentation. Bookmark this page—all your references must be from primary or secondary sources (no google, wikipedia, textbooks, blogs, etc.).
You must use a minimum of 7 references that can be found by searching NCBI.
A workshop is intended for those who are interested in and are in the planning stages of conducting an RNA-Seq experiment. Topics to be discussed will include:
* Experimental Design of RNA-Seq experiment
* Sample preparation, best practices
* High throughput sequencing basics and choices
* Cost estimation
* Differential Gene Expression Analysis
* Data cleanup and quality assurance
* Mapping your data
* Assigning reads to genes and counting
* Analysis of differentially expressed genes
* Downstream analysis/visualizations and tables
To get PhDs, Masters and Bachelors??
To provide solutions to complex problems
To investigate laws of nature
To make new discoveries
To develop new products
To save costs
To improve our life
Human desires
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1. one complete report from all the 4 labs
biology report and need the explanation and answer to help me learn.
Exercises Completed in Module I
Introduction to genomic databases and BLAST: Why do we use BLAST? What does the
output of BLAST searches tell us?
Primer Design and PCR: What is the object of designing good
primers? How will you use the primers?
Molecular Cloning: Why do we clone genes? What do you need to
clone a gene and amplify it?
You have been compiling the “Results” of your report. Let’s put it all
together.
Group Research Report
1) Introduction: Describe the major question, problem, or technical issue addressed. Explain
relevance and background information, including previous work.
2) Materials/Methods: Summarize the materials and methods used to test the hypothesis
for an
assigned gene.
3) Results: Explain the results and how they were obtained. Refer specifically to data in
figures
and/or tables. You will be trained to prepare scientific illustrations for images or
quantitative
data. This section will discuss the methods used (refer to the Methods section of the report).
4) Conclusions and Discussion: Briefly conclude findings and raises future questions.
Summarize
and explain the significance of the work presented in the paper. When appropriate, you are
encouraged to discuss other relevant approaches or articles from the literature.
Discussion
Discussion: Based on the knowledge you have built from the lab
exercises, include a discussion on building a reporter gene or a
transgene: a) define a transgene; b) discuss what gene elements are
necessary for gene expression of artificial genes; c) discuss ideas for
how to use the transgene (see articles posted in Module II in Canvas).
so the report should be a complete report from all 4 labs results. it should have :
2. Introduction ( for all 4 labs results) and ( see the instructions for all 4 labs)
methods (for all 4 labs results) and ( see the instructions for all 4 labs)
Results (Data) (for all 4 labs results) make sure to include table, graph and ( see the
instructions for all 4 labs)
Discussion/Conclusion ( for all 4 labs results) and ( see the instructions for all 4 labs)
Reference(for also 4 labs results)
Grammer
Requirements: depends | .doc file
Biology 366LLab Report Due First Meeting Week 7
Exercises Completed in Module I•Introduction to genomic databases and BLAST: Why do
we use BLAST? What does the output of BLAST searches tell us?•Primer Design and PCR:
What is the object of designing good primers? How will you use the
primers?•MolecularCloning:Why do we clone genes? What
doyouneedtocloneageneandamplifyit?•You have been compiling the “Results” of your
report. Let’s put it all together.
Group Research Report1)Introduction: Describe the major question, problem, or technical
issue addressed. Explain relevance and background information, including previous
work.2)Materials/Methods: Summarize the materials and methods used to test the
hypothesis for an assigned gene.3)Results: Explain the results and how they were obtained.
Refer specifically to data in figures and/or tables. You will be trained to prepare scientific
illustrations for images or quantitative data. This section will discuss the methods used
(refer to the Methods section of the report).4)Conclusions and Discussion: Briefly conclude
findings and raises future questions. Summarize and explain the significance of the work
presented in the paper. When appropriate, you are encouraged to discuss other relevant
approaches or articles from the literature.
Discussion•Discussion: Based on the knowledge you have built from the lab exercises,
include a discussion on building a reporter gene or a transgene: a) define a transgene; b)
discuss what gene elements are necessary for gene expression of artificial genes; c) discuss
ideas for how to use the transgene (see articles posted in Module II in Canvas).
Gene expression in living cells is often difficult to detect because of limited access of
substrates to marker enzymes. Here gene expression in specific neurons of the nematode
Caenorhabditis elegans is monitored by the bright green fluorescence of the green
fluorescent protein (GFP) from the jellyfish Aequorea victoria. The GFP fills entire neurons,
including in one neuron an extended, fanned growth cone visible in the tail end (upper
portion) of the nematode. See page 802. [Photo: Martin
Chalfie]https://www.nobelprize.org/prizes/chemistry/2008/summary/
Group No.:Biology 366L: Spring 2023Grader:Laboratory Report Rubric Total Points
100ExcellentGoodFairNeeds ImprovementIntroduction (26 pts)1. Clearly communicates
the question that is trying to be answered. 2. States a clear a goal or
hypothesis that is predictable and testable. 3.
3. Provides evidence to support hypothesis from background/research. 4.
Incorporated edits to improve introduction from previous report.Some of the "excellent"
items are missing.Methods (8 pts)A description or step-by-step list of how the experiment
was performed. Description, unclear could not be repeated.Results (Data) (26 pts)1. Results
are clearly recorded and organized. 2. Figures are of publish
quality and clearly labeled and easy to follow with written results.
3. Easy for the reader to see trends in the results. 4. Clearly
transitioned and incorporated new data into results.One of the "excellent" items is
missing.Two of the "excellent" items are missing.Three of the "excellent" items are
missing.Discussion/Conclusion (28 pts)1. Summarizes essential data used to draw
conclusions. 2. Discusses application and
implications of results. ("real world connections"). 3. Puts findings
into context of previously published results. 4. Describes future
direction of project. 5. Concludes with an overall statement (e.g., In
summary, this work...). One of the "excellent" items is missing.Two of the "excellent"
items are missing.Three of the "excellent" items are missing.References (6 pts)Clearly
organized.Missing appropriate citations.Format and Grammar (6 pts) Neat organized with
clear headings, few grammatical and spelling errors.Somewhat lacking in organization,
multiple spelling and grammar errors, not neat.Comments:
Transgenes –definition, design and testing
A Typical Eukaryotic GeneThe typical eukaryotic gene is composed of a cis-regulatory
domain as well as the sequences required to encode the protein Start of
transcriptionExons/intronsCis-regulatory region
The cis-regulatory domain contains binding sites for transcription factors –the binding of
these proteins regulates gene expression
In multicellular organisms, genes can be expressed in different cell types (like the
Cionaembryos to the left –green color), at different times and at different levelsThe cis-
regulatory sequences of the gene regulate temporal, spatial and quantitative expression
A transgene combines these cis-regulatory sequences with a reporter gene (like GFP). When
expressed in the cell/tissue/organism it should reflect the expression of the endogenous
gene.To know if your transgene works correctly, you need to have characterize the
expression of the endogenous gene you are interested in studying: i.e. its spatial, temporal
and quantitative expression. This can be analyzed using in situ hybridization (see next
slide).You then generate a transgenic embryo and analyze the expression of the transgene.
If it matches the expression pattern of the endogenous gene, then you have likely captured
the relevant regulatory information on the piece of cis-regulatory sequence you used in
your transgene.
In situ hybridization can be used to localize gene expression in cells, tissues and
organismsThe purple stain shows you where the gene is expressed
Steps to design/test a transgene:•Start with genome sequence 5’ to the start of the gene of
interest –why? This is commonly where most regulatory sequence information
exists•Identify appropriate restriction enzymes to use to clone the DNA fragment•Create a
translation fusion between exon of the gene and the reporter protein –why? Because
4. studies have shown that including an intron in your transgene can enhance its expression.
There can also be regulatory elements located in introns•Express your transgene in your
organism. If it is expressed the same as the exogenous gene, you most likely have all of the
required regulatory elements contained within your fragment. If not, you are missing
important regulatory regions and must locate those. They could be in other introns,
downstream of the gene of further upstream. You would need to design and test additional
transgene reporters.Your goal for this exercise is to design a transgene reporter for the gene
your group has been working on.
Go to ensemble.org and find the Brachyury gene as you have done beforeHere we want to
grad sequence upstream (i.e. 5’) of the gene. The arrow in the name is pointing to the right
so we are in the correct orientation. Click and drag to zoom in a bit more.
Brachyury geneUpstream geneWe want sequence from this region, type the values above to
zoom inType in range 3900-6600
Click on export data to get a fasta-formatted sequence of this region.Note, if your gene is
oriented in the opposite direction, you can still follow these instructions to get your
sequence. In Snapgeneyou can easily reverse your sequence to work with it.
Hit next, then select “text” that will give you the sequence you see to the right. Grab that
sequence…
Using Snapgene, add this sequence as a new sequencePut the sequence in “sequence view”,
we are going to annotate the first couple of coding exons to help us design our transgene.
In ensembl, pull up the Brachyury exon sequencesWe just need to first two exons, copy the
first exon sequence (just the blue sequence is ok)
In Snapgene, navigate to align 2 sequences
The Brachyury sequence you grabbedThe first exon sequence you just copiedAlign them!
Note the endpoints, then let’s annotate the sequence
Select the same region of sequence (2394-2462) then click on add features-> add feature
Name itExon or cds, once you hit translate it will force it to be cdsClick on feature
translation options to select the proper reading frame. This is correct –hint you get compare
to the Brachyury protein sequence to confirm.
Now do the same steps from exon 2Now both exons are annotated and you have the correct
reading frames aligned with you sequence. This will help you with getting the restriction
enzyme site in the proper reading frame.
This is the transgene cloning vectorIt has the following parts: AmpR(selectable marker)Ori
–plasmid origin of replicationMCS –multiple cloning siteCrHistone–histone coding
regionYFP –YFP coding regionCrBetaActin3’ UTR –3’UTRYou will be cloning your genome
fragment into the MCS and cloning it in-frame with the histone coding region. This will
produce a reporter transgene that will express YFP in the nuclei of cells (because YFP is
fused to a histone)
Go to sequence viewNow you can see the enzymes in the polylinkerand the reading frame
relative to the histone sequence (blue box).Now you want to go the Brachyury sequence in
Snapgeneand see if it has any of the polylinkersequences. If it does, skip those and make
note of which polylinkerenzymes you could use to clone your transgene. Note that both
5. PvuIIand BamHIare not in bold, indicating these are not unique sites –don’t use them!To
save you some time, you can go to enzymes-> noncuttersand see a list of enzymes that don’t
cut your sequence (you can export this information to make it easier to search)
These PL enzymes do not cut and are OK to
use:AatIINotIXhoIHindIIISphIPstISalISmaIAcc65iThese enzymes that you can use are listed
from the 5’ to the 3’ of the MCSIf you look at the MCS, you’ll see a stop codon present 5’ of
the HindIIIsite. Therefore, make sure your 3’ most enzyme is between HindIIIand Acc65i.
Let’s use XhoIfor the 5’ end of DNA fragment and Acc65i for the 3’ end of the fragment
Start with the 5’ (easy) endGrab the first 25-30 nucleotides and check against the NEB Q5
DNA polymerase calculator from last labhttps://tmcalculator.neb.com/#!/mainNow add a
few ntplus your RE site for XhoI:5’ (forward) primer:
gcgctcgagCTTCTCATAGAAATACAATACAAGTTACG
The 3’ primer is a bit more complex….You can hover the mouse over the RE site to
determine the sequence and see how it lines up with the coding region.For Acc65i, the site
is GGATCC and this is the reading frame that we to maintain:cgGGTA CcaR V PWhat we
want to do in this case is to find a region in exon 2, find a codon with “G” in position 3 and
then add GTACCgcgto add our RE
Here we use this methionineGATCCAACTGCGATGTATTCCGTCATG gtaccgcg
Add your primers to the Brachyury sequence
Add your primers to the Brachyury sequenceDon’t forget to reverse compliment the reverse
primer!
Now let’s build the reporterUse your primers to generate a PCR fragment of the genomic
region you want to cloneUsing this fragment, subcloneit into the transgene vector with
XhoIand Acc65iUse the same steps you did previously
The resulting clone, check the sequence view.
Here is where your 3’ end of your clone went into the vectorNotice that the reading runs
from exon 2 into the histone sequence without frameshifts
The sequence you just cloned corresponds the region known to be an active regulatory
domain for the CionaBrachyury gene.If you put this transgene into the ascidian Ciona, the
cells where Brachyury is expressed (the notochord cells) would be expressing the transgene
as shown in an image of living embryos above.
AssignmentUsing the Cionagene assigned to your group from last week, build a transgene
following the example in these slidesDocument your steps, screen grabs can be used if
needed:1)What region did you select (like slide #9).2)Show your sequence annotation (like
slide #20). If your gene only has one exon, just use that single exon3)Indicate which
enzymes you can use (like slide 23). If you can’t identify a pair of enzymes, what could you
do?4)Show your forward and reverse primers (like slides 24 and 26)5)Show your
completed transgene (slide 30)6)Show a closeupof the 3’ cloning region indicating that you
cloned your sequence in-frame with the reporter construct (like slide 31).
The Polymerase Chain Reaction (PCR) and PCR-based cloning•PCR has revolutionized the
way in which nucleic acids can be identified, amplified and used in molecular biology
Key milestones in the development of PCRhttps://www.integra-
biosciences.com/japan/en/blog/article/complete-guide-pcrWatson and CrickNobel Prize
6. for DNA structureArthur and SylvyKornbergNobel Prize for DNA replicationHar
KhoranaNobel Prize for genetic codeKleppe, a postdoc of Khorana’s proposed 2 primer
system but never did the experiment!Kary MullisBrock -1969 isolated a thermophile from
Yellowstone NP -Thermus aquaticus. 1976 -Chien and Trelapurified DNA polymerase
showing it was active at 80C, clone reported in 1988Needed to add enzymes each cycle,
needed to move tube by hand to different water baths
•This video provides a brief review of the PCR process:
https://www.youtube.com/watch?v=c07_5BfIDTw
The implementation of PCR required several key developments•Discovery and
characteristic of DNA polymerase enzymes•Generation of oligonucleotides which could be
used as “primers”•Realization that orienting primers opposed to one another could cause
exponential amplification•Isolation and cloning of thermostable DNA polymerase (Taq
polymerase)•Engineering of thermocycler machines that could automatically change
sample temperatures during the process.
Key steps in the PCR cycle: Denaturation, Annealing, Extensionhttps://www.integra-
biosciences.com/japan/en/blog/article/complete-guide-pcr
Primer design•Although designing primers seems straightforward, there are a variety of
issues that one must think about when planning for an experiment. The following video
outlines some of these issues which we will
discuss.•https://www.youtube.com/watch?v=mcOwlFVEino
Primer design considerations –length, GC content and TmGC content, length and melting
temperature. These are related as follows: longer primers and/or those with higher GC
content have higher melting points. Generally, you want to end the 3’ primer sequence with
a “C” or “G” (what is known as a “GC” clamp). This may require you to move the location of
the primer along your sequence to be amplified. Primers should have a Tm within about 5C
of each other and within the range of 65-75C, this will help to avoid mis-
primingOligonucleotides are synthesized 3’ to 5’ and the longer the primer, the lower the
percentage of full-length product in the synthesis reaction% Full length product =
(efficiency)(n-1) where efficiency is the synthesis coupling efficiency (typically about 99%)
and n is the number of cycles. For a 50 ntlong primer:% full product = (0.99)49= 61%. 49
cycles are needed to make a 50-mer
Primer design considerations –sequence composition•Avoid runs of 4 or more bases
(“AAAA” or “CCCC”)•Avoid dinucleotide repeats (i.e. “ATATATAT”)•Avoid intra-or inter-
primer homology as this will cause the primers to bind internally or with each other
interfering with the amplification of your product.Hairpin from internal bindingInter-
primer homologyLastly, if you plan to incorporate restriction enzymes sites for cloning, add
those with 3-5 bp additional sequence to allow efficient enzyme activity
Given the previous constraints, software packages have been developed to assist with
primer designIn this exercise we will be amplifying specific regions of genes and this
typically requires one to hand-design primers sequences. We will use a tool called
PrimerBlastto help us determine what the best design would be.First we will design primer
sets to specifically amplify the coding region of a gene so that it can be cloned into an
expression vector with a specific reading frame.Second, we will work to design primers to
7. make a transgene reporter.The following slides will walk you through the process.
Sequences to useFor simplicity, you will be working with several different sequences from
the ascidian Cionaintestinalis. Recall that you already have worked with Cionasequences at
ensemble, here you will be provided with community-annotated transcripts that include 5’
and 3’ UTRs.The cloning vectors will also be explained and annotatedFirst, let’s translate the
BrachyurycDNA sequence into the correct reading frame.
Use https://web.expasy.org/translate/to translate Make sure you select:the bottom radio
buttonForward strandThen hit translate
Scrolling down shows frame 2 as the correct reading
framecacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacgt
catcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonatgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTP
PSLtgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonHINT –put
your sequences in font Courier New so everything aligns
We want to design PCR primers that will amplify the entire open reading frame from the
start to the stop
codoncacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacg
tcatcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonLet’s make a first pass at making a primer. Grab about 7-8 codons of sequence,
stopping at position 2 of the last codon. Why should you stop there?
Check your primer sequence for use with Q5 DNA polymerase (a common PCR enzyme)
https://tmcalculator.neb.com/#!/mainLooks good, now do the second primer
atgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPPSLtga
cgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonThese look good, let’s
check them for other characteristics
Go to Primer Blast: https://www.ncbi.nlm.nih.gov/tools/primer-blast/BrachyurycDNA
sequenceForward primerReverse primerFor your reverse primer use this reverse-
compliment tool: https://www.bioinformatics.org/sms2/rev_comp.htmlUse these values
Scroll down, remove humans and add CionaintestinalisThis will allow the software to check
your primer design against the genome to help identify off-target hitsHit “get primers” and
wait for search to complete.
Output –not bad, but some 3’ and self complimentarityForward: atgttttcgaagctcagtgcgag3’
self comp, can form hairpin in primerPoly T run, sometimes you can’t avoidClick here for
detailed results
Tweak the forward
primercacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgac
gtcatcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonNew forward: atgttttcgaagctcagtgc. This is shorter so the Tm decreases, make the
reverse primer shorter
tooatgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPPSL
tgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonNew reverse:
cttacgccaccttctttgtga
Primers look good, let’s do a virtual PCR
8. PCR test:
https://www.bioinformatics.org/sms2/pcr_products.htmlBrachyurysequenceForward and
reverse primers (make sure to reverse compliment the reverse primer)
PCR Products results –looks like the correctregion>1359 bpproduct from linear template
KH.S1404.1.v1.A.nonSL1-1 Brachyury, base 26 to base 1384 (Forward -Reverse).
ATGTTTTCGAAGCTCAGTGCGAGTTACAAACCTATAATGACGTCATCAGATAGTAAGTTA
GCAGGTATGACGTCATCAGAATCAATTGAAACATGTGCAGTAAAGATGGCGCTAATAGAG
CATGGTTTATGGTCGAGGTTTCACGCGTTTGTCAACGAGATGATTGTGACAAAAAATGGA
CGACGAATGTTTCCGGTTCTTAAAACATCGATTACGGGACTTGATCCAACTGCGATGTAT
TCCGTCATGCTTGACTTTGTACCCGTGGATAACAATAGATGGAAATATGTGAACGGTGAG
TGGATCCCCGGGGGAAAACCCGAACCCCATGTTTCGTCATGTGCTTATATTCACCCCGAT
TCACCCAACTTTGGTTCGCACTGGATGAAACAACCCGTTGGTTTCAGTCGCGTCAAACTT
ACTAATAAAGCTACAGGGAACCCCCAGCAAATAATGTTGAACTCTCTGCATAAATACGAA
CCCAGGATCCACATCATGCGCGTTGGGGGCGCTGAATCGCAACAAGTCGTTGCCTCGCAT
TCATTTCAGGAGACAAGGTTCATTGCTGTTACTGCTTACCAAAACGAAGATGTTACTTCG
CTCAAGATAAAATATAATCCTTTCGCCAAAGCTTTCCTCGACGCAAAGGAGAGCCGCTCA
GGGAGTGAAAATTATTTTAAAGATTCAACGAAAGCGGGTTCGTCGCAAAATTATTCGAGA
GCGAACACATGGACGGCAAATCAAAGCAACCCAACATTCAATCAATGTCAGTATGAATCA
GGGATTCCCCCATTCCATTACCCCATCCCAAACCAACCGAAAACAACAAGAGAGCGGCGC
ATGAGCCGCACACAAAGAAGCCATCCCTACAAACCATCCACAACGCAAATTTATCAAGAT
TTCCAACCAACCAATTACCCCGCCCTACCAAACGACCAATGGCAATCAAGTATCGAAGGT
CATGAACTCGATGAGGGACACTTTAGTCTCGAACCAGTCTCTGTAGATGACGTCACCGCC
TTAGGATTCGACACCCCGCATCAGGGTTTTGCAACAAACGATCTTCTTTCAATAGAGCCT
TCCTATTCGTTAGATTATCCTCAATTCACGACCACGTGGTCCTACCCGACCAATCACATG
CCAGGATATGTTTCAAACAGTCCAATCAGAAGCCTGGAACAACCGGAGTACTTTTATAGA
GGCTATGACGTATCACAGCAAAATTACGTCACGATGACGTCAGCAGATGTTAGTAATGTG
ACGTCATCATTATACGAAACTCCGTCACCGGGGGTCATGCAAAGGCCACAAGAAGATTTT
TCAATCGCGTATACGCCGCTTACGCCACCTTCTTTGTGACheck the ORF at
https://web.expasy.org/translate/
Looks good!
Now let’s clone this sequence into a bacterial expression vector (plasmid) so we can make
BrachyuryproteinReview that plasmids are circular DNA pieces present in bacteria.
Researchers have adapted these molecules for use in molecular BiologyIf you’d like a
reminder on basic cloning approaches, please review these videos:Restriction enzyme
cloning: https://www.neb.com/tools-and-resources/video-library/cloning-with-
restriction-enzymesOur approach: https://www.neb.com/tools-and-resources/video-
library/overview-of-traditional-cloning
Bacterial vector for making proteinsFree viewer at snapgene.com. This will let you do basic
analysis like see where restriction enzymes cutVector = pTrcHisA, B or C
The polylinker(cloning site) has recognition sites for different restriction enzymesWe need
to identify the correct enzymes to use and add those to our PCR primers.
This vector has three versions which changes the reading frame around the restriction
enzyme sitesABCSince we can use PCR to set the reading frame, we can just focus on one
9. vector, so we will use pTrcHisC
Here reading frame is importantBamHITGGATCCGACCTAGGCW
IBglIIGAGATCTGCTCTATGACD LAcc65i/KpnIGGTACCTAACCCTGGTV PRE sites are in
PURPLECodon is in REDTo express our protein, we need to add the correct RE sites to our
primers with the correct reading frameThe enzymes in our polylinkerare: BamHI, BglII, PstI,
Acc65i, EcoRIand HindIIIOur DNA insert must be oriented ATG -> stop in this direction
Let’s see what enzymes we can use: https://nc3.neb.com/NEBcutter/Paste your FASTA
sequence in, preferably from your PCR productOverviewExamine enzyme listLook at zero
cutters –these are enzymes that don’t cut our PCR product
BamHIcuts our DNABglIIdoes not cutPstIdoes not cutAcc65i does not cutEcoRIdoes not
cutHindIIIcuts our DNALet’s use Acc65i and EcoRIThese let us insert our DNA in the proper
orientationThe enzymes in our polylinkerare: BamHI, BglII, PstI, Acc65i, EcoRIand
HindIIIWhat happens if we can’t usable RE sites??
We need to worry about the reading frame for Acc65i on our clone. Because EcoRIis after
the stop codon, reading frame doesn’t matterAcc65i/KpnIGGTACCTAACCCTGGTV
PcacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacgtcatc
agatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonOur primer needs to preserve this reading frame: G GTA CCT AOur primer sequence
starts with the start codon ATGIf we keep the prolinecodon from the RE and simply add our
primer sequence we are good to go:G GTA CCT atgttttcgaagctcagtgcgV P M F S K L S
ALastly, add 3 nucleotides to the 5Õ end of the primer so the enzyme can cut: CgcG GTA CCT
atgttttcgaagctcagtgcg
The reverse primer is
easieratgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPP
SLtgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonNew reverse:
cttacgccaccttctttgtgaCttacgccaccttctttgtgaGAATTCgcgEcoRIRemember to reverse
compliment:cgcGAATTCtcacaaagaaggtggcgtaaG
Group assignmentEach group will be given a gene to work with, these are provided
separately. I’ve also provided the Brachyurysequence used in the powerpointwalk-through.
1.For your gene, first identify the ORF as on slide 12. Show the correct start and stop
codons2.Design your first round of primers like slides 13-153.Check your primers in Primer
Blast and report the output (slide 18). If you find there are additional matches, you can
always blast your ntsequence at ncbito identify if you are hitting alternative splice forms of
your gene. If you do, will this interfere with your PCR amplification? Why or why not?
4.Tweak primers if needed and report like slides 19-205.Check your PCR product like on
slides 22 and 236.Check your sequence (the PCR product) on NEBCutterand report the
results (i.e. slides 29/30). Which enzymes can you use for your primers. If you can’t use any
enzymes, what could you do? Hint: sequence mutation would be needed in your PCR
product –what would you need to do?7.Report final forward and reverse primer design
(like slides 31-32)
Website referencesORF finder: https://web.expasy.org/translate/Primer Blast:
https://www.ncbi.nlm.nih.gov/tools/primer-blast/Reverse compliment:
https://www.bioinformatics.org/sms2/rev_comp.htmlNEB Q5 calculator:
10. https://tmcalculator.neb.com/#!/mainNEB Cutter: https://nc3.neb.com/NEBcutterPCR
simulation: https://www.bioinformatics.org/sms2/pcr_products.html
Gene orthologues, genome browsers and gene landmarksIn this week’s lab we will build
upon the exercises from last week (extracting sequences from DBs, BLAST searching,
translating)Here we use a test gene called Brachyury to explore genome browsers and gene
structure These exercises will prepare you for the next sequence exercises –designing
primers to clone and mutate genes and build transgene reporters.
Important Links•Ensembl genome website:
https://uswest.ensembl.org/index.html•Ensembl metazoa:
https://metazoa.ensembl.org/index.html•https://www.youtube.com/watch?v=C2g37X_uM
ok•The above video link provides a walk through on how to browse genomes and genes at
ensembl
Brachyury is a transcription factor important for early development –it will be our example
proteinhttps://en.wikipedia.org/wiki/T-box_transcription_factor_TThis is a model of the
Brachyury protein binding to DNA (purple molecule)
Brachyury belongs to a family of genes called Tbox genes.
https://www.pnas.org/doi/10.1073/pnas.1309748110Brachyury itself is widely
distributed among metazoans and even fungiWe will use this characteristic to learn how to
explore orthologues of Brachyury in different animal genomes.
Select animal genomes –based on a sample of Biology faculty model
organismsDrosophila(fruit fly): Bernstein, Cripps, Capelson
https://uswest.ensembl.org/Drosophila_melanogaster/Info/IndexSea Urchin:
SchrankelPlanarian: ZayasAscidian:
Zellerhttps://metazoa.ensembl.org/Lytechinus_variegatus_gca018143015v1/Info/Indexhtt
ps://parasite.wormbase.org/Schmidtea_mediterranea_prjna12585/Info/Indexhttps://usw
est.ensembl.org/Ciona_intestinalis/Info/IndexAll of these animals have much smaller
genomes than vertebrates (mice, humans, fish) making some of our exercises easier to do. C.
elegans: Luallenhttps://uswest.ensembl.org/Caenorhabditis_elegans/Info/Index
Let’s get started! Go to ensemble.orgEnsembl maintains genome assemblies for dozens of
organisms and is a one-stop place for doing comparative genome browsing. Your TAs may
use other genome sites for their own research –ask them about it!
Retrieve the brachyury protein from the mouse genome:Select the mouse genomeType
“brachyury” in the box and hit go
Find the circled link and click it
General information, note the accession number (ENSMU…)Position in genome,
chromosome 17,Nucleotides 8653255 to 8661328Forward strandLinks to similar proteins
Mouse Brachyury on the genome browserA genome browser is typically web-based and
shows a variety of information that you can explore to learn about genes and
genomesBefore moving on, let’s explore the browser interface and the information it
displays.Click here!
Mouse Brachyury on the genome browserClick here!
Detailed viewDirection of transcription ->8 exons, 7 introns1234 56785’UTR3’UTRThe rest
of the exons have coding sequenceClick on cDNA after viewing slide
11. cDNA sequenceNote the sequence is colored coded and the translation is also shown
Exon view. Note the exon sequence is shown separately from the intron sequences. The UTR
sequences are also color coded
Protein view.You can download the Fasta formatted protein sequence
Mouse Brachyury on the genome browserGo back to this viewClick on “region in detail”
Chromosome and positionChromosomal region surrounding the Brachyury gene, ~500
MBZoom controls for belowBrachyury gene
Arrows shift view upstream or downstreamThese are called “tracks” and describe various
kinds of data to display such as predicted/known genes, SNPs, regulatory gene information,
etcT-201 is BrachyurypromoterTry zooming outNote that there are several alternative
splice forms of Brachyury
BrachyurypromoterenhancerIf you wanted to build a transgene, you would need to include
promoter/enhancer information
Go back to this page, click on tab labeled Gene: TIf you click on orthologues, you can get
other proteins that match, but we’ll do a Blast instead
Paste in your mouse Fasta file for Brachyury, select Drosophila, C. elegansand Ciona
intestinalis for other species and then run Blast
Paste in your mouse Fasta file for Brachyury, select Drosophilaand Ciona interstinalis for
other species and then run BlastSearch the protein DBs for your species –why? This will
allow you to BLAST against a protein database that will then allow you to find the
corresponding genomic region. BLASTing against the genome will split your hits up among
the various exons of the gene.
Check your results!
For Ciona, the top hit is the CionaBrachyury geneClick on genomic location
Brachyury!You may need to adjust the view to see this image –zoom out move the window
An easy way to get the view you want is to zoom out, then drag a box about the region you
want to focus on. Select jump to region.
Now you have a centered view of the CionaBrachyury gene. Instead of zooming out and
selecting the region, you can just type in the coordinatesThis small > symbol tells you the
direction of transcription
Now look at the Drosophilahit repeating the steps needed to view the gene plotted on the
genome.This small > symbol tells you the direction of transcriptionNote: although the
Brachyury gene is found in a wide group of organisms, it has been lost in C. elegans and in
the Planarian Schmidtea mediterranea.
Compare the three Brachyury gene structuresmouseDrosophilaCionaHow many exons?
How many BP in each genome? Characterize the UTRs/coding regionsAlternative
transcripts
exonexonexonexonexonexonexonExon-intronboundaryHow to identify intron-exon
boundaries. Each line of nucleotides is the same length, so you can easily line them upThis is
the mouse Brachyury gene
Helpful hints:1)Paste your cDNA sequence into a word or google doc. Take a few minutes to
clean up the view so it is easy to read.2)Make sure you put the text into “courier new” font
and adjust the font size until it looks nice3)Now just search for the sequence from the exon
12. view in your document. The first exon boundary ends at “GAACGGCAG”4)You can use the
amino coordinates to help you find the junctions in your clustalw alignment (next slide)
T-201
MSSPGTESAGKSLQYRVDHLLSAVESELQAGSEKGDPTERELRVGLEESELWLRFKELTN60brach
yury-201 --------------------MTSSDSKLAGMTSSESIETCAVKMALIEHGLWSRFHAFVN40::: :*:*
. :.. . :::.* * ** **: :.*T-201
EMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVTADNHRWKYVNGEWVPGGKPEPQAP120
brachyury-201
EMIVTKNGRRMFPVLKTSITGLDPTAMYSVMLDFVPVDNNRWKYVNGEWIPGGKPEPHVS100**
**************..::****.****.:**** .**:*********:*******:. T-201
SCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGG-GQIMLNSLHKYEPRIHIVRVGGP-
178brachyury-201
SCAYIHPDSPNFGSHWMKQPVGFSRVKLTNKATGNPQQIMLNSLHKYEPRIHIMRVGGAE160**.*
*********:**** **.**:****** .*. ****************:**** T-201 -
QRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERNDHKDVMEEPGDCQ237brach
yury-201
SQQVVASHSFQETRFIAVTAYQNEDVTSLKIKYNPFAKAFLDAKERSGSENYFKDSTKAG220*::::**
.* **:**********::*:******************.. :: ::: .. T-201 Q-PGYSQ-WGWLVPGAG---
TLCPPASSHPQFGGSLSLPSTHGCER-YPALRNHRSSPYP291brachyury-201
SSQNYSRANTWTANQSNPTFNQCQYESGIPPFHYPI--PNQPKTTRERRMSRTQRSHPYK278. .**:
* . :. . * *. * * : *. * *.:** ** T-201 SPYAHRNSSPTYADNSSACLSMLQSHDNWSSLG-
----VPGHTSMLPVSHNASP------340brachyury-201 PSTT-----QIYQDFQPTNYP-
ALPNDQWQSSIEGHELDEGHFSLEPVSVDDVTALGFDT332: * * . : :*:*.* ** *: *** :
T-201 -----------------PTGSSQYPSLWSVSNGTITPGSQTAGVS--
NGLGAQFFRGSPA381brachyury-201 PHQGFATNDLLSIEPSYSLDYPQFTTTWSYPTNHMP-
GYVSNSPIRSLEQPEYFYRGYDV391. *: : ** .. : * : . *:** .T-201
HYTPLTHTVSAATSSSSGSPMYEGAATVTDISDSQYDTAQSLLIASWTPVSPPSM436brachyury-
201 SQQNYVTMT-SADVSNVTSSLYETPS------PGVMQRPQEDFSIAYTPLTPPSL439. . :* *. *
:** : . : *. : ::**::***:Just the mouse (top) and Ciona (bottom) sequences are shown.Use
font “courier new” to get your alignment to look nice.In this example, the amino acids
located at the exon-intron borders have been highlightedNote that some boundaries are
conserved(i.e. same relative positions in proteins, red circles)While others are not (blue
circles)
Analysis and questions for the exampleCompare and contrast your three brachyury genes
(mouse, Ciona, Drosophila):Do the genes have the same numbers of exons in each
species?Do the genes span the same amount of space (length) in the genome?Are the 5’ and
3’ UTR regions the same length? Are the UTRs located on their own exon or on an exon
which contains coding region?Are the exons located at the same positions within each gene?
You will need to extract the exon sequence for each gene for this and compare to the cDNA
sequence for eachPerform a clustalw alignment of your 5 proteins and annotate the
positions of the intron/exon boundaries on the alignment (this is on slide 32 for you)
Data needed for assignments•Each group has a gene to analyze in 5 genomes (Drosophila,
Ciona, C. elegans, Sea Urchin and Planarian. These are listed below.•Use the accession
13. number to find the corresponding mouse protein sequence, use that in a blast search•For
Drosophila, C.elegans and Ciona intestinalis: https://uswest.ensembl.org/index.htmlFrom
that site, select blast and input your protein sequence. Be sure to select all three species so
that you only need to do one blast run for these 3 species•For the sea urchin, use this link:
https://metazoa.ensembl.org/Strongylocentrotus_purpuratus/Info/Indexand select blast
from the top menu•For planarian, use this link:
https://parasite.wormbase.org/Schmidtea_mediterranea_prjna12585/Info/Index/and
select blast from the top menu•Retrieve the protein sequence from each genome and do a
clustalw multiple alignment (we did that last week). This information will be used later as in
the example.•Put screen grabs of the genes for each species in your document (i.e. like on
slide 29)•Retrieve the exon and cDNA sequences for each gene, you will need that to answer
some questions.
If your gene has alternative splice forms, use the longest one with the most exons
Mouse gene assignments by
group1ENSMUSP000000253742ENSMUSP000000411183MGP_WSBEiJ_P00627004ENSMU
SP000000692775ENSMUSP000000060716ENSMUSP000001273967ENSMUSP000000580
208ENSMUSP00000044879
What to turn in:•Compare and contrast your five genes (C. elegans, sea urchin, planarian,
Ciona, Drosophila). Make a figure like on slide 29 and answer the next five questions•Do the
genes have the same numbers of exons in each species?•Do the genes span the same
amount of space (length) in the genome?•Are the 5’ and 3’ UTR regions the same length?
•Are the UTRs located on their own exon or on an exon which contains coding region?•Do
all of the genes have annotated 5’ and 3’ UTRs? If not, why do you think that is?•Perform a
clustalw alignment of your 5 proteins and annotate the positions of the intron/exon
boundaries on the alignment like on slide 32.•Are the exons located at the same positions
within each gene? Why do you think some boundaries are preserved while others are
unique?
Biology 366LLab Report Due First Meeting Week 7
Exercises Completed in Module I•Introduction to genomic databases and BLAST: Why do
we use BLAST? What does the output of BLAST searches tell us?•Primer Design and PCR:
What is the object of designing good primers? How will you use the
primers?•MolecularCloning:Why do we clone genes? What
doyouneedtocloneageneandamplifyit?•You have been compiling the “Results” of your
report. Let’s put it all together.
Group Research Report1)Introduction: Describe the major question, problem, or technical
issue addressed. Explain relevance and background information, including previous
work.2)Materials/Methods: Summarize the materials and methods used to test the
hypothesis for an assigned gene.3)Results: Explain the results and how they were obtained.
Refer specifically to data in figures and/or tables. You will be trained to prepare scientific
illustrations for images or quantitative data. This section will discuss the methods used
(refer to the Methods section of the report).4)Conclusions and Discussion: Briefly conclude
findings and raises future questions. Summarize and explain the significance of the work
presented in the paper. When appropriate, you are encouraged to discuss other relevant
14. approaches or articles from the literature.
Discussion•Discussion: Based on the knowledge you have built from the lab exercises,
include a discussion on building a reporter gene or a transgene: a) define a transgene; b)
discuss what gene elements are necessary for gene expression of artificial genes; c) discuss
ideas for how to use the transgene (see articles posted in Module II in Canvas).
Gene expression in living cells is often difficult to detect because of limited access of
substrates to marker enzymes. Here gene expression in specific neurons of the nematode
Caenorhabditis elegans is monitored by the bright green fluorescence of the green
fluorescent protein (GFP) from the jellyfish Aequorea victoria. The GFP fills entire neurons,
including in one neuron an extended, fanned growth cone visible in the tail end (upper
portion) of the nematode. See page 802. [Photo: Martin
Chalfie]https://www.nobelprize.org/prizes/chemistry/2008/summary/
Group No.:Biology 366L: Spring 2023Grader:Laboratory Report Rubric Total Points
100ExcellentGoodFairNeeds ImprovementIntroduction (26 pts)1. Clearly communicates
the question that is trying to be answered. 2. States a clear a goal or
hypothesis that is predictable and testable. 3.
Provides evidence to support hypothesis from background/research. 4.
Incorporated edits to improve introduction from previous report.Some of the "excellent"
items are missing.Methods (8 pts)A description or step-by-step list of how the experiment
was performed. Description, unclear could not be repeated.Results (Data) (26 pts)1. Results
are clearly recorded and organized. 2. Figures are of publish
quality and clearly labeled and easy to follow with written results.
3. Easy for the reader to see trends in the results. 4. Clearly
transitioned and incorporated new data into results.One of the "excellent" items is
missing.Two of the "excellent" items are missing.Three of the "excellent" items are
missing.Discussion/Conclusion (28 pts)1. Summarizes essential data used to draw
conclusions. 2. Discusses application and
implications of results. ("real world connections"). 3. Puts findings
into context of previously published results. 4. Describes future
direction of project. 5. Concludes with an overall statement (e.g., In
summary, this work...). One of the "excellent" items is missing.Two of the "excellent"
items are missing.Three of the "excellent" items are missing.References (6 pts)Clearly
organized.Missing appropriate citations.Format and Grammar (6 pts) Neat organized with
clear headings, few grammatical and spelling errors.Somewhat lacking in organization,
multiple spelling and grammar errors, not neat.Comments:
Transgenes –definition, design and testing
A Typical Eukaryotic GeneThe typical eukaryotic gene is composed of a cis-regulatory
domain as well as the sequences required to encode the protein Start of
transcriptionExons/intronsCis-regulatory region
The cis-regulatory domain contains binding sites for transcription factors –the binding of
these proteins regulates gene expression
In multicellular organisms, genes can be expressed in different cell types (like the
Cionaembryos to the left –green color), at different times and at different levelsThe cis-
15. regulatory sequences of the gene regulate temporal, spatial and quantitative expression
A transgene combines these cis-regulatory sequences with a reporter gene (like GFP). When
expressed in the cell/tissue/organism it should reflect the expression of the endogenous
gene.To know if your transgene works correctly, you need to have characterize the
expression of the endogenous gene you are interested in studying: i.e. its spatial, temporal
and quantitative expression. This can be analyzed using in situ hybridization (see next
slide).You then generate a transgenic embryo and analyze the expression of the transgene.
If it matches the expression pattern of the endogenous gene, then you have likely captured
the relevant regulatory information on the piece of cis-regulatory sequence you used in
your transgene.
In situ hybridization can be used to localize gene expression in cells, tissues and
organismsThe purple stain shows you where the gene is expressed
Steps to design/test a transgene:•Start with genome sequence 5’ to the start of the gene of
interest –why? This is commonly where most regulatory sequence information
exists•Identify appropriate restriction enzymes to use to clone the DNA fragment•Create a
translation fusion between exon of the gene and the reporter protein –why? Because
studies have shown that including an intron in your transgene can enhance its expression.
There can also be regulatory elements located in introns•Express your transgene in your
organism. If it is expressed the same as the exogenous gene, you most likely have all of the
required regulatory elements contained within your fragment. If not, you are missing
important regulatory regions and must locate those. They could be in other introns,
downstream of the gene of further upstream. You would need to design and test additional
transgene reporters.Your goal for this exercise is to design a transgene reporter for the gene
your group has been working on.
Go to ensemble.org and find the Brachyury gene as you have done beforeHere we want to
grad sequence upstream (i.e. 5’) of the gene. The arrow in the name is pointing to the right
so we are in the correct orientation. Click and drag to zoom in a bit more.
Brachyury geneUpstream geneWe want sequence from this region, type the values above to
zoom inType in range 3900-6600
Click on export data to get a fasta-formatted sequence of this region.Note, if your gene is
oriented in the opposite direction, you can still follow these instructions to get your
sequence. In Snapgeneyou can easily reverse your sequence to work with it.
Hit next, then select “text” that will give you the sequence you see to the right. Grab that
sequence…
Using Snapgene, add this sequence as a new sequencePut the sequence in “sequence view”,
we are going to annotate the first couple of coding exons to help us design our transgene.
In ensembl, pull up the Brachyury exon sequencesWe just need to first two exons, copy the
first exon sequence (just the blue sequence is ok)
In Snapgene, navigate to align 2 sequences
The Brachyury sequence you grabbedThe first exon sequence you just copiedAlign them!
Note the endpoints, then let’s annotate the sequence
Select the same region of sequence (2394-2462) then click on add features-> add feature
16. Name itExon or cds, once you hit translate it will force it to be cdsClick on feature
translation options to select the proper reading frame. This is correct –hint you get compare
to the Brachyury protein sequence to confirm.
Now do the same steps from exon 2Now both exons are annotated and you have the correct
reading frames aligned with you sequence. This will help you with getting the restriction
enzyme site in the proper reading frame.
This is the transgene cloning vectorIt has the following parts: AmpR(selectable marker)Ori
–plasmid origin of replicationMCS –multiple cloning siteCrHistone–histone coding
regionYFP –YFP coding regionCrBetaActin3’ UTR –3’UTRYou will be cloning your genome
fragment into the MCS and cloning it in-frame with the histone coding region. This will
produce a reporter transgene that will express YFP in the nuclei of cells (because YFP is
fused to a histone)
Go to sequence viewNow you can see the enzymes in the polylinkerand the reading frame
relative to the histone sequence (blue box).Now you want to go the Brachyury sequence in
Snapgeneand see if it has any of the polylinkersequences. If it does, skip those and make
note of which polylinkerenzymes you could use to clone your transgene. Note that both
PvuIIand BamHIare not in bold, indicating these are not unique sites –don’t use them!To
save you some time, you can go to enzymes-> noncuttersand see a list of enzymes that don’t
cut your sequence (you can export this information to make it easier to search)
These PL enzymes do not cut and are OK to
use:AatIINotIXhoIHindIIISphIPstISalISmaIAcc65iThese enzymes that you can use are listed
from the 5’ to the 3’ of the MCSIf you look at the MCS, you’ll see a stop codon present 5’ of
the HindIIIsite. Therefore, make sure your 3’ most enzyme is between HindIIIand Acc65i.
Let’s use XhoIfor the 5’ end of DNA fragment and Acc65i for the 3’ end of the fragment
Start with the 5’ (easy) endGrab the first 25-30 nucleotides and check against the NEB Q5
DNA polymerase calculator from last labhttps://tmcalculator.neb.com/#!/mainNow add a
few ntplus your RE site for XhoI:5’ (forward) primer:
gcgctcgagCTTCTCATAGAAATACAATACAAGTTACG
The 3’ primer is a bit more complex….You can hover the mouse over the RE site to
determine the sequence and see how it lines up with the coding region.For Acc65i, the site
is GGATCC and this is the reading frame that we to maintain:cgGGTA CcaR V PWhat we
want to do in this case is to find a region in exon 2, find a codon with “G” in position 3 and
then add GTACCgcgto add our RE
Here we use this methionineGATCCAACTGCGATGTATTCCGTCATG gtaccgcg
Add your primers to the Brachyury sequence
Add your primers to the Brachyury sequenceDon’t forget to reverse compliment the reverse
primer!
Now let’s build the reporterUse your primers to generate a PCR fragment of the genomic
region you want to cloneUsing this fragment, subcloneit into the transgene vector with
XhoIand Acc65iUse the same steps you did previously
The resulting clone, check the sequence view.
Here is where your 3’ end of your clone went into the vectorNotice that the reading runs
from exon 2 into the histone sequence without frameshifts
17. The sequence you just cloned corresponds the region known to be an active regulatory
domain for the CionaBrachyury gene.If you put this transgene into the ascidian Ciona, the
cells where Brachyury is expressed (the notochord cells) would be expressing the transgene
as shown in an image of living embryos above.
AssignmentUsing the Cionagene assigned to your group from last week, build a transgene
following the example in these slidesDocument your steps, screen grabs can be used if
needed:1)What region did you select (like slide #9).2)Show your sequence annotation (like
slide #20). If your gene only has one exon, just use that single exon3)Indicate which
enzymes you can use (like slide 23). If you can’t identify a pair of enzymes, what could you
do?4)Show your forward and reverse primers (like slides 24 and 26)5)Show your
completed transgene (slide 30)6)Show a closeupof the 3’ cloning region indicating that you
cloned your sequence in-frame with the reporter construct (like slide 31).
The Polymerase Chain Reaction (PCR) and PCR-based cloning•PCR has revolutionized the
way in which nucleic acids can be identified, amplified and used in molecular biology
Key milestones in the development of PCRhttps://www.integra-
biosciences.com/japan/en/blog/article/complete-guide-pcrWatson and CrickNobel Prize
for DNA structureArthur and SylvyKornbergNobel Prize for DNA replicationHar
KhoranaNobel Prize for genetic codeKleppe, a postdoc of Khorana’s proposed 2 primer
system but never did the experiment!Kary MullisBrock -1969 isolated a thermophile from
Yellowstone NP -Thermus aquaticus. 1976 -Chien and Trelapurified DNA polymerase
showing it was active at 80C, clone reported in 1988Needed to add enzymes each cycle,
needed to move tube by hand to different water baths
•This video provides a brief review of the PCR process:
https://www.youtube.com/watch?v=c07_5BfIDTw
The implementation of PCR required several key developments•Discovery and
characteristic of DNA polymerase enzymes•Generation of oligonucleotides which could be
used as “primers”•Realization that orienting primers opposed to one another could cause
exponential amplification•Isolation and cloning of thermostable DNA polymerase (Taq
polymerase)•Engineering of thermocycler machines that could automatically change
sample temperatures during the process.
Key steps in the PCR cycle: Denaturation, Annealing, Extensionhttps://www.integra-
biosciences.com/japan/en/blog/article/complete-guide-pcr
Primer design•Although designing primers seems straightforward, there are a variety of
issues that one must think about when planning for an experiment. The following video
outlines some of these issues which we will
discuss.•https://www.youtube.com/watch?v=mcOwlFVEino
Primer design considerations –length, GC content and TmGC content, length and melting
temperature. These are related as follows: longer primers and/or those with higher GC
content have higher melting points. Generally, you want to end the 3’ primer sequence with
a “C” or “G” (what is known as a “GC” clamp). This may require you to move the location of
the primer along your sequence to be amplified. Primers should have a Tm within about 5C
of each other and within the range of 65-75C, this will help to avoid mis-
primingOligonucleotides are synthesized 3’ to 5’ and the longer the primer, the lower the
18. percentage of full-length product in the synthesis reaction% Full length product =
(efficiency)(n-1) where efficiency is the synthesis coupling efficiency (typically about 99%)
and n is the number of cycles. For a 50 ntlong primer:% full product = (0.99)49= 61%. 49
cycles are needed to make a 50-mer
Primer design considerations –sequence composition•Avoid runs of 4 or more bases
(“AAAA” or “CCCC”)•Avoid dinucleotide repeats (i.e. “ATATATAT”)•Avoid intra-or inter-
primer homology as this will cause the primers to bind internally or with each other
interfering with the amplification of your product.Hairpin from internal bindingInter-
primer homologyLastly, if you plan to incorporate restriction enzymes sites for cloning, add
those with 3-5 bp additional sequence to allow efficient enzyme activity
Given the previous constraints, software packages have been developed to assist with
primer designIn this exercise we will be amplifying specific regions of genes and this
typically requires one to hand-design primers sequences. We will use a tool called
PrimerBlastto help us determine what the best design would be.First we will design primer
sets to specifically amplify the coding region of a gene so that it can be cloned into an
expression vector with a specific reading frame.Second, we will work to design primers to
make a transgene reporter.The following slides will walk you through the process.
Sequences to useFor simplicity, you will be working with several different sequences from
the ascidian Cionaintestinalis. Recall that you already have worked with Cionasequences at
ensemble, here you will be provided with community-annotated transcripts that include 5’
and 3’ UTRs.The cloning vectors will also be explained and annotatedFirst, let’s translate the
BrachyurycDNA sequence into the correct reading frame.
Use https://web.expasy.org/translate/to translate Make sure you select:the bottom radio
buttonForward strandThen hit translate
Scrolling down shows frame 2 as the correct reading
framecacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacgt
catcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonatgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTP
PSLtgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonHINT –put
your sequences in font Courier New so everything aligns
We want to design PCR primers that will amplify the entire open reading frame from the
start to the stop
codoncacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacg
tcatcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonLet’s make a first pass at making a primer. Grab about 7-8 codons of sequence,
stopping at position 2 of the last codon. Why should you stop there?
Check your primer sequence for use with Q5 DNA polymerase (a common PCR enzyme)
https://tmcalculator.neb.com/#!/mainLooks good, now do the second primer
atgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPPSLtga
cgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonThese look good, let’s
check them for other characteristics
Go to Primer Blast: https://www.ncbi.nlm.nih.gov/tools/primer-blast/BrachyurycDNA
sequenceForward primerReverse primerFor your reverse primer use this reverse-
19. compliment tool: https://www.bioinformatics.org/sms2/rev_comp.htmlUse these values
Scroll down, remove humans and add CionaintestinalisThis will allow the software to check
your primer design against the genome to help identify off-target hitsHit “get primers” and
wait for search to complete.
Output –not bad, but some 3’ and self complimentarityForward: atgttttcgaagctcagtgcgag3’
self comp, can form hairpin in primerPoly T run, sometimes you can’t avoidClick here for
detailed results
Tweak the forward
primercacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgac
gtcatcagatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonNew forward: atgttttcgaagctcagtgc. This is shorter so the Tm decreases, make the
reverse primer shorter
tooatgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPPSL
tgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonNew reverse:
cttacgccaccttctttgtga
Primers look good, let’s do a virtual PCR
PCR test:
https://www.bioinformatics.org/sms2/pcr_products.htmlBrachyurysequenceForward and
reverse primers (make sure to reverse compliment the reverse primer)
PCR Products results –looks like the correctregion>1359 bpproduct from linear template
KH.S1404.1.v1.A.nonSL1-1 Brachyury, base 26 to base 1384 (Forward -Reverse).
ATGTTTTCGAAGCTCAGTGCGAGTTACAAACCTATAATGACGTCATCAGATAGTAAGTTA
GCAGGTATGACGTCATCAGAATCAATTGAAACATGTGCAGTAAAGATGGCGCTAATAGAG
CATGGTTTATGGTCGAGGTTTCACGCGTTTGTCAACGAGATGATTGTGACAAAAAATGGA
CGACGAATGTTTCCGGTTCTTAAAACATCGATTACGGGACTTGATCCAACTGCGATGTAT
TCCGTCATGCTTGACTTTGTACCCGTGGATAACAATAGATGGAAATATGTGAACGGTGAG
TGGATCCCCGGGGGAAAACCCGAACCCCATGTTTCGTCATGTGCTTATATTCACCCCGAT
TCACCCAACTTTGGTTCGCACTGGATGAAACAACCCGTTGGTTTCAGTCGCGTCAAACTT
ACTAATAAAGCTACAGGGAACCCCCAGCAAATAATGTTGAACTCTCTGCATAAATACGAA
CCCAGGATCCACATCATGCGCGTTGGGGGCGCTGAATCGCAACAAGTCGTTGCCTCGCAT
TCATTTCAGGAGACAAGGTTCATTGCTGTTACTGCTTACCAAAACGAAGATGTTACTTCG
CTCAAGATAAAATATAATCCTTTCGCCAAAGCTTTCCTCGACGCAAAGGAGAGCCGCTCA
GGGAGTGAAAATTATTTTAAAGATTCAACGAAAGCGGGTTCGTCGCAAAATTATTCGAGA
GCGAACACATGGACGGCAAATCAAAGCAACCCAACATTCAATCAATGTCAGTATGAATCA
GGGATTCCCCCATTCCATTACCCCATCCCAAACCAACCGAAAACAACAAGAGAGCGGCGC
ATGAGCCGCACACAAAGAAGCCATCCCTACAAACCATCCACAACGCAAATTTATCAAGAT
TTCCAACCAACCAATTACCCCGCCCTACCAAACGACCAATGGCAATCAAGTATCGAAGGT
CATGAACTCGATGAGGGACACTTTAGTCTCGAACCAGTCTCTGTAGATGACGTCACCGCC
TTAGGATTCGACACCCCGCATCAGGGTTTTGCAACAAACGATCTTCTTTCAATAGAGCCT
TCCTATTCGTTAGATTATCCTCAATTCACGACCACGTGGTCCTACCCGACCAATCACATG
CCAGGATATGTTTCAAACAGTCCAATCAGAAGCCTGGAACAACCGGAGTACTTTTATAGA
GGCTATGACGTATCACAGCAAAATTACGTCACGATGACGTCAGCAGATGTTAGTAATGTG
ACGTCATCATTATACGAAACTCCGTCACCGGGGGTCATGCAAAGGCCACAAGAAGATTTT
20. TCAATCGCGTATACGCCGCTTACGCCACCTTCTTTGTGACheck the ORF at
https://web.expasy.org/translate/
Looks good!
Now let’s clone this sequence into a bacterial expression vector (plasmid) so we can make
BrachyuryproteinReview that plasmids are circular DNA pieces present in bacteria.
Researchers have adapted these molecules for use in molecular BiologyIf you’d like a
reminder on basic cloning approaches, please review these videos:Restriction enzyme
cloning: https://www.neb.com/tools-and-resources/video-library/cloning-with-
restriction-enzymesOur approach: https://www.neb.com/tools-and-resources/video-
library/overview-of-traditional-cloning
Bacterial vector for making proteinsFree viewer at snapgene.com. This will let you do basic
analysis like see where restriction enzymes cutVector = pTrcHisA, B or C
The polylinker(cloning site) has recognition sites for different restriction enzymesWe need
to identify the correct enzymes to use and add those to our PCR primers.
This vector has three versions which changes the reading frame around the restriction
enzyme sitesABCSince we can use PCR to set the reading frame, we can just focus on one
vector, so we will use pTrcHisC
Here reading frame is importantBamHITGGATCCGACCTAGGCW
IBglIIGAGATCTGCTCTATGACD LAcc65i/KpnIGGTACCTAACCCTGGTV PRE sites are in
PURPLECodon is in REDTo express our protein, we need to add the correct RE sites to our
primers with the correct reading frameThe enzymes in our polylinkerare: BamHI, BglII, PstI,
Acc65i, EcoRIand HindIIIOur DNA insert must be oriented ATG -> stop in this direction
Let’s see what enzymes we can use: https://nc3.neb.com/NEBcutter/Paste your FASTA
sequence in, preferably from your PCR productOverviewExamine enzyme listLook at zero
cutters –these are enzymes that don’t cut our PCR product
BamHIcuts our DNABglIIdoes not cutPstIdoes not cutAcc65i does not cutEcoRIdoes not
cutHindIIIcuts our DNALet’s use Acc65i and EcoRIThese let us insert our DNA in the proper
orientationThe enzymes in our polylinkerare: BamHI, BglII, PstI, Acc65i, EcoRIand
HindIIIWhat happens if we can’t usable RE sites??
We need to worry about the reading frame for Acc65i on our clone. Because EcoRIis after
the stop codon, reading frame doesn’t matterAcc65i/KpnIGGTACCTAACCCTGGTV
PcacccgagtgtgatttggaggcagaatgttttcgaagctcagtgcgagttacaaacctataMFSKLSASYKPIatgacgtcatc
agatagtaagttagcaggtatgacgtcatcagaatcaattgaaacatgtMTSSDSKLAGMTSSESIETCStart
codonOur primer needs to preserve this reading frame: G GTA CCT AOur primer sequence
starts with the start codon ATGIf we keep the prolinecodon from the RE and simply add our
primer sequence we are good to go:G GTA CCT atgttttcgaagctcagtgcgV P M F S K L S
ALastly, add 3 nucleotides to the 5Õ end of the primer so the enzyme can cut: CgcG GTA CCT
atgttttcgaagctcagtgcg
The reverse primer is
easieratgcaaaggccacaagaagatttttcaatcgcgtatacgccgcttacgccaccttctttgMQRPQEDFSIAYTPLTPP
SLtgacgtcacaatgcgaatataattatcgattgtttcgtgaaataacattaaaatatgaac-Stop codonNew reverse:
cttacgccaccttctttgtgaCttacgccaccttctttgtgaGAATTCgcgEcoRIRemember to reverse
compliment:cgcGAATTCtcacaaagaaggtggcgtaaG
21. Group assignmentEach group will be given a gene to work with, these are provided
separately. I’ve also provided the Brachyurysequence used in the powerpointwalk-through.
1.For your gene, first identify the ORF as on slide 12. Show the correct start and stop
codons2.Design your first round of primers like slides 13-153.Check your primers in Primer
Blast and report the output (slide 18). If you find there are additional matches, you can
always blast your ntsequence at ncbito identify if you are hitting alternative splice forms of
your gene. If you do, will this interfere with your PCR amplification? Why or why not?
4.Tweak primers if needed and report like slides 19-205.Check your PCR product like on
slides 22 and 236.Check your sequence (the PCR product) on NEBCutterand report the
results (i.e. slides 29/30). Which enzymes can you use for your primers. If you can’t use any
enzymes, what could you do? Hint: sequence mutation would be needed in your PCR
product –what would you need to do?7.Report final forward and reverse primer design
(like slides 31-32)
Website referencesORF finder: https://web.expasy.org/translate/Primer Blast:
https://www.ncbi.nlm.nih.gov/tools/primer-blast/Reverse compliment:
https://www.bioinformatics.org/sms2/rev_comp.htmlNEB Q5 calculator:
https://tmcalculator.neb.com/#!/mainNEB Cutter: https://nc3.neb.com/NEBcutterPCR
simulation: https://www.bioinformatics.org/sms2/pcr_products.html
Gene orthologues, genome browsers and gene landmarksIn this week’s lab we will build
upon the exercises from last week (extracting sequences from DBs, BLAST searching,
translating)Here we use a test gene called Brachyury to explore genome browsers and gene
structure These exercises will prepare you for the next sequence exercises –designing
primers to clone and mutate genes and build transgene reporters.
Important Links•Ensembl genome website:
https://uswest.ensembl.org/index.html•Ensembl metazoa:
https://metazoa.ensembl.org/index.html•https://www.youtube.com/watch?v=C2g37X_uM
ok•The above video link provides a walk through on how to browse genomes and genes at
ensembl
Brachyury is a transcription factor important for early development –it will be our example
proteinhttps://en.wikipedia.org/wiki/T-box_transcription_factor_TThis is a model of the
Brachyury protein binding to DNA (purple molecule)
Brachyury belongs to a family of genes called Tbox genes.
https://www.pnas.org/doi/10.1073/pnas.1309748110Brachyury itself is widely
distributed among metazoans and even fungiWe will use this characteristic to learn how to
explore orthologues of Brachyury in different animal genomes.
Select animal genomes –based on a sample of Biology faculty model
organismsDrosophila(fruit fly): Bernstein, Cripps, Capelson
https://uswest.ensembl.org/Drosophila_melanogaster/Info/IndexSea Urchin:
SchrankelPlanarian: ZayasAscidian:
Zellerhttps://metazoa.ensembl.org/Lytechinus_variegatus_gca018143015v1/Info/Indexhtt
ps://parasite.wormbase.org/Schmidtea_mediterranea_prjna12585/Info/Indexhttps://usw
est.ensembl.org/Ciona_intestinalis/Info/IndexAll of these animals have much smaller
genomes than vertebrates (mice, humans, fish) making some of our exercises easier to do. C.
22. elegans: Luallenhttps://uswest.ensembl.org/Caenorhabditis_elegans/Info/Index
Let’s get started! Go to ensemble.orgEnsembl maintains genome assemblies for dozens of
organisms and is a one-stop place for doing comparative genome browsing. Your TAs may
use other genome sites for their own research –ask them about it!
Retrieve the brachyury protein from the mouse genome:Select the mouse genomeType
“brachyury” in the box and hit go
Find the circled link and click it
General information, note the accession number (ENSMU…)Position in genome,
chromosome 17,Nucleotides 8653255 to 8661328Forward strandLinks to similar proteins
Mouse Brachyury on the genome browserA genome browser is typically web-based and
shows a variety of information that you can explore to learn about genes and
genomesBefore moving on, let’s explore the browser interface and the information it
displays.Click here!
Mouse Brachyury on the genome browserClick here!
Detailed viewDirection of transcription ->8 exons, 7 introns1234 56785’UTR3’UTRThe rest
of the exons have coding sequenceClick on cDNA after viewing slide
cDNA sequenceNote the sequence is colored coded and the translation is also shown
Exon view. Note the exon sequence is shown separately from the intron sequences. The UTR
sequences are also color coded
Protein view.You can download the Fasta formatted protein sequence
Mouse Brachyury on the genome browserGo back to this viewClick on “region in detail”
Chromosome and positionChromosomal region surrounding the Brachyury gene, ~500
MBZoom controls for belowBrachyury gene
Arrows shift view upstream or downstreamThese are called “tracks” and describe various
kinds of data to display such as predicted/known genes, SNPs, regulatory gene information,
etcT-201 is BrachyurypromoterTry zooming outNote that there are several alternative
splice forms of Brachyury
BrachyurypromoterenhancerIf you wanted to build a transgene, you would need to include
promoter/enhancer information
Go back to this page, click on tab labeled Gene: TIf you click on orthologues, you can get
other proteins that match, but we’ll do a Blast instead
Paste in your mouse Fasta file for Brachyury, select Drosophila, C. elegansand Ciona
intestinalis for other species and then run Blast
Paste in your mouse Fasta file for Brachyury, select Drosophilaand Ciona interstinalis for
other species and then run BlastSearch the protein DBs for your species –why? This will
allow you to BLAST against a protein database that will then allow you to find the
corresponding genomic region. BLASTing against the genome will split your hits up among
the various exons of the gene.
Check your results!
For Ciona, the top hit is the CionaBrachyury geneClick on genomic location
Brachyury!You may need to adjust the view to see this image –zoom out move the window
An easy way to get the view you want is to zoom out, then drag a box about the region you
want to focus on. Select jump to region.
23. Now you have a centered view of the CionaBrachyury gene. Instead of zooming out and
selecting the region, you can just type in the coordinatesThis small > symbol tells you the
direction of transcription
Now look at the Drosophilahit repeating the steps needed to view the gene plotted on the
genome.This small > symbol tells you the direction of transcriptionNote: although the
Brachyury gene is found in a wide group of organisms, it has been lost in C. elegans and in
the Planarian Schmidtea mediterranea.
Compare the three Brachyury gene structuresmouseDrosophilaCionaHow many exons?
How many BP in each genome? Characterize the UTRs/coding regionsAlternative
transcripts
exonexonexonexonexonexonexonExon-intronboundaryHow to identify intron-exon
boundaries. Each line of nucleotides is the same length, so you can easily line them upThis is
the mouse Brachyury gene
Helpful hints:1)Paste your cDNA sequence into a word or google doc. Take a few minutes to
clean up the view so it is easy to read.2)Make sure you put the text into “courier new” font
and adjust the font size until it looks nice3)Now just search for the sequence from the exon
view in your document. The first exon boundary ends at “GAACGGCAG”4)You can use the
amino coordinates to help you find the junctions in your clustalw alignment (next slide)
T-201
MSSPGTESAGKSLQYRVDHLLSAVESELQAGSEKGDPTERELRVGLEESELWLRFKELTN60brach
yury-201 --------------------MTSSDSKLAGMTSSESIETCAVKMALIEHGLWSRFHAFVN40::: :*:*
. :.. . :::.* * ** **: :.*T-201
EMIVTKNGRRMFPVLKVNVSGLDPNAMYSFLLDFVTADNHRWKYVNGEWVPGGKPEPQAP120
brachyury-201
EMIVTKNGRRMFPVLKTSITGLDPTAMYSVMLDFVPVDNNRWKYVNGEWIPGGKPEPHVS100**
**************..::****.****.:**** .**:*********:*******:. T-201
SCVYIHPDSPNFGAHWMKAPVSFSKVKLTNKLNGG-GQIMLNSLHKYEPRIHIVRVGGP-
178brachyury-201
SCAYIHPDSPNFGSHWMKQPVGFSRVKLTNKATGNPQQIMLNSLHKYEPRIHIMRVGGAE160**.*
*********:**** **.**:****** .*. ****************:**** T-201 -
QRMITSHCFPETQFIAVTAYQNEEITALKIKYNPFAKAFLDAKERNDHKDVMEEPGDCQ237brach
yury-201
SQQVVASHSFQETRFIAVTAYQNEDVTSLKIKYNPFAKAFLDAKERSGSENYFKDSTKAG220*::::**
.* **:**********::*:******************.. :: ::: .. T-201 Q-PGYSQ-WGWLVPGAG---
TLCPPASSHPQFGGSLSLPSTHGCER-YPALRNHRSSPYP291brachyury-201
SSQNYSRANTWTANQSNPTFNQCQYESGIPPFHYPI--PNQPKTTRERRMSRTQRSHPYK278. .**:
* . :. . * *. * * : *. * *.:** ** T-201 SPYAHRNSSPTYADNSSACLSMLQSHDNWSSLG-
----VPGHTSMLPVSHNASP------340brachyury-201 PSTT-----QIYQDFQPTNYP-
ALPNDQWQSSIEGHELDEGHFSLEPVSVDDVTALGFDT332: * * . : :*:*.* ** *: *** :
T-201 -----------------PTGSSQYPSLWSVSNGTITPGSQTAGVS--
NGLGAQFFRGSPA381brachyury-201 PHQGFATNDLLSIEPSYSLDYPQFTTTWSYPTNHMP-
GYVSNSPIRSLEQPEYFYRGYDV391. *: : ** .. : * : . *:** .T-201
HYTPLTHTVSAATSSSSGSPMYEGAATVTDISDSQYDTAQSLLIASWTPVSPPSM436brachyury-
24. 201 SQQNYVTMT-SADVSNVTSSLYETPS------PGVMQRPQEDFSIAYTPLTPPSL439. . :* *. *
:** : . : *. : ::**::***:Just the mouse (top) and Ciona (bottom) sequences are shown.Use
font “courier new” to get your alignment to look nice.In this example, the amino acids
located at the exon-intron borders have been highlightedNote that some boundaries are
conserved(i.e. same relative positions in proteins, red circles)While others are not (blue
circles)
Analysis and questions for the exampleCompare and contrast your three brachyury genes
(mouse, Ciona, Drosophila):Do the genes have the same numbers of exons in each
species?Do the genes span the same amount of space (length) in the genome?Are the 5’ and
3’ UTR regions the same length? Are the UTRs located on their own exon or on an exon
which contains coding region?Are the exons located at the same positions within each gene?
You will need to extract the exon sequence for each gene for this and compare to the cDNA
sequence for eachPerform a clustalw alignment of your 5 proteins and annotate the
positions of the intron/exon boundaries on the alignment (this is on slide 32 for you)
Data needed for assignments•Each group has a gene to analyze in 5 genomes (Drosophila,
Ciona, C. elegans, Sea Urchin and Planarian. These are listed below.•Use the accession
number to find the corresponding mouse protein sequence, use that in a blast search•For
Drosophila, C.elegans and Ciona intestinalis: https://uswest.ensembl.org/index.htmlFrom
that site, select blast and input your protein sequence. Be sure to select all three species so
that you only need to do one blast run for these 3 species•For the sea urchin, use this link:
https://metazoa.ensembl.org/Strongylocentrotus_purpuratus/Info/Indexand select blast
from the top menu•For planarian, use this link:
https://parasite.wormbase.org/Schmidtea_mediterranea_prjna12585/Info/Index/and
select blast from the top menu•Retrieve the protein sequence from each genome and do a
clustalw multiple alignment (we did that last week). This information will be used later as in
the example.•Put screen grabs of the genes for each species in your document (i.e. like on
slide 29)•Retrieve the exon and cDNA sequences for each gene, you will need that to answer
some questions.
If your gene has alternative splice forms, use the longest one with the most exons
Mouse gene assignments by
group1ENSMUSP000000253742ENSMUSP000000411183MGP_WSBEiJ_P00627004ENSMU
SP000000692775ENSMUSP000000060716ENSMUSP000001273967ENSMUSP000000580
208ENSMUSP00000044879
What to turn in:•Compare and contrast your five genes (C. elegans, sea urchin, planarian,
Ciona, Drosophila). Make a figure like on slide 29 and answer the next five questions•Do the
genes have the same numbers of exons in each species?•Do the genes span the same
amount of space (length) in the genome?•Are the 5’ and 3’ UTR regions the same length?
•Are the UTRs located on their own exon or on an exon which contains coding region?•Do
all of the genes have annotated 5’ and 3’ UTRs? If not, why do you think that is?•Perform a
clustalw alignment of your 5 proteins and annotate the positions of the intron/exon
boundaries on the alignment like on slide 32.•Are the exons located at the same positions
within each gene? Why do you think some boundaries are preserved while others are
unique?
25. Biology 366L 2023Week of January 30th
A brief review •The central dogma of molecular biology•Gene structure•Location of genes
in chromosomes
The central dogma of molecular biology:DNA -> RNA ->
Proteinhttps://www.youtube.com/watch?v=gG7uCskUOrAIn general, information is stored
in the genome as DNAis Transcribedinto RNA and then Translatedinto Protein.
The central dogma of molecular
biologyhttps://www.britannica.com/science/geneInformation flow:DNA -> RNA ->
ProteinIntrons spliced outA more detailed review can be found here:
https://openlab.citytech.cuny.edu/openstax-bio/exam-4/gene-function/
The coding region encodes the amino acids for the corresponding
proteinhttps://www.researchgate.net/figure/The-structure-of-a-gene-and-the-control-of-
gene-expression-in-eukaryotes-Initially-RNA_fig2_263714372Open reading frame
Genes are located along chromosomes within a eukaryotic
organismhttps://sitn.hms.harvard.edu/flash/2012/issue127a/
Our genetic information is stored in our chromosomesGiven either a nucleic acid or protein
sequence, researchers can use various bioinformatic approaches to identify and clone a
gene, build transgene reports, and manipulate gene functionHere we will introduce a
variety of algorithms and databases that are used for such purposes.
Sequence search algorithmsProbably one of the most-utilized algorithms for sequence
searching is BLASTBLAST stands for Basic Local Alignment Search ToolBLAST website:
https://blast.ncbi.nlm.nih.gov/Blast.cgi
There are several “flavors” of BLAST, depending on whether you are searching with nucleic
acid or protein sequences•BlastN –nucleotide vs nucleotide•BlastP –protein vs
protein•BlastX –nucleotide vs protein•tblastN –protein vs translated nucleotide•The above
indicate query (the sequence you have) vs subject (the DB)
You want to start with a FASTA formatted sequence fileFasta files begin with a “>”
character, then a description. The sequence (either nucleotide or protein) follows on
subsequent
lines>avGFPMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW
PTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR
IELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIG
DGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKYou can retrieve
sequences from genbank, to get the GFP sequence above, retrieve the sequence using the
sequence identifier “AAA27722.1” This is also referred to as an accession number.
This link will give you the corresponding nucleotide sequence
Fasta formatted sequence
Use SMS to translate a sequenceUse this accession: L29345.1To retrieve the GFP mRNA
sequenceNote: although there are many websites that can perform sequence manipulations,
etc., many labs have their own local software for performing these functions.Which reading
frame do you need to translate the mRNA into to obtain the GFP protein sequence?
Example BlastP runProtein query sequence in Fasta formatDefault DB is non-redundant
26. proteinsBlastP is selected
BLAST output is scored so you can analyze the resulting “hits” Evalue: smaller = better
hitQuery cover: how much of your query sequence was matchedPer identityAccession: link
to “hit” sequence
Clicking on graphical summary gives you a visual representation of hitsClick on conserved
domains to learn about detected protein domains
GFP has a single, conserved domain
The alignments tab shows you the aligned query and subject sequences
Fluorescent proteins are used for many different research purposesVideo link about GFP
(can show in lab): https://www.youtube.com/watch?v=Z4vJ8rQCNggVideo link for more
GFP information (optional, don’t show in lab):
https://www.youtube.com/watch?v=qK9aYnkIr3w
FPBase –a curated database for fluorescent proteinsExplore if interested
Protein structure -alphafoldAlphafold –computational protein foldingYou can learn more
about alphafold here:https://www.deepmind.com/research/highlighted-
research/alphafoldInput "green fluorescent protein" Aequorea into the search box to
explore
With AlphaFold you can interact with the predicted protein structure
Sequence multiple alignmentsYou may wish to determine how similar different sequences
are to one another, to do that you can use a tool such as ClustaW:
https://www.ebi.ac.uk/Tools/msa/clustalo/Here you can provide multiple sequences to
determine similarities across the sequence. The corresponding document in Canvas has
more specific instructions.
Output codes:* = exact match: = strong conservation. = weak conservation= mismatch
UniProt is a protein repository
Expasy is a portal to access various suites of bioinformatic tools
AssignmentYour instructorwill organize you into working groups. Each group will be
provided an accession # for a gene of interest. You can make a simple group report using
google sheets or the office suite. Use text, screen grabs and pictures in your findings.Given
this accession # please do the following:1)Retrieve the nucleotide and protein sequences,
what species is you sequence from?2)Given the gene name from the sequence file, use
resources at NCBI to determine the function of your gene3)Using the mRNA sequence for
your gene, use SMS to translate this sequence into the correct protein –which reading frame
was needed?4)Perform a Blast search using the nucleotide sequence and then the protein
sequence, report on the top 5 hits. Do they correspond to the same gene in each search?
Why or why not?5)From your protein blast search, identify conserved domains (if any).
What are they and what is their function?6)With the top 5 proteins from your blast search,
perform a multiple alignment using ClustalW. Are these proteins similar? Does the region of
similarity overlap with the regions of conserved domains? Why or why not?7)With your
protein sequence for your gene, locate its predicted structure on AlphaFold–add a screen
grab to your report.