This document discusses advanced biotechnology tools for engineering plasmids. It describes using restriction enzymes to insert genes of interest and antibiotic resistance genes into plasmids. Selection involves growing transformed bacteria on antibiotic plates to identify bacteria that took up the plasmid. Additional techniques described include screening recombinant plasmids, finding genes of interest using probes, making DNA libraries, cDNA libraries, microarrays, and comparing gene expression between conditions.
Biotechnology uses genetic engineering tools and techniques to manipulate DNA and genes. These tools include restriction enzymes that cut DNA at specific sequences, leaving "sticky ends" that allow DNA fragments to be combined. Plasmids and bacterial transformation allow genes to be inserted and copied within bacteria. This allows bacteria to produce proteins from other organisms, like human insulin. Genetic engineering has applications in medicine and agriculture, like producing disease-resistant crops or improving food quality and nutrition.
Gene cloning involves making many identical copies of a gene. The basic steps include isolating DNA from an organism, cutting the DNA into fragments using restriction enzymes, inserting the fragments into plasmids, and transforming the plasmids into bacterial cells. Each bacterial cell will contain copies of the gene of interest, allowing scientists to isolate and study that gene. Viruses and bacteria can act as vectors, or carriers, to transfer genes into cells. Plasmids and bacteriophages are commonly used vectors for cloning genes into bacterial cells.
Recombinant DNA technology allows genes to be moved between species. This document discusses gene cloning using recombinant DNA technology. It describes how restriction enzymes cut DNA into fragments which can then be joined with other DNA fragments using DNA ligase. Plasmids and bacteriophages are commonly used as cloning vectors to replicate and express inserted DNA fragments in host bacteria. The production of recombinant plasmids and transformation of bacteria allows the cloned DNA fragments to be isolated and purified.
Molecular cloning refers to the process of replicating DNA molecules to generate multiple identical copies. It involves isolating a gene or DNA fragment and inserting it into a host organism to generate multiple copies. The key steps include choosing a host and vector, preparing the vector and DNA to be cloned, creating recombinant DNA, introducing it into a host, selecting clones containing the insert, and screening clones. Applications include studying gene expression, producing recombinant proteins, creating transgenic organisms, and developing gene therapies.
The document summarizes the basic steps in a gene cloning experiment. A fragment of DNA containing the gene to be cloned is inserted into a circular DNA molecule called a vector to create a recombinant DNA molecule. The vector then transports the gene into a host cell, usually a bacterium, where the recombinant DNA molecule replicates along with the vector and host cell DNA. As the host cell divides, copies of the recombinant DNA are distributed to new cells, eventually resulting in a clone or colony of identical host cells containing copies of the cloned gene.
Gene cloning in eukaryotes such as Saccharomyces cerevisiae and Pichia pastoris involves inserting foreign DNA into cloning vectors that can replicate in these organisms. Key steps include isolating the cloning vector and gene of interest, inserting the gene into the vector using restriction enzymes and ligase, transforming the vector into host cells, and identifying cell clones carrying the gene. Yeasts like S. cerevisiae are commonly used for eukaryotic gene cloning due to their ability to perform post-translational modifications, though P. pastoris has higher protein yields. Vectors like YACs allow cloning of very large DNA fragments. Eukaryotic expression systems optimize protein production through features like inducible
This document discusses cloning vectors. It begins with a brief history of cloning vectors, noting that the first designed cloning vector was the plasmid pBR322 created in 1977. It then describes the key features of cloning vectors, including an origin of replication, cloning sites, selectable markers like antibiotic resistance genes, and reporter genes. Examples of different types of cloning vectors are also provided, such as plasmids, bacteriophages, cosmids, and artificial chromosomes that can be used in prokaryotes or eukaryotes. The document concludes by differentiating between cloning vectors and expression vectors.
Restriction endonucleases are enzymes that cut DNA at specific sequences. They have been used to map DNA by cutting it into fragments of different sizes that can be separated by gel electrophoresis. More than 3000 restriction endonucleases have been isolated from bacteria and are useful for applications such as cloning DNA fragments into vectors like plasmids. The ability to cut and paste DNA fragments using restriction enzymes and recombinant DNA technology has enabled scientists to study genes and their functions.
Biotechnology uses genetic engineering tools and techniques to manipulate DNA and genes. These tools include restriction enzymes that cut DNA at specific sequences, leaving "sticky ends" that allow DNA fragments to be combined. Plasmids and bacterial transformation allow genes to be inserted and copied within bacteria. This allows bacteria to produce proteins from other organisms, like human insulin. Genetic engineering has applications in medicine and agriculture, like producing disease-resistant crops or improving food quality and nutrition.
Gene cloning involves making many identical copies of a gene. The basic steps include isolating DNA from an organism, cutting the DNA into fragments using restriction enzymes, inserting the fragments into plasmids, and transforming the plasmids into bacterial cells. Each bacterial cell will contain copies of the gene of interest, allowing scientists to isolate and study that gene. Viruses and bacteria can act as vectors, or carriers, to transfer genes into cells. Plasmids and bacteriophages are commonly used vectors for cloning genes into bacterial cells.
Recombinant DNA technology allows genes to be moved between species. This document discusses gene cloning using recombinant DNA technology. It describes how restriction enzymes cut DNA into fragments which can then be joined with other DNA fragments using DNA ligase. Plasmids and bacteriophages are commonly used as cloning vectors to replicate and express inserted DNA fragments in host bacteria. The production of recombinant plasmids and transformation of bacteria allows the cloned DNA fragments to be isolated and purified.
Molecular cloning refers to the process of replicating DNA molecules to generate multiple identical copies. It involves isolating a gene or DNA fragment and inserting it into a host organism to generate multiple copies. The key steps include choosing a host and vector, preparing the vector and DNA to be cloned, creating recombinant DNA, introducing it into a host, selecting clones containing the insert, and screening clones. Applications include studying gene expression, producing recombinant proteins, creating transgenic organisms, and developing gene therapies.
The document summarizes the basic steps in a gene cloning experiment. A fragment of DNA containing the gene to be cloned is inserted into a circular DNA molecule called a vector to create a recombinant DNA molecule. The vector then transports the gene into a host cell, usually a bacterium, where the recombinant DNA molecule replicates along with the vector and host cell DNA. As the host cell divides, copies of the recombinant DNA are distributed to new cells, eventually resulting in a clone or colony of identical host cells containing copies of the cloned gene.
Gene cloning in eukaryotes such as Saccharomyces cerevisiae and Pichia pastoris involves inserting foreign DNA into cloning vectors that can replicate in these organisms. Key steps include isolating the cloning vector and gene of interest, inserting the gene into the vector using restriction enzymes and ligase, transforming the vector into host cells, and identifying cell clones carrying the gene. Yeasts like S. cerevisiae are commonly used for eukaryotic gene cloning due to their ability to perform post-translational modifications, though P. pastoris has higher protein yields. Vectors like YACs allow cloning of very large DNA fragments. Eukaryotic expression systems optimize protein production through features like inducible
This document discusses cloning vectors. It begins with a brief history of cloning vectors, noting that the first designed cloning vector was the plasmid pBR322 created in 1977. It then describes the key features of cloning vectors, including an origin of replication, cloning sites, selectable markers like antibiotic resistance genes, and reporter genes. Examples of different types of cloning vectors are also provided, such as plasmids, bacteriophages, cosmids, and artificial chromosomes that can be used in prokaryotes or eukaryotes. The document concludes by differentiating between cloning vectors and expression vectors.
Restriction endonucleases are enzymes that cut DNA at specific sequences. They have been used to map DNA by cutting it into fragments of different sizes that can be separated by gel electrophoresis. More than 3000 restriction endonucleases have been isolated from bacteria and are useful for applications such as cloning DNA fragments into vectors like plasmids. The ability to cut and paste DNA fragments using restriction enzymes and recombinant DNA technology has enabled scientists to study genes and their functions.
This document discusses cloning vectors and their use in recombinant DNA technology. It defines a cloning vector as a DNA molecule that can accept foreign DNA and replicate within a host cell to produce multiple clones. Examples provided are plasmids, phages, cosmids, and phagemids. Key features of cloning vectors discussed include origins of replication, selectable markers, and restriction enzyme sites. Specific vectors are described in more detail, such as the E. coli plasmid pBR322, phage lambda, cosmids, and phagemids. Cloning vectors allow for the isolation of genes and determination of nucleotide sequences, as well as the investigation of protein function and identification of mutations.
Cloning in gram positive bacteria by neelima sharma,neelima.sharma60@gmail.co...Neelima Sharma
This document discusses cloning in gram-positive bacteria like Bacillus subtilis. Key points include:
1. Vectors for cloning in B. subtilis are often derived from Staphylococcus aureus plasmids which can replicate in B. subtilis.
2. Hybrid plasmids that can replicate in both E. coli and B. subtilis are often used, allowing cloning in E. coli and expression in B. subtilis.
3. Recombinant DNA can be structurally unstable in B. subtilis, so vectors that replicate through the theta mechanism tend to be more stable.
This document provides information about vectors used in molecular cloning. It defines vectors as DNA molecules that can carry foreign genetic material into host cells. The main types of vectors discussed are plasmids, bacteriophages, cosmids, and artificial chromosomes. Plasmids are the most commonly used vectors and can exist in various forms like Ti, F, R, and virulence plasmids. Bacteriophages and viral vectors are also described. The document examines the features of vectors, including origins of replication and selectable markers. It concludes with feedback acknowledging the academic writing course.
This document provides an overview of cloning techniques including restriction enzymes, joining DNA fragments, plasmid vectors, transforming bacteria, and expressing cloned genes. Key points include:
- Restriction enzymes cut DNA at specific sites and were first isolated in 1970, allowing scientists to cut and join DNA fragments.
- In 1972, Berg et al. created the first recombinant DNA molecule by joining fragments of simian virus 40 and lambda phage DNA.
- Boyer and Cohen later isolated the lacZ gene using restriction enzymes and plasmids, demonstrating genes could be amplified in bacteria.
- Plasmids are commonly used as cloning vectors, with features like antibiotic resistance and multiple cloning sites to insert DNA fragments.
Vectors are DNA molecules that can deliver foreign DNA into host cells. The main types of vectors are plasmids, bacteriophages, cosmids, and phagemids. Plasmids are circular, self-replicating DNA molecules that are commonly used as vectors. Key plasmid vectors include pBR322, which contains antibiotic resistance genes, and pUC18/19, which are smaller and contain an ampicillin resistance gene. Bacteriophage vectors like lambda phage and M13 phage can incorporate larger DNA fragments. Cosmids are hybrid vectors that contain phage and plasmid elements, allowing them to replicate like plasmids. Shuttle vectors contain origins of replication from different species, allowing replication in multiple host
Gene cloning involves copying a gene and inserting it into a self-replicating vector to produce multiple copies of the gene. PCR (polymerase chain reaction) is a technique used to amplify specific genes. Both gene cloning and PCR are important for obtaining pure samples of genes and amplifying them for various applications like sequencing, expression of proteins, and genetic engineering.
This document discusses recombinant DNA technology and its various applications. Recombinant DNA is produced by splicing DNA from different sources for purposes such as cloning DNA, genetically engineering organisms, and producing proteins. Key aspects covered include restriction endonucleases, cloning strategies using vectors like plasmids and bacteriophages, cDNA and genomic libraries, DNA sequencing techniques like Sanger sequencing, PCR, expression of eukaryotic proteins in prokaryotes, microarrays, and gene silencing using siRNA.
Gene cloning techniques allow scientists to make multiple copies of gene-sized DNA fragments. The basic cloning process involves inserting a foreign gene into a bacterial plasmid, introducing the recombinant plasmid into bacterial cells, and allowing the bacteria to replicate and produce many copies of the gene. Restriction enzymes cut DNA at specific recognition sites, creating sticky ends that allow insertion of a foreign DNA fragment into a plasmid. Recombinant plasmids are then introduced into bacteria by transformation, allowing clones containing the gene of interest to be identified and isolated.
It is the basics of vector cloning which necessary for every and each student who is intrested in biotechnology. It is only starting, if you want to more than this then please comment on it.
Genetic engineering involves transferring genes between organisms using recombinant DNA techniques. This allows genes to be isolated, cloned, and moved within and between different species. Cloning a gene involves using restriction enzymes to cut DNA at specific sequences, and DNA ligase to join DNA fragments together. Cloned genes have many research uses such as determining gene sequences, altering phenotypes, and obtaining protein products of genes.
This document discusses various cloning vectors that can be used to clone DNA fragments. It describes some key vectors such as plasmids, bacteriophages, cosmids, yeast artificial chromosomes, and bacterial artificial chromosomes. Plasmids are commonly used cloning vectors that can hold inserts of up to 10kb and replicate autonomously in bacteria. Bacteriophages like lambda can accept larger inserts up to 25kb. Cosmids and YACs can hold even larger fragments, up to 40kb and over 2Mb respectively. The choice of vector depends on factors like the insert size, host system, and intended use of the cloned DNA.
Genetic engineering and Recombinant DNAHala AbuZied
Genetic engineering involves altering the DNA of living organisms using biotechnology. It includes techniques like changing single DNA base pairs, deleting or adding genes, or combining DNA from different species. Recombinant DNA technology is used to create recombinant DNA molecules by manipulating DNA in vitro and introducing them into host organisms. This allows bacteria to be engineered to produce human insulin through inserting the human insulin gene into bacterial plasmids. Genomic libraries can be created by ligating fragmented genomic or cDNA into plasmid vectors to transform bacteria and clone the entire genome.
This document provides an overview of DNA cloning. It begins by defining cloning as making identical copies of DNA, genes, or cells. The basic steps of DNA cloning are described, including using a source DNA, vector, restriction enzymes to cut DNA, ligation to join DNA fragments, transformation of host bacteria, and selection of recombinant clones. Common vectors like plasmids are discussed along with selection techniques like blue-white screening. The document emphasizes that the goal is to generate multiple copies of the cloned insert DNA. Examples are given of important medical and agricultural applications of cloning genes.
Molecular (gene) cloning involves extracting a target gene from DNA, inserting it into a vector, and using that vector to make multiple copies of the gene. The vector contains a marker gene and a restriction site that allows the target gene to be inserted. Restriction endonucleases cut the target DNA and vector DNA at specific restriction sites. The cut DNA fragments are then joined together using DNA ligase to form recombinant DNA, which is transferred into a host cell where it can be replicated through the cell's enzymatic machinery, producing many copies of the cloned gene. Molecular cloning has applications in gene study, production of human protein drugs, and vaccine preparation.
The document describes the Gateway cloning system, a molecular biology technique for efficiently transferring DNA fragments between plasmids. It involves site-specific recombination mediated by bacteriophage lambda enzymes and proprietary enzyme mixes. DNA fragments flanked by att sites can be shuttled between donor, entry, and destination vectors for functional analysis and protein expression. The process is based on two recombination reactions - BP reaction inserts DNA into an entry vector, while LR reaction transfers it to an expression vector. This technique provides a rapid and accurate method for cloning genes into multiple vectors.
Recombinant DNA technology involves isolating a specific fragment of DNA containing a desired gene, inserting this fragment into a plasmid or bacterial chromosome, and using the modified DNA to reproduce and synthesize copies of the gene. Key steps include extracting and purifying DNA, using restriction enzymes to fragment the DNA, isolating the fragment containing the target gene, ligating this fragment into a carrier molecule like a plasmid, transforming target cells with the recombinant DNA, and allowing the cells to replicate and express the inserted gene.
Plasmids are commonly used as cloning vectors. They are circular DNA molecules that can replicate independently of the bacterial chromosome. Many plasmid cloning vectors were constructed from bacterial plasmids and contain an origin of replication, selectable marker like antibiotic resistance, and a multiple cloning site to insert DNA fragments. Successful clones can be identified by techniques like blue-white screening where expression of a gene like lacZ allows colonies to be screened blue or white.
The document is a transcript from a talk given by Jonathan A. Eisen at the Lake Arrowhead Microbial Genomes conference in 2010. It includes quotes from past conferences, suggested homework activities related to microbial genomes and Lake Arrowhead, and a discussion of increasing phylogenetic coverage of bacterial and archaeal lineages to fill gaps in genomic sampling across the domains.
Paul Roos Grammar School's choir, The Awesome Foursome, participated in an annual choir festival hosted by Stellenbosch High School on May 10th. The festival featured performances from Rhenish High School, Paul Roos Grammar School, Stellenbosch High School, and the Tygerberg Children's Choir. All choirs delivered performances worthy of the festival's title of "Awesome Foursome".
This document discusses cloning vectors and their use in recombinant DNA technology. It defines a cloning vector as a DNA molecule that can accept foreign DNA and replicate within a host cell to produce multiple clones. Examples provided are plasmids, phages, cosmids, and phagemids. Key features of cloning vectors discussed include origins of replication, selectable markers, and restriction enzyme sites. Specific vectors are described in more detail, such as the E. coli plasmid pBR322, phage lambda, cosmids, and phagemids. Cloning vectors allow for the isolation of genes and determination of nucleotide sequences, as well as the investigation of protein function and identification of mutations.
Cloning in gram positive bacteria by neelima sharma,neelima.sharma60@gmail.co...Neelima Sharma
This document discusses cloning in gram-positive bacteria like Bacillus subtilis. Key points include:
1. Vectors for cloning in B. subtilis are often derived from Staphylococcus aureus plasmids which can replicate in B. subtilis.
2. Hybrid plasmids that can replicate in both E. coli and B. subtilis are often used, allowing cloning in E. coli and expression in B. subtilis.
3. Recombinant DNA can be structurally unstable in B. subtilis, so vectors that replicate through the theta mechanism tend to be more stable.
This document provides information about vectors used in molecular cloning. It defines vectors as DNA molecules that can carry foreign genetic material into host cells. The main types of vectors discussed are plasmids, bacteriophages, cosmids, and artificial chromosomes. Plasmids are the most commonly used vectors and can exist in various forms like Ti, F, R, and virulence plasmids. Bacteriophages and viral vectors are also described. The document examines the features of vectors, including origins of replication and selectable markers. It concludes with feedback acknowledging the academic writing course.
This document provides an overview of cloning techniques including restriction enzymes, joining DNA fragments, plasmid vectors, transforming bacteria, and expressing cloned genes. Key points include:
- Restriction enzymes cut DNA at specific sites and were first isolated in 1970, allowing scientists to cut and join DNA fragments.
- In 1972, Berg et al. created the first recombinant DNA molecule by joining fragments of simian virus 40 and lambda phage DNA.
- Boyer and Cohen later isolated the lacZ gene using restriction enzymes and plasmids, demonstrating genes could be amplified in bacteria.
- Plasmids are commonly used as cloning vectors, with features like antibiotic resistance and multiple cloning sites to insert DNA fragments.
Vectors are DNA molecules that can deliver foreign DNA into host cells. The main types of vectors are plasmids, bacteriophages, cosmids, and phagemids. Plasmids are circular, self-replicating DNA molecules that are commonly used as vectors. Key plasmid vectors include pBR322, which contains antibiotic resistance genes, and pUC18/19, which are smaller and contain an ampicillin resistance gene. Bacteriophage vectors like lambda phage and M13 phage can incorporate larger DNA fragments. Cosmids are hybrid vectors that contain phage and plasmid elements, allowing them to replicate like plasmids. Shuttle vectors contain origins of replication from different species, allowing replication in multiple host
Gene cloning involves copying a gene and inserting it into a self-replicating vector to produce multiple copies of the gene. PCR (polymerase chain reaction) is a technique used to amplify specific genes. Both gene cloning and PCR are important for obtaining pure samples of genes and amplifying them for various applications like sequencing, expression of proteins, and genetic engineering.
This document discusses recombinant DNA technology and its various applications. Recombinant DNA is produced by splicing DNA from different sources for purposes such as cloning DNA, genetically engineering organisms, and producing proteins. Key aspects covered include restriction endonucleases, cloning strategies using vectors like plasmids and bacteriophages, cDNA and genomic libraries, DNA sequencing techniques like Sanger sequencing, PCR, expression of eukaryotic proteins in prokaryotes, microarrays, and gene silencing using siRNA.
Gene cloning techniques allow scientists to make multiple copies of gene-sized DNA fragments. The basic cloning process involves inserting a foreign gene into a bacterial plasmid, introducing the recombinant plasmid into bacterial cells, and allowing the bacteria to replicate and produce many copies of the gene. Restriction enzymes cut DNA at specific recognition sites, creating sticky ends that allow insertion of a foreign DNA fragment into a plasmid. Recombinant plasmids are then introduced into bacteria by transformation, allowing clones containing the gene of interest to be identified and isolated.
It is the basics of vector cloning which necessary for every and each student who is intrested in biotechnology. It is only starting, if you want to more than this then please comment on it.
Genetic engineering involves transferring genes between organisms using recombinant DNA techniques. This allows genes to be isolated, cloned, and moved within and between different species. Cloning a gene involves using restriction enzymes to cut DNA at specific sequences, and DNA ligase to join DNA fragments together. Cloned genes have many research uses such as determining gene sequences, altering phenotypes, and obtaining protein products of genes.
This document discusses various cloning vectors that can be used to clone DNA fragments. It describes some key vectors such as plasmids, bacteriophages, cosmids, yeast artificial chromosomes, and bacterial artificial chromosomes. Plasmids are commonly used cloning vectors that can hold inserts of up to 10kb and replicate autonomously in bacteria. Bacteriophages like lambda can accept larger inserts up to 25kb. Cosmids and YACs can hold even larger fragments, up to 40kb and over 2Mb respectively. The choice of vector depends on factors like the insert size, host system, and intended use of the cloned DNA.
Genetic engineering and Recombinant DNAHala AbuZied
Genetic engineering involves altering the DNA of living organisms using biotechnology. It includes techniques like changing single DNA base pairs, deleting or adding genes, or combining DNA from different species. Recombinant DNA technology is used to create recombinant DNA molecules by manipulating DNA in vitro and introducing them into host organisms. This allows bacteria to be engineered to produce human insulin through inserting the human insulin gene into bacterial plasmids. Genomic libraries can be created by ligating fragmented genomic or cDNA into plasmid vectors to transform bacteria and clone the entire genome.
This document provides an overview of DNA cloning. It begins by defining cloning as making identical copies of DNA, genes, or cells. The basic steps of DNA cloning are described, including using a source DNA, vector, restriction enzymes to cut DNA, ligation to join DNA fragments, transformation of host bacteria, and selection of recombinant clones. Common vectors like plasmids are discussed along with selection techniques like blue-white screening. The document emphasizes that the goal is to generate multiple copies of the cloned insert DNA. Examples are given of important medical and agricultural applications of cloning genes.
Molecular (gene) cloning involves extracting a target gene from DNA, inserting it into a vector, and using that vector to make multiple copies of the gene. The vector contains a marker gene and a restriction site that allows the target gene to be inserted. Restriction endonucleases cut the target DNA and vector DNA at specific restriction sites. The cut DNA fragments are then joined together using DNA ligase to form recombinant DNA, which is transferred into a host cell where it can be replicated through the cell's enzymatic machinery, producing many copies of the cloned gene. Molecular cloning has applications in gene study, production of human protein drugs, and vaccine preparation.
The document describes the Gateway cloning system, a molecular biology technique for efficiently transferring DNA fragments between plasmids. It involves site-specific recombination mediated by bacteriophage lambda enzymes and proprietary enzyme mixes. DNA fragments flanked by att sites can be shuttled between donor, entry, and destination vectors for functional analysis and protein expression. The process is based on two recombination reactions - BP reaction inserts DNA into an entry vector, while LR reaction transfers it to an expression vector. This technique provides a rapid and accurate method for cloning genes into multiple vectors.
Recombinant DNA technology involves isolating a specific fragment of DNA containing a desired gene, inserting this fragment into a plasmid or bacterial chromosome, and using the modified DNA to reproduce and synthesize copies of the gene. Key steps include extracting and purifying DNA, using restriction enzymes to fragment the DNA, isolating the fragment containing the target gene, ligating this fragment into a carrier molecule like a plasmid, transforming target cells with the recombinant DNA, and allowing the cells to replicate and express the inserted gene.
Plasmids are commonly used as cloning vectors. They are circular DNA molecules that can replicate independently of the bacterial chromosome. Many plasmid cloning vectors were constructed from bacterial plasmids and contain an origin of replication, selectable marker like antibiotic resistance, and a multiple cloning site to insert DNA fragments. Successful clones can be identified by techniques like blue-white screening where expression of a gene like lacZ allows colonies to be screened blue or white.
The document is a transcript from a talk given by Jonathan A. Eisen at the Lake Arrowhead Microbial Genomes conference in 2010. It includes quotes from past conferences, suggested homework activities related to microbial genomes and Lake Arrowhead, and a discussion of increasing phylogenetic coverage of bacterial and archaeal lineages to fill gaps in genomic sampling across the domains.
Paul Roos Grammar School's choir, The Awesome Foursome, participated in an annual choir festival hosted by Stellenbosch High School on May 10th. The festival featured performances from Rhenish High School, Paul Roos Grammar School, Stellenbosch High School, and the Tygerberg Children's Choir. All choirs delivered performances worthy of the festival's title of "Awesome Foursome".
Vinted is an online marketplace app that allows users to buy and sell used clothing and accessories. It has seen strong growth in Europe and does $10 million in monthly sales volume. Vinted is looking to Facebook to help with scalability, precise targeting of users, and accurate tracking. Facebook allows Vinted to target users based on interests, device/operating system, lookalikes, and customer acquisition from mobile apps to help further grow its business.
Defrag Panel _Filtering information through a social network _ TamccannT. A. McCann
The document discusses statistics related to online content creation and consumption including that 290,000 words are written per minute worldwide, 12.5 billion words are created per month, and there are 10 million content creators and 300 million readers monthly. It also notes that over 50% of traffic on Gist.com comes from outside North America and 66% is in a language other than English. The document advocates that both timely information and code/people are important to find and understand online content. It provides a proposed formula for calculating a person's importance based on interactions and connections on social media and email.
SSL/TLS Heartbleed.
In questo talk parlo molto velocemente del bug SSL/TLS Heartbleed, bug che ha afflitto dal 2012 al 2014 la cryptolibreria di OpenSSL. Sfruttando il suddetto bug era possibile violare completamente una comunicazione protetta da SSL/TLS. Il talk si conclude spiegando all'utente che il bug è stato risolto grazie al fatto che il progetto di OpenSSL è OpenSource e questo ha facilitato di molto la scoperta e la rilevazione del codice buggato.
Минуло два года с тех пор , как Михаил Столяров и Олег Гарин вернулись из Зоны . Столяров продолжает служить в СБУ , Гарин удачно женился и занялся бизнесом. Однако в мире вновь начинают происходить необъяснимые события : терпят крушения самолеты и поезда , пропадают люди . В одной из таких аварий погибает жена Олега Гарина , и он уже не в состоянии больше закрывать глаза на эти происшествия . С ним связывается подполковник Столяров и подтверждает его догадку : череда катастроф – не что иное , как признаки воздействия пси – оружия . Гариным движет жажда мести , а Столяровым – профессиональный , офицерский долг , и оба прекрасно знают , где следует искать разработчиков этого страшного оружия . Итак , они возвращаются в Зону ..
Увлекательное чтение Вам гарантировано ..
Este documento describe Suite-EBS, una plataforma omnicanal que fusiona un e-commerce, terminal punto de venta y ERP. Suite-EBS ayuda a los comercios a vender más al administrar todos sus canales de venta desde una única aplicación. Ofrece tienda online, gestión de pedidos, inventario, facturación y más para adaptarse a los hábitos de compra omnicanal de los consumidores.
Чему мы научились разрабатывая микросервисы?Vadim Madison
Доклад с конференции Backend Conf 2016
Начав разработку нового продукта через микросервисы, мы неожиданно для себя обнаружили, что микросервисы — это не просто "вместо одного большого приложения теперь пишем много маленьких". При разработке большой системы она сама собой через какое-то время становится набором отдельных сервисов, которые должны взаимодействовать между собой, поэтому стабильная работа сервисов и их взаимодействие не стало чем-то новым. Неожиданностью стало то, что система стала значительно более динамической, она стала постоянно изменяться отдельными небольшими частями, сервисы стали часто перезапускаться, а количество запущенных нод сервисов стало расти по экспоненте.
Очень быстро стал актуальным вопрос конфигурирования — если раньше, выкатив новую версию монолита с единым конфигом, мы применяли правки на всю систему сразу, то с микросервисами все сложнее — пара сотен работающих нод и всем нужно применить изменения. Требования к деплою также поменялись — он стал частью процесса разработки, а тестирование стало частью деплоя. Количество необходимого ПО для функционирования системы также стало некоторым сюрпризом.
В докладе я расскажу о том, как в итоге это работает у нас, как мы решили такие вопросы как:
- конфигурирование сервисов;
- интеграция между собой;
- тестирование;
- версионирование;
- масштабирование.
Расскажу, какие тулзы мы в итоге используем, а от каких отказались.
AWS IoT is a managed cloud platform that lets connected devices easily and securely interact with cloud applications and other devices. In this session, we will discuss how constrained devices can leverage AWS IoT to send data to the cloud and receive commands back to the device using the protocol of their choice. We will discuss how devices can connect securely using MQTT and HTTP protocols, and how can developers and businesses can leverage the AWS IoT Rules Engine, Thing Shadows, and accelerate prototype development using AWS IoT Device SDKs. We will cover major hardware platforms from Arduino, Marvell, Dragonboard and MediaTek.
A prezentációmban Forgós Sándor e-learninges tananyagát használtam fel. Szó esik benne a rádiózás illetve a televíziózás kialakulásáról. Fontosabb évszámokról, illetve személyekről.
Moving DNUG Usergroup von on-premise in die IBM Connections CloudSynCoTec
Vortrag gehalten am 10.03.2016 auf der ics.ug in Hamburg.
Zum Inhalt: Ende des Jahres 2015 wurde die DNUG Geschäftsstelle aufgelöst und die bestehende on-premise Domino Infrastruktur in die Cloud und somit in eine virtuelle Geschäftsstelle umgezogen. In einem ersten Schritt wurden die vorhandenen Mailboxen mithilfe des IBM Mail Onboarding Managers in eine IBM SmartCloud Notes Hybrid Umgebung migriert. Wer sich also derzeit oder in Zukunft mit einer Mailbox-Migration in die IBM Cloud beschäftigen möchte bzw. muß, kommt um dieses Tool nicht herum und erfährt in dieser Session, wie es funktioniert und insbesondere auch das, was nicht funktioniert: #lessonslearned und #bestpractices.
Informal Job Sector or the Blue Collar Job Market makes a significant portion of Indian economy.
There is no centralized system to organize the informal job to arrange to offer better quality permanent jobs to them.
The document summarizes the process of transforming E. coli bacteria to produce fluorescent proteins. It will be done by inserting a plasmid containing a gene for a fluorescent protein into E. coli. The plasmid also contains an antibiotic resistance gene so that transformed bacteria can be selected by growing them in media containing antibiotics. The foreign DNA from the plasmid will be transcribed and translated within the bacterial cells, causing them to produce and glow with the fluorescent protein. This demonstrates how altering an organism's genotype through molecular biology techniques can change its phenotype.
This document discusses genomics and genome sequencing. It provides an overview of the history of genome sequencing including early organisms sequenced like bacteriophage. It describes how genomes are sequenced through library construction, cloning, and strategies like Sanger sequencing. Applications of genome sequencing are also mentioned such as predicting genes, studying genome organization and evolution, and understanding the genetic basis of disease.
Genetic engineering involves the synthesis and manipulation of genes. Scientists can synthesize genes in the lab and create recombinant DNA by combining DNA fragments from different sources. This allows for the transfer of genes between organisms. Common techniques include using restriction enzymes to cut DNA at specific sites, inserting the DNA into vectors like plasmids, and cloning the chimeric molecules in host cells. Applications include producing recombinant proteins like vaccines, hormones, and antibodies.
1. Molecular cloning involves cutting DNA from one organism and inserting it into a vector that can replicate in a host organism, allowing the DNA fragment to be amplified. Recombinant DNA technology uses restriction enzymes to cut DNA into fragments that are then ligated into cloning vectors like plasmids or bacteriophages.
2. After transforming host bacteria with the recombinant vector, clones containing the inserted DNA fragment can be selected for and amplified. Colonies containing the insert are identified through antibiotic resistance or colorimetric markers present on the vector.
3. cDNA libraries provide a way to clone and study eukaryotic genes. mRNA is isolated and reverse transcribed into cDNA, which is then ligated into vectors and
Recombinant DNA technology involves intentionally modifying organisms' genomes for practical purposes such as eliminating undesirable traits, combining beneficial traits from different organisms, or creating organisms that synthesize useful products. The key steps involve isolating a gene of interest, inserting it into a plasmid, introducing the plasmid into bacteria, and harvesting copies of the gene or its protein products. Common tools used include mutagens, reverse transcriptase to synthesize cDNA, synthetic nucleic acids, restriction enzymes to cut DNA at specific sites, vectors to deliver genes into cells, and gene libraries containing collections of cloned genes.
Cloning involves creating identical copies of DNA fragments. Molecular cloning uses restriction enzymes to cut DNA into fragments which are then joined to plasmid vectors via ligation. The recombinant plasmids are introduced into host bacteria via transformation. This allows the DNA fragment to be amplified in large quantities for further study and manipulation. Common vectors used in cloning include plasmids, bacteriophages, cosmids, and YACs which can accommodate varying sizes of DNA inserts.
1. Bacteria and viruses come in various shapes and sizes, with bacteria being 1-5 micrometers on average and viruses requiring a living host cell to reproduce.
2. Bacteria are classified as either eubacteria or archaebacteria based on their cell structure, while viruses contain either DNA or RNA surrounded by a protein capsid.
3. Bacteria and viruses can cause diseases through various mechanisms, such as releasing toxins or disrupting the host cell, and are controlled through vaccination, sterilization, antibiotics, and proper hygiene.
Bacteria are the oldest living structures on Earth and can be categorized into three kingdoms - Archaebacteria, Eubacteria, and Eukaryotes. Archaebacteria inhabit extreme environments and have cell walls that do not contain peptidoglycan. Eubacteria are more diverse and have cell walls containing peptidoglycan. Bacteria exist in various shapes, groupings, and sizes from 0.5 to 10 microns. They reproduce through binary fission and exchange genetic information through transformation, conjugation, and transduction. Bacteria play important economic roles in nitrogen fixation, nutrient recycling, food production, and medicine.
Plasmids are small, circular DNA molecules that can replicate independently of the host chromosome. They are commonly used in molecular biology to clone and amplify genes. The summarized document describes the key functional elements found in yeast overexpression plasmids, including selection markers like URA3, origins of replication, promoters, and tags. It also summarizes how plasmids are constructed through molecular cloning and purified from bacteria using their unique physical properties like supercoiling and renaturation compared to host chromosomal DNA and proteins. Purification involves alkaline lysis to break open cells, neutralization to allow plasmid renaturation, and multiple wash steps on a silica resin column to isolate pure plasmid DNA.
The document discusses using functional genomic approaches like quantitative PCR (qPCR), proteomics, and next-generation sequencing (NGS) to better understand interactions between shellfish, pathogens, and the environment. It provides case studies on using these approaches to discover genes in naturally disease-resistant oysters and clams and a pathogenic Vibrio bacteria. NGS was found to be effective for gene discovery and expression analysis when a reference genome is available, while proteomics identified pathogen virulence pathways but faces barriers. The goal is to improve shellfish health and predict disease outbreaks under environmental change.
The document provides an overview of cell structure and function. It describes the key organelles found in eukaryotic cells like the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, lysosomes, and cytoskeleton. It explains how these organelles allow cells to carry out essential processes like protein production, energy production, waste removal, cell signaling, and cell division. The roles of membranes, enzymes, and transport systems within organelles are also summarized.
Chapter 4 Explain How Bacterial Plasmids Differ From...Melissa Luster
Bacterial plasmids differ from chromosomes in that they are not essential for bacterial growth or metabolism. They are extrachromosomal DNA that can contain genes allowing bacteria to survive in stressful conditions. This document discusses the process of using PCR to isolate the LacZ gene from E. coli and ligate it to a plasmid vector for future experiments. It describes the primer design, PCR reaction setup, and gel electrophoresis to confirm successful amplification of the target gene fragment.
The document discusses various vector systems used for molecular cloning, including plasmids like pBR322 and pUC, phages such as λ phage and cosmids, and phagemids. λ phage and cosmids can accept large DNA inserts up to 20-50kb, making them useful for genomic libraries. M13 phages produce single-stranded recombinant DNA useful for sequencing and mutagenesis, while plasmids called phagemids also generate single-stranded DNA with helper phages.
DNA cloning allows for the replication and amplification of specific DNA fragments. It involves inserting DNA fragments into vector molecules, such as plasmids, which are then introduced into host cells. The vector carries the DNA fragment into the host cell and acts as a replicating molecule. Restriction enzymes and ligases are used to cut out the DNA fragment and insert it into the vector. The cells are then grown on selective media so that cells containing the vector and insert are selected for. This allows unlimited copies of the DNA fragment of interest to be produced for study and manipulation.
Vectors are DNA molecules that can carry foreign DNA fragments into host cells. There are two major classes of vectors: plasmids and phages. Plasmid vectors like pBR322 were some of the earliest cloning vectors and have replication origins, antibiotic resistance genes, and multiple cloning sites. PUC plasmids are derived from pBR322 and use blue-white screening. Lambda phages can accommodate larger DNA fragments than plasmids. Cosmids and phagemids have characteristics of both plasmids and phages, allowing larger DNA fragments to be cloned and packaged. M13 phages produce single-stranded DNA clones. Different vector types are suited for various cloning and expression purposes.
Transformation and transfection allow the genetic alteration of cells through the introduction of foreign DNA. Transformation refers specifically to bacteria, where naked DNA fragments can be taken up through natural competence or artificial methods like heat shock or electroporation. Transfection applies to eukaryotic cells, using techniques like lipofection to introduce DNA through membrane pores. Common methods to transform plants include Agrobacterium infection, particle bombardment, and electroporation. These techniques generate genetically modified cells and organisms.
The document discusses cloning vectors. It defines a vector as a DNA molecule used to transport exogenous DNA into a host cell. Cloning refers to creating clones of organisms, cells, or DNA fragments. The process of molecular cloning involves digesting DNA fragments with restriction enzymes, ligating the target fragment into a vector, and introducing the construct into a host cell. Common vectors include plasmids, bacteriophages, cosmids, BACs, YACs, and retroviral vectors. Each has advantages like replication ability and ability to incorporate large DNA inserts that make them suitable for cloning different sizes of DNA fragments.
Biotechnology refers to the use of living organisms or their components to develop products and processes. It has applications in fields like agriculture, medicine, and industry. Modern biotechnology techniques include genetic engineering and aseptic techniques. Genetic engineering involves altering genetic material through techniques like recombinant DNA, gene transfer into host organisms, and gene cloning. It allows scientists to modify organisms for useful purposes. Restriction enzymes, vectors, DNA polymerase and ligase are important tools used in genetic engineering and recombinant DNA technology.
This document discusses host cells and vectors used in gene cloning. It describes various prokaryotic and eukaryotic host cells, including E. coli, yeast, and mammalian cells. It also discusses the key features and types of vectors, including plasmids, bacteriophages, cosmids, and phagemids. Plasmids are the most commonly used prokaryotic vectors and come in various types including low-copy and high-copy plasmids. Common plasmid vectors discussed include pBR322, pUC18, and commercially available vectors. Bacteriophages like lambda phage and M13 phage are also described as viral vectors.
Bacterial heredity and variation can occur through several mechanisms. Genetic variation arises from mutations in bacterial chromosomes and genetic elements like plasmids, bacteriophages, and transposable elements. Gene transfer and recombination can also introduce variation as bacteria can undergo transformation, conjugation, and transduction to exchange genetic material. This allows bacteria to evolve new traits like antibiotic resistance or changes in virulence over multiple generations.
This document provides a review for a Physical Science final exam, outlining 9 competencies covered on the exam. It includes 75 multiple choice and short answer questions testing understanding of concepts in motion, waves, electricity, thermodynamics, atomic structure, nuclear processes, bonding, and acids/bases. Sample questions assess knowledge of the scientific method, graphing, Newton's laws, energy transformations, electromagnetic radiation, the periodic table, nuclear reactions, and chemical equations.
This document provides 42 multi-part physics problems involving Newton's laws of motion. The problems cover concepts such as force, mass, acceleration, weight, and their relationships. Some sample answers are provided. The problems involve calculating unknown values like force, mass, or acceleration given information about real-world scenarios involving objects in motion or at rest under the influence of various forces.
1. This document discusses different types of waves including transverse, longitudinal, and electromagnetic waves. It defines key wave properties such as amplitude, wavelength, frequency, period, and wave speed.
2. Frequency is defined as the number of vibrations per second, measured in Hertz (Hz). Period is the time for one full vibration. Frequency and period are inversely related.
3. Examples are provided to demonstrate calculating wave properties like frequency, period, wavelength, and wave speed from information given about the wave.
This document discusses electrical power and energy. It explains that power is calculated as current multiplied by voltage, and is measured in watts. It asks the reader to calculate the power needed to operate a clock radio drawing 0.05 amps from a household circuit. The document also explains that electrical energy is provided by power companies and sold to homeowners in units of kilowatt-hours, which is 1000 watts delivered for one hour. It provides an example of calculating the electrical energy used and cost for a 1200W toaster oven used for 15 minutes.
This document explains the differences between alternating current (AC) and direct current (DC). It defines AC as an electric current that periodically reverses direction and changes its magnitude continuously with time in contrast to DC, which flows in one direction. The document also outlines the key characteristics of series and parallel electric circuits. Series circuits have the same current flowing through all elements and the total voltage is divided among the elements. Parallel circuits have the same voltage across each element and the total current is the sum of the currents in the individual branches. The document concludes by noting that fuses are used to prevent circuit overloading by melting and breaking the circuit if too much current passes through.
This document provides an Ohm's Law worksheet with 6 practice problems calculating voltage, current, and resistance using the equations: I = V/R, R = V/I, and V = IR. Students are asked to use these equations to find the missing value in each circuit scenario, such as calculating the voltage applied to a light bulb with a known current and resistance.
This document contains a worksheet on Ohm's Law with 14 problems. The worksheet provides the three forms of Ohm's Law and asks students to calculate values like voltage, current, and resistance using circuits with resistors and batteries. Students are asked to determine unknown values, total resistances, and currents in various circuit diagrams applying the relationships defined by Ohm's Law.
This document provides an Ohm's Law worksheet with 6 practice problems calculating voltage, current, and resistance using the equations: I = V/R, R = V/I, and V = IR. Students are asked to use these equations to find the missing value in each circuit scenario, such as calculating the voltage applied to a light bulb with a known current and resistance.
This document discusses resistance and Ohm's Law. It describes the key parts of Ohm's Law including volts, amps, and resistance. It also explains how to calculate an unknown value using two known values and Ohm's Law. Examples are provided to demonstrate calculating current and resistance using Ohm's Law. The document also discusses how resistance affects current and electric shock, and provides examples of calculating current through the body at different resistances and voltages.
Static electricity and electrical currantssbarkanic
This document defines static electricity and current electricity. It explains that static electricity is caused by an imbalance of electric charges, usually through rubbing materials together, while current electricity involves the controlled flow of electrons. It distinguishes conductors that allow electron flow from insulators that do not, and describes how static charges build up and arc in lightning.
This document covers acids and bases, including definitions, properties, examples and the pH scale. It also discusses acid rain, its effects and causes. For radioactivity, it defines different types and compares the strong force to the electric force in alpha and beta equations. It explains transmutation, half-life, fission and chain reactions. Additionally, it outlines nuclear power plants, how they create electricity from fission, reasons for past meltdowns and pros and cons of nuclear power. Finally, it addresses the big bang theory, evidence supporting it, the potential end of the universe, star formation, star types and life cycles.
This document discusses chemical equations and reactions. It explains that chemical equations are used to represent chemical reactions, and that they consist of reactants on the left side of the arrow yielding products on the right. It also describes how to balance chemical equations by adjusting coefficients so that the same number of each type of atom is on both sides of the equation. Balancing chemical equations ensures conservation of mass during chemical reactions.
Naming and writing compounds and moleculessbarkanic
This document provides instructions for writing formulas and naming ionic compounds, covalent molecules, and polyatomic ions. It explains that for ionic compounds, you write the symbols of the ions and use the crossover method to determine subscripts before naming the compound by writing the cation name followed by the anion name with "ide." For covalent molecules, Greek prefixes indicate subscripts and the name is written by specifying each element followed by the number of atoms. Polyatomic ions are also named and included in ionic compounds by looking up their formula and charge. Examples and practice problems are provided to demonstrate the process.
1) The document provides instructions for drawing Lewis structures to show ionic and covalent bonding between various elements. Students are asked to draw Lewis structures for pairs of elements, and indicate electron transfers or sharing to write chemical formulas. 2) For ionic bonds, students should draw Lewis structures, arrows to show electron transfer, charges for each ion, and chemical formulas. 3) For covalent bonds, the instructions are to draw Lewis structures, circles around shared electrons, bond structures, and chemical formulas.
The document discusses atomic spectra and the Bohr model. It explains that atoms can absorb and emit light at specific frequencies, and this atomic spectrum acts as a fingerprint that can be used to identify elements. The Bohr model describes electrons occupying different energy shells around the nucleus, and electrons absorbing and emitting energy by jumping between shells and releasing light. The document also briefly mentions flame tests and spectroscopes as methods to observe atomic spectra.
Ernest Rutherford (1871-1937) was a notable British physicist and chemist who made seminal contributions to the development of the modern atomic model. Through his gold foil experiment in 1911, Rutherford was able to formulate the Rutherford model of the atom, which established that atoms have a small, positively charged nucleus surrounded by low-mass electrons. For this breakthrough discovery, Rutherford received numerous honors including the Nobel Prize in Chemistry in 1908. His work fundamentally changed scientific understanding of atomic structure.
Lise Meitner was an Austrian/German physicist born in 1878 who made significant contributions to nuclear physics. She received her doctorate in 1905 as the second woman to earn a PhD from the University of Vienna. In 1938, Meitner, Otto Hahn, and Fritz Strassmann discovered nuclear fission when bombarding uranium with neutrons. This splitting of uranium atoms led to additional neutrons and the potential for an explosive chain reaction. Sadly, her discovery was later used in 1945 for the atomic bomb dropped on Hiroshima. Meitner received several honors for her work, including the Max Planck medal in 1949.
Murray Gell-Mann was born in 1929 and is still living. He graduated valedictorian from Columbia Grammar School and attended Yale University at age 15. Gell-Mann won the 1969 Nobel Prize in Physics. In 1964, he discovered the quark, which makes up protons and neutrons in the nucleus. Quarks have never been isolated due to their small size of 10-15 mm. Gell-Mann is also interested in activities like bird watching and collecting antiques.
Democritus was a Greek philosopher born around 460-457 BC in Abdera, Thrace. He developed the first atomic theory, proposing that all matter is made up of indivisible atoms moving through empty space. Democritus believed that atoms were the fundamental building blocks of the natural world and that their behavior determined natural phenomena. He and his mentor Leucippus are considered the founders of atomic theory. Democritus was highly respected in his lifetime for making discoveries and predictions that were later proven true.
1. A Little More Advanced
Biotechnology Tools
Better Plasmids
AP Biology 2007-2008
2. Engineered plasmids
Building custom plasmids
restriction enzyme sites
antibiotic resistance genes as a selectable marker
EcoRI
BamHI HindIII
restriction sites
Selectable marker
antibiotic resistance
gene on plasmid
ampicillin plasmid
resistance
selecting for
successful
transformation ori amp
AP Biology resistance
successful uptake
3. Selection for plasmid uptake
Antibiotic becomes a selecting agent
only bacteria with the plasmid will grow
on antibiotic (ampicillin) plate
only transformed
all bacteria grow bacteria grow
a
a a a a
a a a
a
a a a a a
a a
a
LB plate LB/amp plate
AP Biology
cloning
4. Need to screen plasmids
Need to make sure bacteria have
recombinant plasmid
restriction sites inserted
EcoRI all in LacZ gene gene
BamHI of interest
HindIII
LacZ gene broken
LacZ gene
lactose → blue color lactose → white color
X
recombinant
plasmid plasmid
amp amp
resistance resistance
origin of
AP Biology
replication
5. Screening for recombinant plasmid
Bacteria take up plasmid Bacteria take up recombinant plasmid
Functional LacZ gene Non-functional LacZ gene
Bacteria make blue color Bacteria stay white color
Which colonies
do we want?
AP Biology
7. Finding your gene of interest
DNA hybridization
find sequence of DNA using a labeled probe
short, single stranded DNA molecule
complementary to part of gene of interest
labeled with radioactive P32 or fluorescent dye
heat treat DNA in gel
unwinds (denatures) strands
wash gel with probe
probe hybridizes with denatured DNA
labeled probe
genomic DNA G A T C A G T A G
C T A G T C A T C
AP Biology
8. Southern blotting
restriction digest gel electrophoresis blot DNA off of gel
onto filter paper
expose filter paper to
wash filter with labeled probe
AP Biology
X-ray film
9. Edwin Southern
Southern blotting
gel of genomic DNA Southern blot Southern blot
IDing one gene illustration
AP Biology
10. DNA libraries
Cut up all of nuclear DNA
from many cells of an
organism
restriction enzyme
Clone all fragments into
many plasmids at same time
“shotgun” cloning
Create a stored collection of
DNA fragments
petri dish has a collection
of all DNA fragments from
the organism
AP Biology
11. Making a DNA library 2
1
all DNA from many cells engineered plasmid
of an organism is cut with selectable marker
with restriction enzymes & screening system
gene of interest
3
all DNA fragments
4 inserted into many
clone plasmids plasmids
into bacteria
AP Biology
12. But how
do we find
DNA library colony with our
gene of interest
recombinant plasmids in it?
inserted into bacteria gene of interest
DNA Library
plate of bacterial colonies
?
storing & copying all genes
from an organism (ex. human)
AP Biology
13. Find your gene in DNA library
Locate Gene of Interest
to find your gene you need some of
gene’s sequence
if you know sequence of protein…
can “guess” part of DNA sequence
“back translate” protein to DNA
if you have sequence of similar gene from
another organism…
use part of this sequence
Which
bacterial colony
?
has our gene?
Like a needle
AP Biology in a haystack!
14. Colony Blots 4
Locate
1 Cloning
- expose film
- plate with bacterial - locate colony on plate
colonies carrying from film
recombinant plasmids
plate
plate + filter film
2 3
Replicate plate Hybridization
- press filter paper onto - heat filter paper to
plate to take sample of denature DNA
cells from every colony filter - wash filter paper with
radioactive probe
which will only attach
AP Biology to gene of interest
15. Problems…
Human Genome library
are there only genes in there?
nope! a lot of junk!
human genomic library has more “junk”
than genes in it
Clean up the junk!
if you want to clone
a human gene into
bacteria, you can’t
have…
introns
AP Biology
16. How do you clean up the junk?
Don’t start with DNA…
Use mRNA
copy of the gene without the junk!
But in the end, you need DNA to clone into
plasmid…
How do you go from RNA → DNA?
reverse transcriptase from RNA viruses
retroviruses
reverse
AP Biology transcriptase
17. cDNA (copy DNA) libraries
Collection of only the
coding sequences of
expressed genes
extract mRNA from
cells
reverse transcriptase
RNA → DNA
from retroviruses
clone into plasmid
Applications
need edited DNA for
expression in bacteria
human insulin
AP Biology
18. Where do we go next….
DNA RNA protein trait
When a gene is turned on, it creates a trait
want to know what gene is being expressed
extract mRNA from cells How do you match mRNA
mRNA = active genes back to DNA in cells???
reverse
AP Biology transcriptase
19. slide with spots of DNA
Microarrays each spot = 1 gene
Create a slide with a sample of each gene from the
organism
each spot is one gene
Convert mRNA → labeled cDNA mRNA → cDNA
mRNA from cells
reverse
transcriptase
AP Biology
20. slide with spots of DNA
Microarrays each spot = 1 gene
Labeled cDNA hybridizes with DNA on slide
each yellow spot = gene matched to mRNA
each yellow spot = expressed gene
mRNA → cDNA cDNA matched to genomic DNA
AP Biology
21. Application of Microarrays “DNA Chip”
2-color fluorescent tagging
Comparing treatments or conditions =
Measuring change in gene expression
sick vs. healthy; cancer vs. normal cells
before vs. after treatment with drug
different stages in development
Color coding: label each condition with different color
red = gene expression in one sample
green = gene expression in other sample
yellow = gene expression in both samples
AP Biologyblack
= no or low expression in both
22. I may be very selective…
But still Ask Questions!
EcoRI
BamHI HindIII
restriction sites
plasmid
ori amp
AP Biology resistance 2007-2008
Editor's Notes
How do we know what’s the right combination of genes on a plasmid? Trail and error research work. selectable markers high copy rate convenient restriction sites There are companies that still develop plasmids, patent them & sell them. Biotech companies (ex. New England BioLabs)
Northern blot: RNA on filter & DNA probe measures gene expression because grabbing mRNA from cell -- only expressed genes Western blot: protein on filter labeled directly
human genome = 3 billion bases fragments are cut to ~5000 bases therefore ~ 600,000 fragments per cell. But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes.
human genome = 3 billion bases fragments are cut to ~5000 bases therefore ~ 600,000 fragments per cell. But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes.
Complementation if you have a mutant that lacks YFG, you can transform bacteria with plasmids from the library until one “cures” (complements) the mutation
Could you imagine how much that first insulin clone was worth to Genentech? One little piece of DNA in a plasmid worth billions! It put them on the map & built a multi-billion dollar biotech company.
Developed by Pat Brown at Stanford in late 1980s Realized quickly he needed an automated system: robot spotter Designed spotter & put plans on Internet for benefit of scientific community.
It’s all about comparisons! Powerful research tool.