Pbr322 and puc8 plasmids
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One of the first plasmids to be used in recombinant genetics was called pBR322. It is approximately 4300 bp in length and has two antibiotic resistance genes: Ap (Ampicillin) and Tc (Tetracycline). Bacteria cells that are successfully transformed with this plasmid are able to grow in the presence of both ampicillin and tetracycline antibiotics
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Shuttle vector
1. Yeast plasmids like the 2 micron circle have been extensively studied and developed into yeast cloning vectors.
2. Shuttle vectors like YEp vectors contain selectable marker genes like LEU2 and bacterial plasmid origins of replication like pBR322, allowing them to replicate in both E. coli and yeast.
3. The 2 micron circle is a 6kb endogenous yeast plasmid that replicates autonomously through an ARS sequence and is maintained at 50-100 copies per cell.
Cosmid
Cosmids are hybrid cloning vectors that combine features of plasmids and bacteriophages. They contain approximately 200 base pairs of DNA from the lambda phage, including the cos site sequence, which allows the vector to be packaged into phage particles and transduced into bacteria like a phage. Cosmids can accommodate large foreign DNA inserts of 35-45 kilobase pairs and are commonly used to construct genomic libraries.
Bacteriophage vectors
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
Dna sequencing
DNA sequencing is a process to determine the order of nucleotides in a DNA molecule. It was discovered in the 1970s by scientists like Frederick Sanger who developed the chain termination method. This method involves DNA replication with modified nucleotides that cause the growing DNA strand to terminate at that point. The fragments are then separated by size to reveal the sequence. Automated sequencing now uses fluorescent dyes and capillary electrophoresis for faster and higher throughput sequencing. DNA sequencing has applications in medicine, forensics, and agriculture.
Prokaryotic genome organization
Prokaryotic genomes are typically organized as single circular chromosomes that are condensed into a nucleoid region within the cell. DNA supercoiling, which involves the over- or under-winding of DNA strands, facilitates compaction of prokaryotic genomes and enables DNA metabolism. Topoisomerases regulate DNA supercoiling by introducing temporary breaks in DNA strands, allowing strands to pass through one another and relieve torsional stress that builds during processes like transcription and replication. The two major types of topoisomerase are types I and II, which introduce single-strand or double-strand breaks, respectively, in regulating supercoiling levels.
P br322
pBR322 is a 4,361 base pair plasmid vector originally constructed in 1977 for use in cloning experiments. It contains genes conferring resistance to ampicillin and tetracycline, which allow selection of recombinant clones, as well as an E. coli origin of replication. Recombinant selection involves insertional inactivation of the tetracycline resistance gene, rendering clones sensitive to tetracycline but resistant to ampicillin. pBR322 was widely used for cloning due to its small size, two selectable markers, and ability to be amplified in host cells. However, it is limited by its mobility between cells and small carrying capacity.
bacterial artificial chromosome & yeast artificial chromosome
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
pUC18 vector
pUC plasmids are small, high copy number cloning vectors developed by Messing and colleagues at the University of California. They contain a multiple cloning site within the lacZ gene, allowing for rapid visual detection of inserts through blue/white screening. Clones with inserts disrupt the lacZ gene and appear white on media containing X-gal, while empty vectors appear blue. The pUC plasmids are useful cloning vectors due to their high copy number, single antibiotic resistance gene, and ability to easily screen for recombinant clones.
Recommended
Shuttle vector
1. Yeast plasmids like the 2 micron circle have been extensively studied and developed into yeast cloning vectors.
2. Shuttle vectors like YEp vectors contain selectable marker genes like LEU2 and bacterial plasmid origins of replication like pBR322, allowing them to replicate in both E. coli and yeast.
3. The 2 micron circle is a 6kb endogenous yeast plasmid that replicates autonomously through an ARS sequence and is maintained at 50-100 copies per cell.
Cosmid
Cosmids are hybrid cloning vectors that combine features of plasmids and bacteriophages. They contain approximately 200 base pairs of DNA from the lambda phage, including the cos site sequence, which allows the vector to be packaged into phage particles and transduced into bacteria like a phage. Cosmids can accommodate large foreign DNA inserts of 35-45 kilobase pairs and are commonly used to construct genomic libraries.
Bacteriophage vectors
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
Dna sequencing
DNA sequencing is a process to determine the order of nucleotides in a DNA molecule. It was discovered in the 1970s by scientists like Frederick Sanger who developed the chain termination method. This method involves DNA replication with modified nucleotides that cause the growing DNA strand to terminate at that point. The fragments are then separated by size to reveal the sequence. Automated sequencing now uses fluorescent dyes and capillary electrophoresis for faster and higher throughput sequencing. DNA sequencing has applications in medicine, forensics, and agriculture.
Prokaryotic genome organization
Prokaryotic genomes are typically organized as single circular chromosomes that are condensed into a nucleoid region within the cell. DNA supercoiling, which involves the over- or under-winding of DNA strands, facilitates compaction of prokaryotic genomes and enables DNA metabolism. Topoisomerases regulate DNA supercoiling by introducing temporary breaks in DNA strands, allowing strands to pass through one another and relieve torsional stress that builds during processes like transcription and replication. The two major types of topoisomerase are types I and II, which introduce single-strand or double-strand breaks, respectively, in regulating supercoiling levels.
P br322
pBR322 is a 4,361 base pair plasmid vector originally constructed in 1977 for use in cloning experiments. It contains genes conferring resistance to ampicillin and tetracycline, which allow selection of recombinant clones, as well as an E. coli origin of replication. Recombinant selection involves insertional inactivation of the tetracycline resistance gene, rendering clones sensitive to tetracycline but resistant to ampicillin. pBR322 was widely used for cloning due to its small size, two selectable markers, and ability to be amplified in host cells. However, it is limited by its mobility between cells and small carrying capacity.
bacterial artificial chromosome & yeast artificial chromosome
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
pUC18 vector
pUC plasmids are small, high copy number cloning vectors developed by Messing and colleagues at the University of California. They contain a multiple cloning site within the lacZ gene, allowing for rapid visual detection of inserts through blue/white screening. Clones with inserts disrupt the lacZ gene and appear white on media containing X-gal, while empty vectors appear blue. The pUC plasmids are useful cloning vectors due to their high copy number, single antibiotic resistance gene, and ability to easily screen for recombinant clones.
Gene library
A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
Cosmid Vectors, YAC and BAC Expression Vectors
1. Cosmid vectors are hybrid vectors derived from plasmids that contain the cos site from bacteriophage lambda, allowing them to clone DNA fragments up to 40 kb in size.
2. Yeast artificial chromosomes (YACs) are engineered yeast chromosomes that can clone very large DNA fragments, averaging 200-500 kb but up to 1 MB, taking advantage of yeast cell machinery.
3. Bacterial artificial chromosomes (BACs) are DNA constructs based on fertility plasmids that can clone up to 300 kb fragments and address issues with YAC stability and recombination.
Insertional inactivation
Insertional inactivation is a technique used in genetic engineering where a fragment of foreign DNA inserts into the genome of a host cell. This insertion disrupts or inactivates an existing gene, such as one that confers antibiotic resistance. Screening methods rely on insertional inactivation to detect recombinant cells. For example, blue/white screening uses disruption of the lacZ gene to distinguish cells with and without recombinant DNA insertion.
P uc vectors
pUC vectors are plasmids derived from pBR322 that have a higher copy number of 500-600 copies per cell. They contain an ampicillin resistance gene for selection, as well as the lacZ' gene containing multiple cloning sites. When a gene of interest is inserted, it disrupts the lacZ' gene, allowing for blue-white screening on media containing IPTG and X-gal to identify recombinant colonies that appear white instead of blue. pUC vectors offer advantages over pBR322 such as high copy number and easy selection, though they cannot accommodate inserts larger than 15kb.
Screening and selection of recombinants
The document discusses various methods for screening and selecting recombinant cells. Direct selection methods include antibiotic resistance screening and blue-white color screening. Indirect selection methods include screening by nucleic acid hybridization, colony hybridization, immunological assays, and detecting protein/enzyme activity. These screening methods allow identification of recombinant cells that contain the gene of interest from a mixture of transformed cells.
Plasmid
This document provides an overview of plasmids. It defines plasmids as small, circular, extrachromosomal DNA molecules that can replicate independently in bacteria. Plasmids contain genes that provide benefits to bacteria like antibiotic resistance. They are transferred between bacteria through processes like transformation, transduction, and conjugation. Plasmids are classified based on their functions and are important tools in biotechnology as they allow cloning, protein production, and other applications.
C dna
This document discusses cDNA and genomic libraries. It defines cDNA as complementary DNA synthesized from mRNA, and genomic libraries as collections of DNA fragments representing an organism's entire genome. It describes how cDNA libraries are constructed by synthesizing cDNA from purified mRNA, and genomic libraries by partially digesting genomic DNA and inserting fragments into cloning vectors. It also explains how these libraries are screened using probes to identify clones containing genes of interest or encoding specific proteins.
Types of vectors, Prokaryotic and Eukaryotic
vectors types
prokaryotic and eukaryotic
prokaryotic vectors= 1. ti-plasmid 2. pUC18 3. pBR322 4. lambda phage
Eukaryotic vectors= 1.BAC 2. YAC
by
Malaka Madhusudhana Reddy
Research Scholar
Dept.of :: Zoology
Yogivemana University, kadapa.
Andhrapradesh, India.
Sv 40
Simian virus 40 (SV40) is a DNA virus that can cause tumors in monkeys and humans, and it was first identified as a contaminant in polio vaccines in the 1960s. SV40 has been widely used as a cloning vector due to its ability to efficiently deliver genes into a variety of cells without killing the host cell or eliciting an immune response. Future research prospects for SV40 vectors include developing recombinant versions for gene transfer applications and furthering understanding of related retroviruses.
Phagemid vector
This document discusses the phagemid vector pBluescript, which can be used in either the positive or negative orientation. pBluescript is a phagemid vector that can be used to clone DNA fragments for sequencing, mutagenesis, protein expression or other molecular biology experiments. References for further information about pBluescript are provided.
Bacterial genomic organization
A detail comparison between the genomic organization between prokaryotic genome and eukaryotic genome.
Blotting techniques
This document describes three types of blotting techniques - Southern blotting, Northern blotting, and Western blotting. Southern blotting is used to detect DNA fragments separated by agarose gel electrophoresis. Northern blotting detects specific RNA sequences separated by gel electrophoresis. Western blotting identifies proteins separated by SDS-PAGE gel using an antibody probe. The document provides detailed procedures and applications for each type of blotting.
Selection & Screening of Recombinant cells & expression of recombinant (2) (1)
This document summarizes various methods for selecting and screening recombinant clones after introducing recombinant DNA into host cells. It discusses direct selection using antibiotic resistance genes, insertional inactivation by inserting DNA into antibiotic resistance genes, and blue-white screening using beta-galactosidase activity. It also covers colony hybridization using radioactive probes and immunological tests using antibodies to identify antigen-expressing colonies. Finally, it briefly discusses protein expression in different systems like bacteria, insect cells, and mammalian cells.
Transposones
This document discusses transposons, which are DNA segments that can move within a genome. Transposons carry genes and can generate DNA rearrangements that impact cell survival and evolution. They encode transposase proteins that catalyze the transposition process. There are different types of transposons based on their mechanism of movement, including cut-and-paste transposons, replicative transposons, and retrotransposons. Examples like Tn3 and bacteriophage Mu are provided. Transposons can cause mutations and have played a significant role in genome alteration and evolution over time.
Plasmid as a Cloning Vector
Plasmids are circular DNA molecules that can exist independently of the bacterial chromosome and are used as cloning vectors. Cloning vectors are DNA molecules that can carry foreign genetic material into a host cell. Plasmids have features like cloning sites, selectable markers, and reporter genes that make them useful for cloning. Common types of cloning vectors include plasmids, bacteriophages, cosmids, and artificial chromosomes. Plasmids are advantageous as cloning vectors because they are small, can replicate independently of the host, and often confer antibiotic resistance, allowing for selection of transformed cells.
DNA Libraries
DNA libraries contain cloned DNA fragments from an organism's genome or cDNA from mRNA. Genomic libraries represent entire genomes, while cDNA libraries represent expressed genes. Genomic libraries are constructed by isolating chromosomal DNA, fragmenting it, cloning the fragments into vectors, and screening clones. cDNA libraries are constructed by synthesizing cDNA from mRNA, cloning the cDNA into vectors, and screening clones. Both library types allow identification of specific DNA or cDNA sequences through hybridization or other screening methods.
DNA microarray
DNA microarrays allow analysis of gene expression across thousands of genes simultaneously. They consist of DNA probes attached to a solid surface in an organized grid pattern, with each spot representing a single gene. Samples are labeled with fluorescent dyes and hybridized to the chip. Complementary sequences pair via hydrogen bonds, while non-specific sequences are washed away. The signal intensity at each spot indicates the amount of target sequence present and thus gene expression levels. DNA microarrays have applications in clinical diagnosis, drug discovery, and other fields by profiling gene expression patterns.
Genomic and c dna library
Genomic and cDNA libraries are constructed to isolate genes of interest from organisms. Genomic libraries contain total chromosomal DNA while cDNA libraries contain mRNA from specific cell types. DNA is digested and ligated into vectors to clone fragments. Libraries are screened using probes and PCR to identify clones containing genes of interest. cDNA libraries are useful for studying eukaryotic gene expression as they contain mRNA from specific cells. Thousands of clones may need to be screened to have high probability of isolating a particular gene fragment.
Phage vector bacteriophage
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
MODIFYING ENZYMES
This document discusses various enzymes used for genetic engineering and DNA manipulation. It describes restriction endonucleases and DNA ligase which cut and join DNA fragments. It also discusses other DNA modifying enzymes like nucleases which degrade DNA, and polymerases which synthesize DNA copies. Specific enzymes covered in detail include DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, terminal transferase, T4 DNA ligase, and T4 RNA ligase.
The term transformation refers to the uptake and expression of DNA fro.pdf
The term transformation refers to the uptake and expression of DNA from the surrounding
medium by cells. Fred Griffith demonstrated transformation in 1928 when he exposed mice to
two different strains of the bacterium Streptococcus pneumoniae. When he injected mice with a
mix of killed, virulent S-strain bacteria and live, nonvirulent R-strain bacteria, the mice died of
pheumonia, and live S-strain bacteria were found in their bodies. Clearly, the R-strain bacteria
had picked up DNA from the dead Sstrain, and in so doing they were transformed into a virulent
strain. The tranformation was possible because Streptococcus bacteria are competent. Competent
bacterial cells are those that have the ability to pick up DNA from the environment. Many genera
of bacteria are naturally competent. But competency may be influenced by various factors,
including temperature changes and the concentration of different ions present in the
environment. E. coli is not a naturally competent species, but it can be induced to "pick up".
DNA to a limited extent after treatment with calcium ( Ca ++ ) ions and by "heat-shock" or short
exposure to higher temperatures ( 4 2 C ) . You will use plasmid DNA to transform E . coli.
Plasmids are selfreplicating, double-stranded, circular DNA molecules that are maintained in
bacteria as independent extra-chromosomal entities. Virtually all bacterial genera have plasmids.
Some plasmids carry information for their own transfer from one cell to another (F plasmids),
others may encode resistance to antibiotics. Plasmids can range in size from less than 1 to more
than 500 kb . Some plasmids (low-copy plasmids) are present in low numbers, 1 to 4 copies per
cell, while others are present in 10 to 100 copies per cell, and are called high-copy-number
plasmids. Plasmids are good vectors to introduce DNA into bacterial cells, for cloning or
amplification purposes. The plasmid you will be using for E . coli transformation is called
pBSKS. It is an artificial plasmid generated by using recombinant DNA techniques. It has a gene
for resistance to the antibiotic ampicillin. Normally, this antibiotic does not allow E . coli
bacteria to grow. However, transformed bacteria containing the plasmid will be resistant to the
antibiotic and will grow in selective medium containing ampicillin. The strain of E. coli you will
use is called JM109. This particular strain is L a c - because it carries a mutant allele of the lacZ
gene encoding galactosidase. The bacteria produces a defective form of the enzyme and would
not survive in a medium in which lactose were used as the only carbon source. The pBSKS
plasmid contains a segment of the lacZ gene. This fragment is capable of complementing the
lacZ mutant allele in the chromosomal DNA. This is an example of intragenic complementation.
JM109 cells transformed with pBSKS produce a functional form of -galactosidase and would
survive on medium containing lactose as the sole carbon source. You could test if your c.
Lab Differential Expression Differential gene expression provides th.pdf
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
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A gene library is a large collection of DNA fragments cloned from an organism. It contains genomic DNA or cDNA sequences. Gene libraries are constructed using molecular tools like restriction enzymes and ligases to cut and paste DNA fragments into vectors such as plasmids, phages, or artificial chromosomes. The choice of vector depends on the size of the genome being cloned. Libraries allow screening to identify genes of interest through techniques like hybridization or expression screening. cDNA libraries contain only expressed sequences without introns, making them preferable for cloning eukaryotic genes in prokaryotes.
Cosmid Vectors, YAC and BAC Expression Vectors
1. Cosmid vectors are hybrid vectors derived from plasmids that contain the cos site from bacteriophage lambda, allowing them to clone DNA fragments up to 40 kb in size.
2. Yeast artificial chromosomes (YACs) are engineered yeast chromosomes that can clone very large DNA fragments, averaging 200-500 kb but up to 1 MB, taking advantage of yeast cell machinery.
3. Bacterial artificial chromosomes (BACs) are DNA constructs based on fertility plasmids that can clone up to 300 kb fragments and address issues with YAC stability and recombination.
Insertional inactivation
Insertional inactivation is a technique used in genetic engineering where a fragment of foreign DNA inserts into the genome of a host cell. This insertion disrupts or inactivates an existing gene, such as one that confers antibiotic resistance. Screening methods rely on insertional inactivation to detect recombinant cells. For example, blue/white screening uses disruption of the lacZ gene to distinguish cells with and without recombinant DNA insertion.
P uc vectors
pUC vectors are plasmids derived from pBR322 that have a higher copy number of 500-600 copies per cell. They contain an ampicillin resistance gene for selection, as well as the lacZ' gene containing multiple cloning sites. When a gene of interest is inserted, it disrupts the lacZ' gene, allowing for blue-white screening on media containing IPTG and X-gal to identify recombinant colonies that appear white instead of blue. pUC vectors offer advantages over pBR322 such as high copy number and easy selection, though they cannot accommodate inserts larger than 15kb.
Screening and selection of recombinants
The document discusses various methods for screening and selecting recombinant cells. Direct selection methods include antibiotic resistance screening and blue-white color screening. Indirect selection methods include screening by nucleic acid hybridization, colony hybridization, immunological assays, and detecting protein/enzyme activity. These screening methods allow identification of recombinant cells that contain the gene of interest from a mixture of transformed cells.
Plasmid
This document provides an overview of plasmids. It defines plasmids as small, circular, extrachromosomal DNA molecules that can replicate independently in bacteria. Plasmids contain genes that provide benefits to bacteria like antibiotic resistance. They are transferred between bacteria through processes like transformation, transduction, and conjugation. Plasmids are classified based on their functions and are important tools in biotechnology as they allow cloning, protein production, and other applications.
C dna
This document discusses cDNA and genomic libraries. It defines cDNA as complementary DNA synthesized from mRNA, and genomic libraries as collections of DNA fragments representing an organism's entire genome. It describes how cDNA libraries are constructed by synthesizing cDNA from purified mRNA, and genomic libraries by partially digesting genomic DNA and inserting fragments into cloning vectors. It also explains how these libraries are screened using probes to identify clones containing genes of interest or encoding specific proteins.
Types of vectors, Prokaryotic and Eukaryotic
vectors types
prokaryotic and eukaryotic
prokaryotic vectors= 1. ti-plasmid 2. pUC18 3. pBR322 4. lambda phage
Eukaryotic vectors= 1.BAC 2. YAC
by
Malaka Madhusudhana Reddy
Research Scholar
Dept.of :: Zoology
Yogivemana University, kadapa.
Andhrapradesh, India.
Sv 40
Simian virus 40 (SV40) is a DNA virus that can cause tumors in monkeys and humans, and it was first identified as a contaminant in polio vaccines in the 1960s. SV40 has been widely used as a cloning vector due to its ability to efficiently deliver genes into a variety of cells without killing the host cell or eliciting an immune response. Future research prospects for SV40 vectors include developing recombinant versions for gene transfer applications and furthering understanding of related retroviruses.
Phagemid vector
This document discusses the phagemid vector pBluescript, which can be used in either the positive or negative orientation. pBluescript is a phagemid vector that can be used to clone DNA fragments for sequencing, mutagenesis, protein expression or other molecular biology experiments. References for further information about pBluescript are provided.
Bacterial genomic organization
A detail comparison between the genomic organization between prokaryotic genome and eukaryotic genome.
Blotting techniques
This document describes three types of blotting techniques - Southern blotting, Northern blotting, and Western blotting. Southern blotting is used to detect DNA fragments separated by agarose gel electrophoresis. Northern blotting detects specific RNA sequences separated by gel electrophoresis. Western blotting identifies proteins separated by SDS-PAGE gel using an antibody probe. The document provides detailed procedures and applications for each type of blotting.
Selection & Screening of Recombinant cells & expression of recombinant (2) (1)
This document summarizes various methods for selecting and screening recombinant clones after introducing recombinant DNA into host cells. It discusses direct selection using antibiotic resistance genes, insertional inactivation by inserting DNA into antibiotic resistance genes, and blue-white screening using beta-galactosidase activity. It also covers colony hybridization using radioactive probes and immunological tests using antibodies to identify antigen-expressing colonies. Finally, it briefly discusses protein expression in different systems like bacteria, insect cells, and mammalian cells.
Transposones
This document discusses transposons, which are DNA segments that can move within a genome. Transposons carry genes and can generate DNA rearrangements that impact cell survival and evolution. They encode transposase proteins that catalyze the transposition process. There are different types of transposons based on their mechanism of movement, including cut-and-paste transposons, replicative transposons, and retrotransposons. Examples like Tn3 and bacteriophage Mu are provided. Transposons can cause mutations and have played a significant role in genome alteration and evolution over time.
Plasmid as a Cloning Vector
Plasmids are circular DNA molecules that can exist independently of the bacterial chromosome and are used as cloning vectors. Cloning vectors are DNA molecules that can carry foreign genetic material into a host cell. Plasmids have features like cloning sites, selectable markers, and reporter genes that make them useful for cloning. Common types of cloning vectors include plasmids, bacteriophages, cosmids, and artificial chromosomes. Plasmids are advantageous as cloning vectors because they are small, can replicate independently of the host, and often confer antibiotic resistance, allowing for selection of transformed cells.
DNA Libraries
DNA libraries contain cloned DNA fragments from an organism's genome or cDNA from mRNA. Genomic libraries represent entire genomes, while cDNA libraries represent expressed genes. Genomic libraries are constructed by isolating chromosomal DNA, fragmenting it, cloning the fragments into vectors, and screening clones. cDNA libraries are constructed by synthesizing cDNA from mRNA, cloning the cDNA into vectors, and screening clones. Both library types allow identification of specific DNA or cDNA sequences through hybridization or other screening methods.
DNA microarray
DNA microarrays allow analysis of gene expression across thousands of genes simultaneously. They consist of DNA probes attached to a solid surface in an organized grid pattern, with each spot representing a single gene. Samples are labeled with fluorescent dyes and hybridized to the chip. Complementary sequences pair via hydrogen bonds, while non-specific sequences are washed away. The signal intensity at each spot indicates the amount of target sequence present and thus gene expression levels. DNA microarrays have applications in clinical diagnosis, drug discovery, and other fields by profiling gene expression patterns.
Genomic and c dna library
Genomic and cDNA libraries are constructed to isolate genes of interest from organisms. Genomic libraries contain total chromosomal DNA while cDNA libraries contain mRNA from specific cell types. DNA is digested and ligated into vectors to clone fragments. Libraries are screened using probes and PCR to identify clones containing genes of interest. cDNA libraries are useful for studying eukaryotic gene expression as they contain mRNA from specific cells. Thousands of clones may need to be screened to have high probability of isolating a particular gene fragment.
Phage vector bacteriophage
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
MODIFYING ENZYMES
This document discusses various enzymes used for genetic engineering and DNA manipulation. It describes restriction endonucleases and DNA ligase which cut and join DNA fragments. It also discusses other DNA modifying enzymes like nucleases which degrade DNA, and polymerases which synthesize DNA copies. Specific enzymes covered in detail include DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, terminal transferase, T4 DNA ligase, and T4 RNA ligase.
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Selection & Screening of Recombinant cells & expression of recombinant (2) (1)
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Similar to Pbr322 and puc8 plasmids
The term transformation refers to the uptake and expression of DNA fro.pdf
The term transformation refers to the uptake and expression of DNA from the surrounding
medium by cells. Fred Griffith demonstrated transformation in 1928 when he exposed mice to
two different strains of the bacterium Streptococcus pneumoniae. When he injected mice with a
mix of killed, virulent S-strain bacteria and live, nonvirulent R-strain bacteria, the mice died of
pheumonia, and live S-strain bacteria were found in their bodies. Clearly, the R-strain bacteria
had picked up DNA from the dead Sstrain, and in so doing they were transformed into a virulent
strain. The tranformation was possible because Streptococcus bacteria are competent. Competent
bacterial cells are those that have the ability to pick up DNA from the environment. Many genera
of bacteria are naturally competent. But competency may be influenced by various factors,
including temperature changes and the concentration of different ions present in the
environment. E. coli is not a naturally competent species, but it can be induced to "pick up".
DNA to a limited extent after treatment with calcium ( Ca ++ ) ions and by "heat-shock" or short
exposure to higher temperatures ( 4 2 C ) . You will use plasmid DNA to transform E . coli.
Plasmids are selfreplicating, double-stranded, circular DNA molecules that are maintained in
bacteria as independent extra-chromosomal entities. Virtually all bacterial genera have plasmids.
Some plasmids carry information for their own transfer from one cell to another (F plasmids),
others may encode resistance to antibiotics. Plasmids can range in size from less than 1 to more
than 500 kb . Some plasmids (low-copy plasmids) are present in low numbers, 1 to 4 copies per
cell, while others are present in 10 to 100 copies per cell, and are called high-copy-number
plasmids. Plasmids are good vectors to introduce DNA into bacterial cells, for cloning or
amplification purposes. The plasmid you will be using for E . coli transformation is called
pBSKS. It is an artificial plasmid generated by using recombinant DNA techniques. It has a gene
for resistance to the antibiotic ampicillin. Normally, this antibiotic does not allow E . coli
bacteria to grow. However, transformed bacteria containing the plasmid will be resistant to the
antibiotic and will grow in selective medium containing ampicillin. The strain of E. coli you will
use is called JM109. This particular strain is L a c - because it carries a mutant allele of the lacZ
gene encoding galactosidase. The bacteria produces a defective form of the enzyme and would
not survive in a medium in which lactose were used as the only carbon source. The pBSKS
plasmid contains a segment of the lacZ gene. This fragment is capable of complementing the
lacZ mutant allele in the chromosomal DNA. This is an example of intragenic complementation.
JM109 cells transformed with pBSKS produce a functional form of -galactosidase and would
survive on medium containing lactose as the sole carbon source. You could test if your c.
Lab Differential Expression Differential gene expression provides th.pdf
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
Lab Differential Expression Differential gene expression provides .pdf
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
E. coli plasmids based vectors
Plasmids are extrachromosomal DNA molecules found in bacteria that can replicate independently of the bacterial chromosome. They contain an origin of replication and may have genes for antibiotic resistance, virulence factors, or degradation of molecules. Plasmids can be classified based on their ability to integrate into chromosomes, copy number, ability to conjugate, and compatibility with other plasmids. Common plasmids used for cloning include pBR322, pUC, and pGEM3Z. These vectors contain antibiotic resistance genes and can be used to select for recombinant plasmids using antibiotic selection or blue/white screening of beta-galactosidase activity. pGEM3Z also allows for in vitro transcription of cloned genes.
Proposal march 2012
This study aims to analyze conserved amino acids in GAPDH proteins from tropical plants Oxalis corniculata and Plectranthus amboinicus. GAPDH is important for energy production. Previous work cloned GAPDH genes from these plants and Myrtaceae psidium. Psidium showed no cloning and was eliminated. The current work will sequence the cloned GAPDH inserts and analyze conserved amino acids related to catalytic function through bioinformatics. This adds to knowledge of important plant genes and their evolution.
Methods of screening
This document discusses various methods for screening recombinant clones, including:
1. Blue-white screening using X-gal to detect the presence or absence of insertions disrupting beta-galactosidase activity.
2. Insertional inactivation screening by inserting DNA fragments to disrupt antibiotic resistance or repressor genes.
3. Antibiotic sensitivity screening to detect circularized recombinant plasmids capable of conferring antibiotic resistance.
4. Auxotrophic yeast strain screening using yeast vectors containing selectable marker genes to complement host mutations.
5. Reporter gene assays using enzymes like luciferase, GFP, etc. fused to promoters to identify positive recombinant clones.
Analysis of recombinants.pptx
1) There are several methods for selecting and screening recombinant transformants, including blue-white screening using X-gal, insertional inactivation of antibiotic resistance genes, and using reporter genes like luciferase or GFP.
2) Gene transfer in animals can be done using viral vectors like retroviruses which incorporate the transgene into their genome, or using non-viral methods like microinjection, gene guns, electroporation, and ultrasound to deliver naked DNA into cells.
3) Stable transfection integrates transferred DNA into the host chromosome for permanent expression, while transient transfection expresses DNA temporarily without integration.
Cloning vectors
This document discusses gene cloning and various cloning vectors. It provides details about plasmid vectors such as pBR322 and pUC18/19. pBR322 was one of the first widely used E. coli cloning vectors, containing genes for ampicillin and tetracycline resistance. It served as a model for prokaryotic transcription and replication studies. The document discusses the structure, properties, replication and uses of pBR322. It also describes the expression vector pUC18/19, which is derived from pBR322 and contains a multiple cloning site for inserting genes between the beta-lactamase gene and a fragment of the lacZ gene.
Transplastomics
A transplastomic plant is a genetically modified plant in which the new genes have not been inserted in the nuclear DNA but in the DNA of the chloroplasts.
Alakesh
This document summarizes transplastomic engineering, which involves genetically engineering chloroplasts. It discusses how transgenes can be integrated into the chloroplast genome via homologous recombination. The first successful transformations occurred in algae and tobacco in the 1990s. Methods for transformation include biolistic transformation using microcarriers coated with DNA and PEG-mediated transformation of protoplasts. The document outlines construction of transgenes for optimal expression and applications like herbicide resistance, pathogen resistance, and producing biopharmaceuticals in chloroplasts. Transplastomic engineering offers advantages over nuclear transformation like high levels of transgene expression.
Honours Report Draft COMPLETE
This document describes experiments aimed at isolating bacteriophage mutants with altered tolerance for plating when the gpD capsid protein is fused to foreign proteins. The author isolated "IPDF" mutants of phage i434Dam123 that could not plate when gpD-fusions were expressed from a plasmid. These IPDF mutants were then used to select "supIPDF" mutants that regained the ability to plate equally in the presence or absence of gpD-fusions. Sequencing showed the mutations responsible for the IPDF and supIPDF phenotypes were located outside of the D and E genes. This suggests there are extragenic mutations that can enhance or suppress the toxicity of gpD-fusions towards viable phage
Protoplast isolation and fusion
Introduction
History
Importance of protoplast
Sources of protoplast
Isolation of protoplast
Viability test
Fusion of protoplast
Selection of hybrid cells
Genetic consequences
Symmetric & asymmetric Hybrids
Cybrids
Advantages
Disadvantages
Conclusion
References
Plasmid Lab Report
This document summarizes a plasmid lab report that used pUC19 plasmids as the vector for E. coli transformation due to its small size, high uptake efficiency, and fast replication time. Key features of pUC19 include an origin of replication and multiple cloning sites. Transformed E. coli were able to grow on agar plates containing ampicillin due to the plasmid containing an ampicillin resistance gene. Non-recombinant E. coli colonies were blue due to expression of the lacZ gene, while recombinant colonies were white due to insertion of the CIH-1 gene within the multiple cloning sites.
Microbiology antibiotic
1. The document discusses various antibiotics and their mechanisms of action on bacterial cells, focusing on how they inhibit important metabolic processes.
2. Key antibiotic classes are described that inhibit cell wall synthesis, protein synthesis, DNA replication, and folic acid biosynthesis. Examples like penicillins, aminoglycosides, fluoroquinolones, and sulfonamides are provided.
3. The major sites of antibiotic action discussed are the bacterial cell wall, cytoplasmic membrane, DNA, ribosomes, and metabolic pathways. How each antibiotic class interferes with these sites is explained.
PBP2a
a presentation introduced as a step of internship MCRI for Pharmaceutical Chemistry Department in Faculty Of Pharmacy, ASU.
299860 633981096231012500
The document discusses different types of vectors used in recombinant DNA technology, specifically focusing on expression vectors and plasmid vectors. It defines an expression vector as a plasmid used to introduce a gene into a target cell so that the encoded protein is produced. Plasmid vectors are commonly used expression vectors that contain regulatory sequences to efficiently transcribe the gene. Key components of expression vectors that allow for transcription and translation are also outlined. The document further discusses features of common plasmid vectors like pBR322, pUC, and Ti plasmid vectors used for plant transformation.
Debanjan summer
This document summarizes work done to clone and isolate the promoter region of the oncostatin-M (OSM) gene. The OSM promoter region was amplified from genomic DNA and inserted into a TA cloning vector. The promoter region was then subcloned into a pEGFP-1 expression vector using EcoRI and BamHI restriction enzymes. The pEGFP1-OSM promoter plasmid was transformed into E. coli cells and a positive colony was selected via colony PCR. Finally, the pEGFP1-OSM promoter plasmid was isolated in large quantities using a PEG/LiCl method for future reporter assays to study OSM gene transcription regulation.
DNA Vaccine + Nanoparticles
The document discusses developing a DNA vaccine for fish using chitosan nanoparticles to deliver plasmid DNA encoding the OMP38 gene of Vibrio anguillarum. Key points:
- Chitosan nanoparticles were developed to deliver the pVAOMP38 plasmid and protect it from nuclease degradation. Studies showed the nanoparticles maintained plasmid integrity.
- The pVAOMP38 plasmid was transfected into seabass kidney cells in vitro and shown to express.
- Fish were vaccinated by feeding with chitosan-pVAOMP38 nanoparticles, chitosan-empty vector, or chitosan alone. The fish were later challenged with V. anguillarum to evaluate vaccine efficiency.
Similar to Pbr322 and puc8 plasmids (20)
The term transformation refers to the uptake and expression of DNA fro.pdf
The term transformation refers to the uptake and expression of DNA fro.pdf
Lab Differential Expression Differential gene expression provides th.pdf
Lab Differential Expression Differential gene expression provides th.pdf
Lab Differential Expression Differential gene expression provides .pdf
Lab Differential Expression Differential gene expression provides .pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
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Bone Tissue Engineering
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Industrial Production of Insulin
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Pbr322 and puc8 plasmids
- 1. Plasmids - pBR322 and pUC8 pBR322 Plasmid One of the first plasmids to be used in recombinant genetics was called pBR322. It is approximately 4300 bp in length and has two antibiotic resistance genes: Ap (Ampicillin) and Tc (Tetracycline). Bacteria cells that are successfully transformed with this plasmid are able to grow in the presence of both ampicillin and tetracycline antibiotics.
- 2. pUC8 Plasmid The pUC8 plasmid was designed by scientists and contains the lac z gene. To produce the plasmid, the pBR322 plasmid was cut in half with EcoR I and the section containing the ampicillin resistance gene was combined with a DNA fragment containing the lac z gene. As a result, the plasmid provides a transformed cell with both ampicillin resistance and the ability to utilize lactose as a food source, since the lac z gene produces B- galactosidase (degrades lactose). Since the pUC8 plasmid was produced using EcoR I, this enzyme cannot be used to insert desired genes when producing recombinant plasmids for use in genetic engineering. This plasmid was originally used in conjunction with a special mutant strain of E. coli called JM101, which has a mutation in its chromosomal lac z gene and cannot survive on media containing only lactose as a food source. If JM1010 bacteria were successfully transformed with the pUC8 plasmid, they gained the ability to use lactose as a food source. Therefore, only successfully transformed bacteria would survive when grown on media containing only lactose and ampicillin.
- 3. Improving the use of pUC8 While it was possible to isolate the bacteria transformed with pUC8 on lactose and ampicillin containing media, a problem existed. During the process of producing recombinant pUC8 plasmids with additional genes of interest inserted in them, some plasmids did not successfully incorporate the desired gene and closed back up into non- recombinant plasmids. When bacteria cells were exposed to the mixture of plasmids, the non-recombinant plasmids were taken into cells along with the recombinant plasmids. Unfortunately, the non-recombinant plasmids also gave cells the ability to metabolize lactose and survive in the presence of ampicillin. Non-Transformed No Plasmid Dies from Ampicillin and lack of Food The solution to the problem of separating the JM101 cells transformed with recombinant plasmids from the JM101 cells transformed with non-recombinant plasmids was solved using the chemicals called X-Gal and IPTG. IPTG acts as an inducer that causes B- galactosidase to bind to X-Gal. When X-Gal is broken down, it turns blue in color. Thus, all the JM101 bacteria that had the plasmid with an active lac z gene (producing B- Galactosidase) would appear blue. The key to using this was to insert the additional desired gene in a location that would disrupt the lac z gene in the plasmid and use media containing lactose, ampicillin, and other nutrients. By doing so, all the bacteria with the recombinant plasmid would appear white and all the bacteria with the non-recombinant plasmid would appear blue. The non-transformed bacteria would be killed by the antibiotic and fail to grow. An additional benefit was that scientists were not restricted to using E. coli from strain JM101. The technique could be successfully employed with any type of bacteria cells that was used. Non-Transformed Transformed Non-Recombinant Plasmids Survives –does not produce desired protein Transformed Recombinant Plasmids Survives - produces desired protein Transformed Non-Recombinant Plasmids Blue in Color (X-Gal is Degraded) Transformed Recombinant Plasmids White in Color (X-Gal is not Degraded) Die