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SUBMITTED BY: ZIKRA BAKHTAWAR SAEED
REG NO: FA21-RBT-030
SUBMITTED TO: DR. JAMSHAID HUSSAIN
COURSE TITLE: GENETIC ENGINEERING(BTY631)
SUBMITTED ON: 4
TH
NOV,2021
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TOPIC: E. COLI STRAIN DH5 Alpha
TABLE OF CONTENT:
Historical Background
Need to construct strain DH5 Alpha
Introduction
Genetic Mutations of DH5 Alpha and its role
i. recA1 mutation
ii. endA1mutation
iii. lacZΔM15 mutation
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HISTORICAL BACKGROUND:
E.coli strains are widely used for certain applications e.g. introduction of biological
parts, obtaining biological information, constructing biotechnological materials and
for generating energy etc.
In 1920, K-12 strain was isolated from patient and leads to development of E.coli
strain MG1655 and its products ( DH10 and DH5 Alpha ). Its competent cells were
genetically engineered by Douglas Hanahan, an American biologist.
NEED TO CONSTRUCT DH5 ALPHA:
Many E.coli strains were in used but the need to construct DH5 Alpha was due to
its activity to maximize the efficiency of transformation, high-throughput cloning
and also cloning of unstable DNA.
In 1920, K-12 strain was isolated from patient and leads to development of E.coli
strain MG1655 and its products ( DH10 and DH5 Alpha ). Its competent cells were
genetically engineered by Douglas Hanahan, an American biologist.
INTRODUCTION:
E.coli DH5 Alpha strain is non-pathogenic and mesophilic
bacterium of family Enterobacteriaceae.
It is a commonly used strain uses as platform in recombinant DNA
technology for laboratory cloning, sub-cloning and other synthetic
biology applications. GoldBio DH5 Alpha competent cells i.e.
chemical competent or electrocompetent cells shoud use for
cloning purposes.
This strain is also efficient for cloning unmethlylated DNA from
PCR or cDNA.
Recombination strain E.coli DH5 Alpha is also regarded as
important instrument for stable amplification of DNA materials
during plasmid propagation.
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GENETIC MUTATIONS AND ITS ROLE:
Different genetic mutations of DH5 Alpha strain which makes it unique
and favourable for cloning procedures are:
recA1 mutation
lacZM15 mutation
endA1mutation
1. recA1 mutation:
It is a type of single point missense mutation which replaces glycine 160
with aspartic acid in recA polypeptide chain. This mutation causes to
deactivate recombinase and in turn inactivates homologous recombination.
So the mutant recA gene limits recombination of plasmid with E.coli
genome to stabilize inserted plasmid.
It also reduces the plasmid multimerization and several deletion.
2. lacZM15 mutation:
Delta(lacZ) M15 is an alpha acceptor which enables blue-white screening
(allows for rapid and convenient detection of recombinant bacteria) of
transformed cells along with lacZ based vectors.
The other role of LacZM15 mutation disables lacZ activity in E.coli DH5
Alpha produces an inactive form of B-galactosidase.
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3. endA1mutation:
In endA1 mutation, an intracellular endonuclease is inactivated that prevents
it from degrading the incorporated plasmid. The degradation of
endonuclease also ensures the increased plasmid transfer rates.
endA1 mutation also enhances the quality of the plasmid DNA isolations.
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REFRENCES:
https://microbewiki.kenyon.edu/index.php/DH5-Alpha_E.coli
https://en.wikipedia.org/wiki/DH5-Alpha_Cell
https://academic.oup.com/femsle/article/362/11/fnv073/490744
https://openwetware.org/wiki/E._coli_genotypes
https://www.thermofisher.com/order/catalog/product/18258012

Unformatted Attachment Preview

SUBMITTED BY: ZIKRA BAKHTAWAR SAEED REG NO: FA21-RBT-030 SUBMITTED TO: DR. JAMSHAID HUSSAIN COURSE TITLE: GENETIC ENGINEERING(BTY631) SUBMITTED ON: 4TH NOV,2021 TOPIC: E. COLI STRAIN DH5 Alpha TABLE OF CONTENT: ▪ ▪ ▪ ▪ Historical Background Need to construct strain DH5 Alpha Introduction Genetic Mutations of DH5 Alpha and its role i. recA1 mutation ii. endA1mutation iii. lacZΔM15 mutation ❖ HISTORICAL BACKGROUND: E.coli strains are widely used for certain applications e.g. introduction of biological parts, obtaining biological information, constructing biotechnological materials and for generating energy etc. In 1920, K-12 strain was isolated from patient and leads to development of E.coli strain MG1655 and its products ( DH10 and DH5 Alpha ). Its competent cells were genetically engineered by Douglas Hanahan, an American biologist. • NEED TO CONSTRUCT DH5 ALPHA: Many E.coli strains were in used but the need to construct DH5 Alpha was due to its activity to maximize the efficiency of transformation, high-throughput cloning and also cloning of unstable DNA. In 1920, K-12 strain was isolated from patient and leads to development of E.coli strain MG1655 and its products ( DH10 and DH5 Alpha ). Its competent cells were genetically engineered by Douglas Hanahan, an American biologist. ➢ ➢ ➢ ➢ ❖ INTRODUCTION: E.coli DH5 Alpha strain is non-pathogenic and mesophilic bacterium of family Enterobacteriaceae. It is a commonly used strain uses as platform in recombinant DNA technology for laboratory cloning, sub-cloning and other synthetic biology applications. GoldBio DH5 Alpha competent cells i.e. chemical competent or electrocompetent cells shoud use for cloning purposes. This strain is also efficient for cloning unmethlylated DNA from PCR or cDNA. Recombination strain E.coli DH5 Alpha is also regarded as important instrument for stable amplification of DNA materials during plasmid propagation. ❖ GENETIC MUTATIONS AND ITS ROLE: Different genetic mutations of DH5 Alpha strain which makes it unique and favourable for cloning procedures are: • recA1 mutation • lacZM15 mutation • endA1mutation 1. recA1 mutation: ➢ It is a type of single point missense mutation which replaces glycine 160 with aspartic acid in recA polypeptide chain. This mutation causes to deactivate recombinase and in turn inactivates homologous recombination. So the mutant recA gene limits recombination of plasmid with E.coli genome to stabilize inserted plasmid. ➢ It also reduces the plasmid multimerization and several deletion. 2. lacZM15 mutation: ➢ Delta(lacZ) M15 is an alpha acceptor which enables blue-white screening (allows for rapid and convenient detection of recombinant bacteria) of transformed cells along with lacZ based vectors. ➢ The other role of LacZM15 mutation disables lacZ activity in E.coli DH5 Alpha produces an inactive form of B-galactosidase. 3. endA1mutation: ➢ In endA1 mutation, an intracellular endonuclease is inactivated that prevents it from degrading the incorporated plasmid. The degradation of endonuclease also ensures the increased plasmid transfer rates. ➢ endA1 mutation also enhances the quality of the plasmid DNA isolations. REFRENCES: ▪ ▪ ▪ ▪ ▪ https://microbewiki.kenyon.edu/index.php/DH5-Alpha_E.coli https://en.wikipedia.org/wiki/DH5-Alpha_Cell https://academic.oup.com/femsle/article/362/11/fnv073/490744 https://openwetware.org/wiki/E._coli_genotypes https://www.thermofisher.com/order/catalog/product/18258012 Name: Description: ...
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