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Disruption of ompT in Escherichia Coli using phage λ Red recombinase

Dahlman, Anna (2019) MOBK01 20182
Degree Projects in Molecular Biology
Popular Abstract
ompT - a challenging gene to disrupt in Escherichia coli

Our goal in all its simplicity was to delete a gene in two strains of E.coli. ompT is a gene encoding a protease, which is a protein that break down other proteins. We wanted to disrupt ompT and see what effect it could have on recombinant protein production. A method for achieving this is homologous recombination via a system called λ Red phage. This method exchanges one homologous sequence to another. What ompT is exchanged for, is an antibiotic marker. Thus making it easy to see if the exchange, i.e. disruption of ompT, was successful since they will then grow on antibiotics if resistance has been incorporated.

Gene for antibiotic resistance for both chloramphenicol and... (More)
ompT - a challenging gene to disrupt in Escherichia coli

Our goal in all its simplicity was to delete a gene in two strains of E.coli. ompT is a gene encoding a protease, which is a protein that break down other proteins. We wanted to disrupt ompT and see what effect it could have on recombinant protein production. A method for achieving this is homologous recombination via a system called λ Red phage. This method exchanges one homologous sequence to another. What ompT is exchanged for, is an antibiotic marker. Thus making it easy to see if the exchange, i.e. disruption of ompT, was successful since they will then grow on antibiotics if resistance has been incorporated.

Gene for antibiotic resistance for both chloramphenicol and kanamycin was amplified via polymerase chain reaction (PCR). The product of PCR was analyzed in a gel electrophoresis. E.coli was cultured and made competent, which means that you make the bacteria more prone to take up foreign DNA. The bacteria was electroporated with the amplified antibiotic, forcing them to incorporate this, a process called transformation. This procedure also included the removal of another antibiotic resistance, ampicillin. The λ Red method in general is a very intriguing method for investigating gene expression.

Did we manage to disrupt ompT?

It was harder than previously thought to obtain desired results. Amplifying the DNA for antibiotic resistance was easy, likewise growing and making E.coli competent. The transformation seemed to be successful, but as far science goes, one can never be confident enough without repeating the try several times. The experiment was repeated with chloramphenicol several times without managing to disrupt ompT. Finally, kanamycin was used instead of chloramphenicol as a resistance marker. And the attempt went faultlessly.

So yes we did manage to disrupt ompT, although a bit later than expected. The difference in effectiveness between the two antibiotics is still unclear. Perhaps further investigation on this area within gene disruption ought to be done. What can be concluded though is that when it comes to disrupting ompT, homologous recombination via λ Red phage is a successful method. But when it comes to choosing antibiotic as resistance marker, kanamycin is a preferable choice over chloramphenicol.

The second part of the investigation was never performed. Initially, the aim was to disrupt ompT and then investigate the effects on recombinant protein production, in which the latter was never started. Further experiments ought to repeat this experiment and further investigate the disruptions effect on protein production in the organism.

Bachelor’s project in Molecular Biology 15 credits autumn 2018.
Department of Biology, Lund University

Supervisor: Claes von Wachenfeldt (Less)
Please use this url to cite or link to this publication:
author
Dahlman, Anna
supervisor
organization
course
MOBK01 20182
year
type
M2 - Bachelor Degree
subject
language
English
id
8972963
date added to LUP
2019-03-14 09:22:04
date last changed
2019-03-14 09:22:04
@misc{8972963,
  author       = {{Dahlman, Anna}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Disruption of ompT in Escherichia Coli using phage λ Red recombinase}},
  year         = {{2019}},
}