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Disruption of ompT in Escherichia coli using phage λ Red recombinase

Dunér, Emma (2019) MOBK01 20182
Degree Projects in Molecular Biology
Popular Abstract
Removal of a gene in E. coli bacteria

Although bacteria are commonly associated with something negative and disgusting it is not the whole truth. It is true that some bacteria can have negative consequences, but as with most things it is unfair to generalize. Bacteria are actually is fascinating organisms. There are many uses for bacteria as well, such as production of protein and other genetic work. A certain strain of the bacteria Escherichia coli (E. coli) is commonly used for protein production. This strain lacks a protease that is present in other strains. Thus, the aim has been to remove the protease from the strain in which it is present, and see if it can be useful in protein production.

The removal of the proteas is done by... (More)
Removal of a gene in E. coli bacteria

Although bacteria are commonly associated with something negative and disgusting it is not the whole truth. It is true that some bacteria can have negative consequences, but as with most things it is unfair to generalize. Bacteria are actually is fascinating organisms. There are many uses for bacteria as well, such as production of protein and other genetic work. A certain strain of the bacteria Escherichia coli (E. coli) is commonly used for protein production. This strain lacks a protease that is present in other strains. Thus, the aim has been to remove the protease from the strain in which it is present, and see if it can be useful in protein production.

The removal of the proteas is done by removing the gene that encodes the proteas.
A gene contains information to generate a specific protease. This can be thought of as a recipe and specific cookie for that recipe. Imagine the gene being the recipe, and the protease the cookie. So, if no such recipe is present, the specific cookie cannot be made. Likewise, if no such gene is present the protease cannot be generated.

How is the removal achieved?

The method that has been used is based on recombination, which essentially is substitution. The gene encoding the protease will be replaced by a gene encoding antibiotic resistance. The antibiotic resistance gene is present in a plasmid. Plasmids are circular fragments of DNA containing genes (fig.1a). To insert the antibiotic resistance gene into the bacteria we mixed the bacteria and the plasmids in a tube and then exposed them to an electrical field (fig.1b). This sensitizes the bacteria and makes the bacteria more permeable, hence facilitating the entrance of the plasmid with the gene.

Next the bacteria were spread on a plate containing nutrients and the antibiotics. Bacterial growth on this plate indicates that bacteria has acquired the antibiotic resistance gene, they are resistant to the antibiotics. To confirm whether the gene was replaced and removed we sent a sample for sequencing.

As we supposed from the growth on the plate, the bacteria cells had obtained the antibiotic resistance gene. However, the sequencinging revealed that the gene encoding the protease was still present in the bacteria. This can possibly mean that the resistance gene was inserted somewhere else, rather than substituted the protease encoding gene. Ergo, we were able to insert the antibiotic resistance gene but not remove the protease encoding gene.

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

Supervisors: Claes von Wachenfeldt and Vinardas Kelpsas
Department of Biology, Lund university (Less)
Please use this url to cite or link to this publication:
author
Dunér, Emma
supervisor
organization
course
MOBK01 20182
year
type
M2 - Bachelor Degree
subject
language
English
id
8972960
date added to LUP
2019-03-14 08:59:49
date last changed
2019-03-14 08:59:49
@misc{8972960,
  author       = {{Dunér, Emma}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Disruption of ompT in Escherichia coli using phage λ Red recombinase}},
  year         = {{2019}},
}