Creating A Gene Deletion in Scherichia coli

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Brian J. Beames and Dr. William R. McCleary, Microbiology

Introduction

Two component systems are an integral part of a bacterial cell. They consist of a histidine kinase and a response regulator. The histidine kinase is a receptor protein that signals from the outside of the cell to the inside. Once a ligand binds to the histidine kinase, it autophosphorylates on the inside of the cell and activates the response regulator by phosphorylating it. Once the response regulator is activated, it can then phosphorylate another protein of the cell and that protein can carry out its function. An example of a two component system is the Pho system. PhoB is a response regulator that is phosphorylated by PhoR, a histidine kinase. When PhoB is phosphorylated, it binds to a region of DNA called a promoter that activates the trascription of a gene called phoA. The PhoA protein is an alkaline phosphatase which takes up phosphate from the environment when phosphate is in low quantities.

Hypothesis

A gene mutation created in the laboratory on a plasmid can be transferred into the chromosome. This was done in Escherichia coli producing a strain that has a precise deletion of phoB. This was accomplished through homologous recombination techniques. Several tests were performed to ensure that the right strain was obtained. One test is an alkaline phosphatase assay which detects the activity of enzymes. Another test that was performed was PCR which detects the presence of the phoB gene. The last test that was performed was a Western Blot which detects the presence of PhoB protein.

Experimental Design

An E. coli bacterium (ANCK10) has a plasmid (pMP2) which was brought into the cell through transformation. This plasmid has a phoB deletion and DNA homologous to the ends of the phoB gene (the homologous DNA allows the plasmid to be integrated into the chromosome). The plasmid also has a gene that codes for antibiotic resistance against chloramphenicol and is a temperature sensitive vector that will integrate into the DNA when grown at 45 degrees Celsius. Integration comes about and was done through cross-over at sites on the DNA homologous to the chromosome. Integration also helped to transfer the mutation into the chromosome.

After the plasmid was integrated into the DNA, the next goal was to excise the plasmid so that the phoB mutation remains on the chromosome. This was accomplished through growing the bacterium at 28 degrees Celsius in liquid media. In doing this, two results happened. The first is that the plasmid contained the same genetic material as was started out with. The second result was that the strain desired was obtained. One thing to note is that even though the desired strain was obtained, the original copy of the phoB gene was still integrated into the plasmid due to the way the series of cross-overs occurred. It was important when performing this procedure that the bacteria was grown at the exponential phase to create optimum conditions for excision of the plasmid. Bacteria grow slower when the plasmid is integrated into the chromosome whereas bacteria that have excised the plasmid will grow faster.

After a series of dilutions, the bacteria were grown at 28 degrees Celsius on low phosphate agar to differentiate between the cells that integrated the mutations into the chromosome and those that have not. In normal cells, PhoA cleaves an organophosphate. The mutant with the phoB gene absent doesn=t have the PhoB protein to bind to the phoA promoter. Hence, it cannot turn on the phoA gene to produce the PhoA protein. As a result, the bacteria cannot cleave the organophosphate. Those colonies that can cleave the organophosphate will turn blue, whereas the colonies that can=t cleave the organophosphate will be white. Difficulty arose because the white colonies have a blue center due to the acid phosphatases cleaving the organophosphate. This confusion was solved by comparing the phoB mutant (deletion of phoB gene) to a cell that had both phoB and phoR, which is next to the phoB gene, deleted in the chromosome (strain ANCH1). After isolating the colonies, the the plasmid with the phob gene was cured (curing the plasmid is to rid the cell of the plasmid) so that the phoB gene was not in the cell. This was done by growing the bacteria in conditions in which the plasmid is not needed for the bacteria to live, i.e. growing in liquid culture without chloramphenicol.

To ensure the right strain was obtained, an alkaline phosphatase assay was performed to detect the presence of PhoA. PhoA was found to be absent. PCR was also performed to detect the presence of the phoB gene. No amplification of the phoB gene was found. This indicates that the phoB gene was not present in the cell. In addition to the alkaline phosphatase assay and PCR, a Western Blot was performed to detect the presence of the PhoB protein. A lack of the protein in the Western Blot indicated that the experiment was a success.

References

  • Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D. Molecular Biology of the Cell Third Edition. Pgs. 773-778, 1994.
  • Bloomfield, I. C., Vaughn, V., Rest, R. F., and Eisenstein, B. I. Allelic Exchange in Escherichia
    coli Using the Bacillus subtilis sacB gene and a Temperature-sensitive pSC101 Replicon. Molecular Microbiology, 5(6):1447-1457, 1991.
  • Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P., and Kushner, S. R. New Method for Generating Deletions and Gene Replacements in Escherichia coli. Journal of Bacteriology, 171(9):4617-4621, 1989.
  • Stryer, L. Biochemistry Fourth Edition. Pgs. 326-332, 1998.
  • Acknowledgment of Dr. William R. McCleary for his support and dedication. 30