You have an Hfr strain of E. coli that contains wild-type genes for maltose catabolism and...

You have an Hfr strain of E. coli that contains wild-type genes for maltose catabolism and alanine biosynthesis. You want to transfer these two genes into an F- strain that cannot use maltose and is auxotrophic for alanine, by conjugation. Your Hfr strain also has a tetracycline resistance gene located distal to the origin of transfer. You have a P1 phage suspension and any ONE strain of E. coli given in the MBIO 2020 lab manual at your disposal. Outline briefly how you would use these materials to carry out a successful conjugation and transfer the two genes of interest. You do not need to include experimental details, but you should give a medium that could be used to select for the successful recombinants after each gene transfer that you use. In the box below, briefly answer the following questions:

Why can’t you just conjugate the two strains as they are? (ie. why do you even need the P1 phage to carry out this experiment?)

What ONE additional E. coli strain from the lab manual would you use?

Outline a brief plan to transfer the mal and ala genes from the Hfr strain to the F- strain. Do not include experimental details (reagents, incubation times, number of test tubes, etc). DO INCLUDE an appropriate selection step for every gene transfer experiment that you use.

Solutions

Expert Solution

The conjugation is the way of transforming a small stretch of DNA from one bacteria to another by conjugation tube. The transduction is the way by which DNA is transferred by a virus. P1 phage is a lytic phage that has a special efficiency of integrating chromosomal DNA from one bacteria and introducing that DNA into another. So if we can grow that P1 lytic phage in an HFR+ strain and then infect the HFR- strain with that P1 phage that was grown in HFR+ strain, the DNA from HFR+ strain will get introduced in HFR- strain. One more important thing is that the DNA will be integrated into the chromosome after phage transduction.

After this tranduction you can use selection media that will have tetracycline and other metabolites that will help to grow only the bacteria which has the DNA and can actively express the protein fro that DNA. This way it will help to select the transduced bacterial strains.

gladiator answered 4 months ago

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