You have available an Hfr lac - ala + vitB + tet s strain of E. coli and an F -

Question

You have available an Hfr lac-ala+vitB+tetsstrain of E. coli and an F-lac+ala-vitB-tetsstrain. You also have P1 transducing phage and a tube of plasmids with a gene for tetracycline resistance. These are the ONLY supplies you can use (besides media and reagents). Describe how you can isolate a pure strain of E.coli lac+ala+vitB+tetr, where all genes of interest are present in the chromosome of the bacterium. Include the gene transfer steps you would use, as well as all media that you would use to select for successful gene transfers.

Can you please explain to me how you know which steps come first? I know it will involve transformation, transduction, and conjugation but other than that I don't know where to go from.

Explanation / Answer

The strains available are:

1 Hfr lac-ala+ vitB+ tets

2) F-lac+ ala-vitB- tets

Since donor cell is Hfr strain, conjugation may not yield F+ cells.

Step 1:

Procedure:

In first step transduction with P1 phage is carried out. Cultures of donor cell strain is inoculated in media, Lactose broth and incubated overnight. The media are supplemented with MgCl2 and CaCl2, to facilitate infectivity by P1 phage. The culture is aerated at for about one to two hours. When turbidity is observed, P1 phage is added. The culture tubes are monitored to observe lytic activity of phage (observing formation of bacterial cell debris or 37°C decrease in turbidity). To terminate the transduction process, add chloroform and vortex, (chloroform should later be allowed to evaporate out of the tubes).

The recipient cells are separately cultured in LB media, overnight. The lysate from the donor cell tube, is added to the recipient cell broth and incubated at 37°C for about one to two hours. The Culture may be further supplemented with LB and sodium citrate, to allow genetic expressions, followed by plating foe selection.

Selection:

The cells are checked for the transfer of ala+ and vitB+ from donor, while recipient cells are lac+. Thus, selection procedure should involve selection of strains of E. coli with lac+ala+vitB+tets.

This may be achieved by using X-Gal media with alanine and vitamin and screened for blue colonies. Or, separate screening for lactose positive ca be screened in X-Gal ad blue colonies further inoculated in minimal media with vitamin B and alanine can be used.

Strains now are lac+ala+vitB+tets. Hence, now the tetracycline resistant gene needs to be incorporated. Since, we have plasmid with tetracycline resistant gene, process of transformation may be used.

Step 2:

Procedure:

The transformation solution with CaCl2 and plasmid. The colonies from step 1 are inoculated in the solution. The transformation is carried out in ice bath, followed by heat shock at 42°C.

Selection:

Selection may be done by using lactose agar or nutrient agar plates with antibiotic tetracycline.

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