While working in Dr. Strap’s lab you identify a number of E. coli mutants that o

Question

While working in Dr. Strap’s lab you identify a number of E. coli mutants that only grow in the presence of cysteine. Your supervisor suggests that the next step is for you to map the cys gene you have discovered relative to other known mutations. To do this, you start with bacterial strains: Hfr cys- gal+ his+ lac+ pur+ trp+ tons F- cys+ gal- his- lac- pur- trp- tonR cys- his- pur- trp- = auxotrophic for cysteine, histidine, purines, tryptophan. lac- gal- = cannot metabolize lactose or galactose. tons = sensitive to phage T1. a. If you want to select for cys+, lac+ and tonR recombinants resulting from a cross between the Hfr and F- strains, what media would you need to plate the bacteria on? That is, what nutrients would the minimal media need to be supplemented with? b. If you supplement the minimal media with glucose, histidine, purines and T1 phage, what recombinant classes will you grow? c. You next perform an interrupted mating experiment using the two strains and of the tonR colonies you obtain the colonies with the following phenotypes: cys+ 0 min cys- 25 min gal+ 20 min his+ 40 min lac+ 10 min pur+ 15 min trp+ 25 min Based on these results draw a map of the bacterial chromosome and the relative position of each of the markers and the distances between them in minutes. d. To determine a more precise position for the cys gene your supervisor insists that you should use P1 co-transduction. To do this, you use a phage P1 grown on a strain of E. coli which is cys+ tonR trp- to infect a recipient strain of E. coli which is cys- tons trp+. These bacteria are then plated on complete media with phage T1. You obtain 50 tonR transductants which you go on to test on minimal media for the other markers. cys+ trp+ 3 cys+ trp- 10 cys- trp+ 24 cys- trp- 13 Based on these results, what is the co-transduction frequency between cys and ton? Between ton and trp? Which of the markers is closest to ton? What is the correct gene order?

Explanation / Answer

a) cys+, lac+ and tonR recombinants will be selected on minimal media containing cysteine, lactose and phages. The recombinants containing tonS, were allowed to grow on media having cysteine, purine, histidine, lactose, galactose, tryptophan, will not grow as they will be sensitive to phages due to the presence of tonS gene. The media containing cys, lac will allow the bacterial growth only if they have tonR gene which are resistant to phages. Therefore, ton gene is basically used for the selection of particular recombinants in the presence or absence of nutrients.

b) The minimal media containing glucose, histidine, purines and T1 phage, will allow the growth of recombinants with genes - glu+ hist+ pur+ tonR

c) cys+-----10 min---lac+---15 min---pur+----20 min---gal+---25 min--- trp+ cys- -----40 min---- his+

d) co-transduction frequency between cys and ton = cys+ + ton+/ total recombinants*100

co-transduction frequency between cys and ton = 13/50 *100 = 26%

  co-transduction frequency between ton and trp = 27/50 *100 = 54%

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