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) The media should have lactose ( lac+ not sensitive to lactose), TI phages and lack cystienes (cys+ not autotrophic to cystienes)

B) gal+ His- Pur- TonR

C) map is

lac+ <-> pur+ <-> gal+ <-> cys-/trp+ <-> his+

5 5 5 15 (in min)

D) all 50 colonies are TonR

Cys+ colonies are 13

Therefore frequency of TonR and Cys+ = 13/50 =0.26

Trp+ colonies are 27

Therefore frequency of TonR and Trp+ = 27/50 =0.54

Based on co transduction frequency , closes to TonR is Cys

And the order of the genes are Cys Ton Trp

Related Questions

Navigate

• Browse (All)
• Browse W
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.