In a controlled mating experiment, researchers crossed an E.coli Hfr strain of g
ID: 60295 • Letter: I
Question
In a controlled mating experiment, researchers crossed an E.coli Hfr strain of genotype leu+arg+his+ade-tetS with an F- strain of genotype leu-arg-his-ade+tetR. Conjugation was allowed to proceed for 20 minutes and then interrupted. Exconjugants were then plated on A, B, C or D type agar (see Table 1 for agar composition) and assessed for growth (see Table 2).
Table 1. Agar types consist of minimal medium and various combinations of nutrient supplements or antibiotics (plus “+” indicates addition of a component while minus “-“ indicates that component is not added).
Agar Type
Leu
Arg
His
Ade
Tet
Alleles Selected
A
+
-
+
+
+
B
+
+
-
+
+
C
-
+
+
+
+
D
+
+
+
-
+
Table 2. Exconjugant growth on agar types A through C.
Agar Type
# Colonies Observed
A
197
B
333
C
50
a) Why is the antibiotic tetracyclin (Tet) added to each type of agar? (1 point)
b)In the spacer provided in Table 1, indicate which alleles are being selected for on each agar type (A through D). (2 points)
c) What is the order of transfer of leu, arg, and his genes from the Hfr strain to F-recipient? Explain how you were able to infer gen transfer order. (2 points)
d) Fifty individual colonies from each agar plate type: 50 from A, 50 from B and 50 from C were picked and tested for growth on agar type D. Of the transferred colonies, 5 of 50 colonies from agar type A grew on D, 15 of 50 agar type B colonies grew on D and 27 of 50 colonies from agar grow on D. Based on this data, which of three genes, leu, arg or his is closest to ade on the bacterial chromosome? Explain your reasoning. (2 points)
Agar Type
Leu
Arg
His
Ade
Tet
Alleles Selected
A
+
-
+
+
+
B
+
+
-
+
+
C
-
+
+
+
+
D
+
+
+
-
+
Explanation / Answer
a) Tetracycline has been added to ensure that only those bacteria grow in which conjugation has taken place as the first gene which would be transferred from an Hfr to an F- bacteria would be a Tets gene.
b) Agar type A Arg
B His
C Leu
D Ade
c) The order of transfer is Leu-Arg-His. The closer the gene will be towards the origin of transfer the more exconjugants will it produce.
d) Leu is closest to Ade gene as the agar plates didn't contain Leu so the overall growth of exonjugants was less. However, bacteris from C grew most on D which suggests that Leu gene is close to Ade.