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Mapping mutants-how to interpret expreiment results.

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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Mapping mutants-how to interpret expreiment results.

Postby turlaa » Wed May 19, 2010 10:40 pm

Hi!
I got some microbial genetics practicals in this semester and all I got are results without any interpratation. Now I try to make some excersises to understand this subject but I don't think I can sort it myself. Therfore I kindly ask for help with my excersices or if you know any good literature which would help me get a grasp of what is going on I would be thankful! This is on undergraduate level.

Task
A bacteriophage lysate prepared on E. coli strain SY23 (galK argC) was employed to transduce E. coli strain DD3 (trpF tolB thiA).
Following brief incubation with the bacteriophage, samples were plated out on two different selective media:

(a) glucose minimal agar containing arginine and tryptophan and

(b) glucose minimal agar containing arginine and thiamin.

The colonies recovered on these selective media were then replica plated onto a variety of other media to screen for the inheritance of the non-selected markers as indicated in Table 1 below.

Use these data to explore the relative positions of the galK, argC, trpF, tolB & thiA genes on the bacterial chromosome and devise one additional experiment, indicating the media to be used for selection and screening that would clarify any uncertainties that remain following your analysis of the data provided in Table 1.

Table 1 Screening of recombinants for the inheritance of non-selected markers

Selective medium a- Minimal agar with arginine and tryptophan
Screening medium Number of colonies
Glucose MA + arg 337
Glucose MA + colicin B 54
Glucose MA + arg + colicin B 140
Glucose MA 57

Selective medium b-arginine and thiamin
Screening medium Number of colonies
Glucose MA + arg 437
Glucose MA 168
Galactose MA + arg 288
Galactose MA 163

galK unable to use galactose as sole source of carbon for energy and growth
argC requires arginine
trpF requires tryptophan
tolB tolerant of colicin B
thiA requires thiamin
turlaa
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Re: Mapping mutants-how to interpret expreiment results.

Postby turlaa » Wed May 19, 2010 10:51 pm

So what guessed already
Selective medium a- Minimal agar with arginine and tryptophan
Screening medium Number of colonies
Glucose MA + arg 337 Galk argC trpfF
Glucose MA + colicin B 54 GalK tolB trpF argC
Glucose MA + arg + colicin B 140 GalK argC tolB TrpF
Glucose MA 57 GalK trpF argC

Selective medium b-arginine and thiamin
Screening medium Number of colonies
Glucose MA + arg 437 galK ArgC ThiA
Glucose MA 168 galK Arg C ThiA
Galactose MA + arg 288 ArgC ThiA
Galactose MA 163 ArgC ThiA

So those are mutant present in those colonies. And this mutants are most likely there as it is impossible to be sure considering that selective medium contains arginine and tryptophan or thiamin. I'm sorry I don't really know how to start solving this task. I guess that when I will sort out % or recombination I will be able to map gene...
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Postby turlaa » Thu May 20, 2010 12:03 am

so can I say that for group growing on medium A
all colonies are galK mutants 100%
None of them is ThiA 0%
377 colonies are 100%
320 are ArgC mutants ( 377 colonies growing on glu MA + Arg - 57colonies growing on gluMA) =85%
237 are tolB mutants (377-54 colonies growing on MA+colicinB)=67% and now I just use those % as length of chromosome. I.e. all chromosome has lenght 100 so 67 is close to the midddle etc?
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