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Screening of a mutation?

Genetics as it applies to evolution, molecular biology, and medical aspects.

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Screening of a mutation?

Postby JessicaDonaldson » Tue Feb 01, 2011 5:26 am

Suppose you isolate a mutation in a forward genetic screen in e.coli and you identify the gene as ybc1 by sequencing. ybc1 appears to confer ability to grow in the absence of thiamine. How could you confirm the identity of ybc1?

Im confused because would it be completely wrong to answer do a screen and plate on plates with thiamine and if they grow then its there and if they dont grow then its not?
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Postby JackBean » Tue Feb 01, 2011 5:39 am

not really, if you grew it on plate with thiamine, all bacteria would grow. On the other hand, if you used plates w/o thiamine, only those, who are able to synthesise it (AKA those with ybc1 mutation) would grow

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Postby canalon » Tue Feb 01, 2011 10:25 pm

The plates should be a defined Minimal medium (MM) of course (like M9 glucose) But I am slightly confused as most E. coli will grow on M9 Glucose w/o the need to add thiamine anyway. But if the parental strain do not you might be able to select that way.

On the other hand if you are looking for bacteria that suddenly become unable to synthesize thiamine from scratch, you will have to do replica plating on MM and MM+Thiamine. If a clone grow only on the supplemented plate, you know that your gene has been disrupted.

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