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Verification of mutant


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#1 Nhung Nguyen

Nhung Nguyen

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Posted 29 October 2015 - 03:06 AM

Hi all,

I am currently doing experiment with gene mutation and expression on a host as yeast Sachraromyces cereviciae. I am looking for a mutant which encodes an enzyme with ability to resistant in a toxin.

 

To make a mutant, I use 2 methods of mutagenesis which are EPPCR and using mutator strain C.coli XL1 red.

The steps that I have been followed after I get the mutant plasmid are:

1. Transform the mutant plasmid (in case of XL1 red) or EPPCR fragment + vector backbone (in case of mutagenesis by EPPCR) to yeast Sacharomyces cereviciae.

2. Wash the transformant by media and spread on toxin plate --> After several days of incubation, there are some colonies on the toxin plates. They are named ''Candidates''. 

3. Innoculate the candidate (single colony) on appropriate media, then spot it in toxin plate (ca. 30.000 cells/spot). Result: I got the spot with positive result! But it is not ended here. I have to do following steps and cannot explain the result!

 

4. Extract DNA from the candiate (which give positive result in step 3)

5. Transform the DNA to E.coli electro competent cell DH10B

6. Miniprep E.coli transformant

7. Retransform to yeast, then I take the transformant to spot in toxin plate as mentioned in step 3.

RESULTS: No colonies grow on the spotting plate (even I have about 200 candidates obtained from Step 3). But all of these candidates contain plasmid mutated by EPPCR. The plasmid mutated by XL1 Red is on progress at step 6. I hope that for this kind of mutantation, I can get the positive result in the second test on toxin).

 

Can anyone explain to me why the candidates grow on toxin in step 3 but after retransformed and tested in toxin in the second time, the function is lost? Does it mean that there are more than one possibility (not only the mutant) for yeast transformant to grow on Step 3? 

If so, does it happen in the case of mutation by XL1 Red and transforme the entire mutant plasmid to yeast?

 

Thank you so much for your time to read and I hope to receive your discussion.

Best wishes,

Nhung


Edited by Nhung Nguyen, 29 October 2015 - 03:25 AM.





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