Question ? for you to help me..!

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Nmr
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Question ? for you to help me..!

Post by Nmr » Thu Oct 21, 2010 2:11 am

What should I do if I want to mutate a protein but I do not have the gene sequence? All I have is the native protein from a wild-type bacteria?

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canalon
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Post by canalon » Thu Oct 21, 2010 2:22 am

1- add mutagens in the media and grow the bacteria, or use UV. Mutations will appear everywhere and maybe in your protein. If you know more about your protein and what kind of mutation you want, you can try to devise a selection step to weed the different mutants out.

2- Sequence the protein. expensive, needs specialized material, but doable...

3- Chop up the DNA of your bacteria in big pieces, clone and look which clone has the protein. Purify vector and chop in smaller pieces repeat until you either do not get the protein anymore, or the fragment is reasonably small for sequence
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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JackBean
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Post by JackBean » Thu Oct 21, 2010 5:50 am

simply sequence your protein. Nowadays it's not so complicated, especially with so many sequenced genomes, which should be able to help you find some homologues.
After you get the protein sequence, find the DNA sequence, here you have several choices. And then you have plenty ways to make mutated protein...
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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