For my bacteria gene, after I got a sequence from the company I compared with the original gene from the gene bank. The result was that my gene differed by just one nucleotide, so that changed 1 amino acid (from Leucine to Proline). Is my gene okay? Can I use that for protein expression? Thanks in advance.
What you found is a SNP difference. Most SNPs accumulate in noncoding regions and in regions of the coding sequence that don't change the amino acid sequence. Most SNPs when they do accumulate in the coding region, they become deleterious, we don't see that much. From your question: you found from sequence that the amino acid changed, from L to P. Well leucine is a hydrophobic amino acid and so is proline; however, proline does cause kinks in folding. Do you have the structure of the protein? P could change how helix look, and etc. Thus could change protein folding and thus make your protein less functional. So to answer your question is your gene ok? Yes, I doubt the gene expression is affected in the transcription level or is affected at the translational level. I would think at the protein functional level probably affected. To answer you second question, can I use that for expression protein? I dont quick understand this, but I think you are asking can I use this for looking at gene expression?
Yes if there is truly an expression difference, but I doubt it. This SNP difference could affect a different gene. I has been shown many times that regions within bacterial genes regulate expression of other genes. Anyways food for thought. Hope this helps.
You need to figure out that whether the sequencing result is correct or your gene cloned is indeed changed. How long is your gene? How many reactions were carried out in your sequencing?
Generally, the change from L to P will affect the structure intensively, because proline can introduce a turn in 3D structure.
Whereas, a site-directed mutagenesis reaction is easy to perform, such as overlapping extension PCR. Just change it back.
Hope it helps. Good luck.
Many thanks to both of you, i still have doubt about this problem, so i was isolated another colony and sent again for sequencing, now i am waiting for the result .
Thanks again ^^
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