What exactly does it mean by cloning a toxic gene - (Oct/17/2007 )
Forgive my elementary question, but what exactly does it mean by cloning a toxic gene? For example, I am trying to clone a gene that undergoes different rearrangements, deletions, and mutations. How is this reported to occur? How does the E. coli cut out sections? Any general reading I can do (books, articles)?
I am clearly befuddled.....
well i dont know if i understood your Q right or not but i will try to answer anyway
if you mean you get strange results * strange protein* from cloning ur E coli may be this is due to your protein codons is rare codons to the E coli so you will get some problem in ur expression like:
Interrupted translation, Frame shifting, Misincorporation of amino acids or even inhibition of protein synthesis and cell growth.
If your question is about cloning not expressing in E coli may be you can give me an idea about ur E coli type is it recA or not endA or not .
hope this help
Thanks for your response. I'm cloning into a non-expression vector (pENTR). I made a version of the gene I'm cloning with no stop codon and one with a stop codon. Interestingly, the clones with a stop codon were fine, but the ones without the stop codon underwent various changes. In fact, the changes aren't consistent (i.e., some clones have a 9bp deletion, some rearranged a 147bp section, and some added a stop codon). I've tried 4 different competent cells AND culturing at 28 degrees C, but I can't seem to get the correct sequence. I should note that I'm using an Invitrogen clone as template for the PCR and my primers contain NOTI & ASCI sites for forward and reverse, respectively.