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Help complementing my mutant in trans


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#1 nathankho

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Posted 07 October 2009 - 11:03 AM

I'm working with E. coli and have made an insertional mutant using the lambda red recombinase system, and inserted a kanamycin resistance casette into my gene of interest to generate this mutant. Now I wish to complement the mutant in trans.

The plasmid I want:
Low copy # to ensure expression of my gene doesnt get too crazy
an antibiotic marker other than ampicillin or kanamycin since this mutant is already resistant to both

I just want to PCR out the protein encoding region of my gene of interest, cut both ends using different RE (ill be generating my own RE sites using primers) and insert it into the plasmid MCS. I just need one that has a ribosome binding site, e. coli promoter etc upstream of it to ensure that the gene will be expressed once i stick it back into the host...or am I overthinking things.

Could I, let's say, just get pBR322, and insert my gene into the ampicillin resistance gene (at EcoRI and PstI sites) in the same orientation as the original ampicillin gene, thereby just using the ampicllin promoter to drive the expression of my gene?

All this Shine-dalgarno, RBS, and Promoter stuff is so confusing!

#2 fishdoc

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Posted 07 October 2009 - 12:13 PM

I'm working with E. coli and have made an insertional mutant using the lambda red recombinase system, and inserted a kanamycin resistance casette into my gene of interest to generate this mutant. Now I wish to complement the mutant in trans.

The plasmid I want:
Low copy # to ensure expression of my gene doesnt get too crazy
an antibiotic marker other than ampicillin or kanamycin since this mutant is already resistant to both

I just want to PCR out the protein encoding region of my gene of interest, cut both ends using different RE (ill be generating my own RE sites using primers) and insert it into the plasmid MCS. I just need one that has a ribosome binding site, e. coli promoter etc upstream of it to ensure that the gene will be expressed once i stick it back into the host...or am I overthinking things.

Could I, let's say, just get pBR322, and insert my gene into the ampicillin resistance gene (at EcoRI and PstI sites) in the same orientation as the original ampicillin gene, thereby just using the ampicllin promoter to drive the expression of my gene?

All this Shine-dalgarno, RBS, and Promoter stuff is so confusing!



I use pBBR1 to complement my mutants. There are 4 types of antibiotic markers available, chlor, kan, amp, and tet, I think. As for the promoter, I always try to clone enough of my gene to include its native promoter so that it's induced from the plasmid when it's induced naturally. However, I think pBBR1 has the lac promoter, so you could express it from there by inducing with IPTG if you'd like.




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