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cloning into vectors with lacZ and gene knockout

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#1 donny



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Posted 04 September 2010 - 05:07 AM


I have an expression vector that has an MCS in a lacZ gene (ala pUC19). I'll be cloning my gene into the MCS site but I have a few questions.
1) Does my gene have to be in frame with the lacZ to be translated properly?
2) Do I have to include an ATG to my gene?
3) Would the expressed protein be a fusion protein with the N-terminal part of LacZ?

Also, another question regarding gene knockout from E coli. I read about the method using RED recombinase by Wanner. Most publications use the template plasmids (ie pKD3, pKD4 and pKD13) and recommended primer design to generate the PCR fragments but some papers simply design their own primers to insert a antibiotic-resistant gene (along with some upstream sequence) into the gene to be disrupted. I checked and found that the antibiotic-resistant gene is not in-frame with the gene that was deleted. That is, the disrupted gene look like this:
ATG.........(upstream sequence...ATG-inserted antibiotic-resistant gene).......TAA
How would the inserted inserted antibiotic-resistant gene be expressed to confer resistance? Through some promoters and ribosome binding sites in the upstream sequence?

Also, my co-supervisor said they don't use the recombinase method anymore. Instead, they are using transduction for gene knockout. But I thought transduction is used to transfer genes between cells. Can transduction be used to transfer a gene from a PCR fragment into the chromosome?

Pls help.Thanks!

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