Dear All,
I am currently working on a project based around an antibiotic resistance enzyme and I got some really interesting results. I am looking to confirm whether the enzyme under investigation can cleave the same antibiotics in vitro and in vivo. Therefore I am looking for a plasmid which will have constitutive expression of gene of interest, however I seem unable to find one.
Could any of you be kind enough and point me to a place where I could buy a plasmid capable of constitutive expression of a gene.
You could just use any of the standard cloning vectors like pUC18/19 or Bluescript, or pGEM etc, that have the lac promoter on them (and do NOT have lacI gene on them, most of these do not) and use an E. coli strain that does not overexpress lac repressor. For example DH5alpha would work fine. This would for all practical purposes be constitutive.
Dear Michall
you can check on the Addgene website
https://www.addgene.org/
good luck
Manuele
The very reason the cloning vectors are designed to not constitutionally express the gene is so that the genes can be controlled by the researcher. Try looking at what you are doing from a biosafety perspective, and you might want to go for a controllable promoter.
I routinely clone resistance genes into the pcr-blunt II vector (Zero Blunt TOPO PCR Cloning Kit). If you transform this vector in E.coli TOP10 cells, your gene will be constitutively expressed. I use this system to measure MICs conferred by different resistance genes/influence of different mutations on the original MIC.
Just be aware that your PCR products need blunt ends and you might need to include the promotor region.
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