Using pcr2.1 Topo (cloning) vector as expression, Huh? - possible? I heard from a PhD student seminar... (May/31/2011 )

Dear All,

I am confused.

I need your opinion in this. I attended a PhD student research seminar about quorum sensing and quorum quenching. I do not hear wrongly: She uses pcr2.1 Topo (a cloning vector, not expression vector), cloned in a quorum sensing gene from acinetobacter, and express in E.coli DHalpha (clone 1). She then use an RE, cut in the middle of the cloned quorum quenching gene, and inserted a "tetracycline gene" to "disrupt the cloned gene", and shows tetracycline gene expression (by surviving) in media with tetracycline antibiotics (clone 2). She then compare the biofilm formation of "Clone1" and "clone2",(mind you, is in E.coli DHalpha) and found that "clone1" have a higher biofilm formation than "clone2". So, she concluded that Acinetobacter/Organism with that quorum sensing gene able to form biofilm for survival in antibiotics.

I do not agree with her experimental design, and think that her approach was flawed. In my opinion, DHalpha is not mean for expression, and 2nd expression vector should not be use in expressing protein. 3rd, the gene disruption method she use is not necessary for comparison of biofilm formation. 4th, she use E.coli as comparison rather than Acinetobacter.

Since she is a PhD student, and I even asked one of the lecturer there and he thinks there is no problem (he even says pcr 2.1 topo is Expression vector), perhaps I am wrong. And since I am wrong, can anyone please correct my misconception.

Many thanks.
Adrian

There is little special about an "expression vector" which distinguishes it from a cloning vector. They sometimes contain a built-in promoter, either inducible with IPTG or arabinose, for example, or a T7 promoter for use with T7 polymerase containing strains such as (DE3) strains. They are sometimes lower copy number than the cloning vectors, which tend to be high copy number. But a vector is a vector, and if you make a construct with a promoter and a coding region, it will express. More problematic is the choice of strain, and you are right that DH5a is a lousy expression strain. And who knows what she is trying to prove by looking at biofilm formation of E. coli cells expressing some random gene from Acinetobacter.