We have antibiotic resistance genes cloned into the pET(+)-21 vector and transformed in DE3 E. coli cells. I realize that this system is used for recombinant expression of proteins, however we want to use this system to determine antibiotic susceptibility patterns of the E. coli cells due to expression of the insert. Preliminary experiments induced with IPTG ranging from 0.4 - 1 mM indicated either no resistance patterns where expected, or single colonies forming around the antibiotic disc. We assume this is due to the formation of inclusion bodies due to hyperexpression of this vector-host system.
Can anyone please provide some insight on how to get this system to express at functional protein levels so that antibiotic susceptibility screening can be performed?
Your assumption that inclusion body formation is the reason why you are not seeing resistance may not be a valid explanation. There are a variety of reasons why a protein might not be expressed, some as simple as your insert has picked up a mutation. Even if most of the protein were in inclusion bodies I would expect that you see some resistance since you don't require much protein to confer resistance (with typical antibiotic resistance genes).
You should be able to observe reasonable levels of resistance even without any IPTG induction, so you might wish to use that as your preliminary screen. You might also want to retransform your plasmid (I presume you have sequenced it? ) and test a number of colonies.
Are you working with any unusual antibiotic resistance genes?
Michael J. Benedik thanks for the feedback. Yes, we sequenced and confirmed the vector inserts and yes we do see some resistance profiles for some genes, but become altered when IPTG concentrations are changed or even incubation temperatures. Also, as you mention, we do see some resistance for some antibiotics without induction due to lack of non-zero expression. We ran all experiments with all appropriate controls and a reasonable explanation could be exclusion bodies which is why it is one of the strategies we are exploring.
You might also be seeing some toxicity issues, for example if your resistance gene were a efflux pump or a secreted protein, you might be exceeding the capacity of the cellular translocation machinery to handle high levels of the protein.
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