I have cloned the beta galactosidase gene into a pQE-tagzyme based vector. The expression system in IPTG inducible with a T5 based promoter. Hence the promoter should be recognized by the host RNA polymerase.
Post cloning I have checked the expression and the activity of the enzyme in E.coli top10 cells as well as BL21(DE3) cells. The enzyme seems to express and seems to break down ONPG.
But the expression or the activity cannot be seen when the vector was transformed and subsequently induced in E.coli K12 cells. Any suggestions are welcome.
DH10B is supposed to have the lacZΔM15 mutation that prevents B-gal function without complementation by the LacZα peptide. Top10 is probably genetically identical to DH10B. I'm honestly a little surprised the enzyme even works at all to be honest. And did you see no enzyme activity in MG1655 or was it just not any higher than wild-type? Because MG1655 should be wild-type for all genes in the lac operon, so it should express no matter what. You might want to double check your strains - X-Gal also usually gives a clearer result than ONPG so it might be worth testing with that.
The cells used for amplifying the gene were a gift from another lab, so there could be a chance of being another e.coli strain. That may explain the discrepancy as the enzyme works very well.
In that case I would definitely streak them out onto IPTG + X Gal plates allow 24 hours for growth at 37°, then maybe incubate a couple hours at 4° before checking. DH10B/Top10 should give white colonies, MG1655 should give blue colonies (might not be as intensely blue as a normal blue-white screen using a plasmid)
If you don't have X-Gal available, MacConkey agar will also show lactose fermentation. You can also try ONPG again if you don't have access to MacConkey agar either, I just find it difficult to check ONPG on normal growth media because the yellow color doesn't contrast with the agar very well.
If you have streptomycin available, you can also check for growth on streptomycin. DH10B/Top10 are highly resistant to streptomycin due to a mutation in rpsL, MG1655 lacks the mutation and is strep sensitive.
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