I'm growing E. coli with an empty plasmid (no gene) for the standardization of the growth rate of other constructs containing different recombinant genes. I found that when I start the induction with IPTG, there is some toxicity production that more or less stops the growth of E. coli containing the empty plasmid.
I tried to grow E. coli with the empty plasmid without inducing with IPTG, but the growth rate is lower than in the majority of my constructs containing different recombinant genes. Does anyone have any explanation or any suggestions about how I can grow my positive control?
Yes, the empty plasmid when induced with IPTG seems to express toxic genes for E. coli. so is not possible, at least pET plasmid to use it empty. See this paper:
Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels
Bruno Miroux, John E. Walker
"Surprisingly, none of the cells containing the ‘‘empty’’ plasmids produced colonies in the presence of IPTG, except for pET 17b, which gave very small colonies, demonstrating that the plasmids themselves are intrinsically toxic to E. coli BL21(DE3) host cells."
Cheers
Are the recombinant genes related to the consumption of the carbon source? I assume the pH is adjusted equally so that shouldn't be a problem. I don't think your possitive control has to show the highest growth rate.
My simple guess is that your control colony was not a "happy" one. I would just try to make them fit again and check if they form normal colonies on a LB plate. We found that common E.coli strains (BL21, DH5alpha, NEB10beta, etc) with AmpR need up to 150-200ug/ml ampicillin for example to not form satellite colonies (that may have no vector in it) or overgrow (ditto). So just go 1-2 times over LB plates + enough antibiotic to get a healthy colony. Good luck!
Ok, my strain is the BL21(DE3). I checked before with a colony PCR, but I can try to plate again and verify. Thanks
I have transformed BL21 cells with an empty pET21 vector several times with no IPTG induction and never noticed any growth problems. So I will eliminate the possibility of the empty vector causing this problem. You should probably try 1-2 more colonies. Make sure your antibiotic concentration is appropriate, because too much might hinder the growth, check your LB media once again. Good luck.
I wonder if the bacterial strain behaves identically with other vector, or is dependent vector pET19b. Have you tested this?
I got the same thing with E. coli transfromed with different kind of empty vectors: pET22 and pET15b. They grow normally at standard conditions but once I induce with IPTG, their OD600 can only reach more or less 1 (in LB) while other transformants with gene can grow much better. Have you figured out your question so far? Thanks
Yes, the empty plasmid when induced with IPTG seems to express toxic genes for E. coli. so is not possible, at least pET plasmid to use it empty. See this paper:
Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels
Bruno Miroux, John E. Walker
"Surprisingly, none of the cells containing the ‘‘empty’’ plasmids produced colonies in the presence of IPTG, except for pET 17b, which gave very small colonies, demonstrating that the plasmids themselves are intrinsically toxic to E. coli BL21(DE3) host cells."
Cheers
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