This may sound stupid/trivial to some of you, but I am not quite clear on this. I will explain myself.
Let's suppose that I have created a recombinant plasmid into which I cloned the gene coding for a bacteriocin under the control of a lac operator (bacteriocins are secreted by bacteria in order to inhibit growth of other bacteria which compete for the same niche) and then I insert this recombinant plasmid into a competent E. coli strain. I will then add IPTG and my E.coli will start to produce the recombinant protein. But how can I be sure that my bacteriocin will not "kill" the bacteria that produce it? In more general terms, how can I be sure that my microorganism (bacterium or fungus) will not be in any way harmed by the biomolecule that I have engineered them to produce in large quantities?
Thank you in advance!
In fact any recombinant protein could be "toxic" for the E.coli cells for different reasons. You just have to do one expression and to check. To minimize the toxic effect of the recombinant protein, one may try to use single copy expression vector ... also strict promotor , to inhibit the basal expression that some inducable systems have; also try induction for shorter time ... try different E.coli strains as host ... good luck !
You can never be sure that "my microorganism (bacterium or fungus) will not be in any way harmed by the biomolecule that I have engineered them to produce in large quantities". Indeed it is a known problem.
There are many solutions, using Glucose to inhibit leaks from the Plac promoter, opting for an expression system like the BL21/pLys any many others. Google: 'Overexpression toxic proteins'
Yoram
I would say first try to test if it is indeed toxic. And if it is you can try to search what mechanism does the strain that originally produces this bacteriocin is using to resist its action, and then try to mimic this action in your e coli chassis.
Maybe a long shot, but if nothing works you can try to put a enteropeptidase site before the sequence of your protein and before that site a protein clump domain and hope that the bacteriocin get clumped and can't act or at least lose some toxicity. After purification you would digest them with enteropetidase to get your pure bacteriocin.
If you are using the T7 system with IPTG you can add 1% glucose throughout (LB agar for transformations and all subsequent media) as Yoram has already suggested. This will effectively block expression from the T7 system especially if you use something like LysS/E strains until the point of induction-it will at least allow you to get some biomass before the expressed toxic protein begins to kill your cells.
Auto-induction may also help as the protein is not expressed until the cells are at, or near, saturation so they are no longer in growth phase when induced (the phase where classically you would add IPTG) .
Another option may be to try to push them into inclusion bodies as rapidly as possible (high IPTG and 37°C for 3-4hrs may be enough) that would essentially partition your toxic protein from the cytoplasm-in fact this is the whole point of inclusion bodies, to protect the cells from toxic or mis-folded protein. The obvious disadvanatge is that you then have to find an efficient method to re-fold your protein.
it help if you understand how the biomolecule is harming or stressing the host ..very often it is not obvious, but we can use omics tools like RNSseq or MS to elucidate possible causes of the metabolic stress. this will sometimes lead to insights and solutions if we can identify the problem..eg..co-factors limitiation or membrane stress..
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