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Re: [ccp4bb] Codon Optimized Expression |
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CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 02 February 2008Subject: Re: Codon Optimized Expression From: David M Shechner shechner {- at -} MIT {- dot -} EDU Date: 2008-02-02 I'm also among the "strongly opposed" camp regarding mixing colonies for expression. Namely, when expressing proteins which might be slightly toxic to their host cells - either as a direct toxin, or by somehow altering the host's gene expression - we've observed a *strong* colony-to-colony variability in expression level. Our sense here (and we've done no direct investigations to the matter, but rather are going on what collaborators have told us), is that if the gene is toxic, the small amount of leaky synthesis that occurs prior to induction (perhaps even too little to make out on a gel) might serve as a strong enough selective pressure to turn off its expression. This could be through mutation of the gene itself, the lysogen, or any number of uncharacterized and otherwise unlikely epigenetic phenomena. We also see that those cells which *do* express the target gene tend to grow more slowly, which is circumstantial evidence supporting our model. Namely, if the cell hasn't somehow altered its expression to turn off the transgene, then the leaky expression during lag- and early log- phases causes a mild toxicity which slows growth. Of course, once you hit mid log and induce, you expect growth to slow down substantially, so there's really no difference post induction. However, I'd guess - as is consistent with my own experiments - that mixing a bunch of random colonies would allow the anti-expression selected individuals to outgrow their slower, protein-expressing counterparts, and take over the medium. One thing you can do is - as others have suggested - is restreak from your glycerol stocks, pick ~10 colonies, and in parallel test their expression on a 5-10mL scale. Save a bit from each prior to induction and propagate that in uninduced culture so that they'll grow as a starter stock while you check induction on a gel. Then, only play with the guys that induce. In general, we also see that letting the starter culture overgrow *too* much can be bad. For genes which express robustly, it's not a problem, but for our problem proteins, we never let the overnight starter go for more than 12 hours before inoculating growth cultures. Another thing to consider is just cloning the gene - as is (if the codon usage isn't a *serious* problem - i.e., requiring a rare codon tRNA or what have you) - into a vector which allows tighter expression control. If it's lag-phase toxicity that's selecting against expression and killing your protein growth, then a vector which shows less 'leakiness' in early growth might circumvent the problem altogether. Your choice of growth medium might help in this regard, too - though I've never checked directly. Also, try inducing overnight at 15C to room temp, rather than at 37. Who knows? Best of luck, Dave Shechner Quoting Artem Evdokimov > In my humble opinion starting with a mixed culture is a bad idea, because: > > Successful expressors have heightened metabolic load even in the absence of > inducing agents. Therefore, during cultivation their 'expression-less' > companions will progress more rapidly and undergo more cell divisions - > leading to an increased fraction of 'empty' cells in the resulting culture. > Furthermore, even after induction the 'expression-less' cells will continue > to grow, thus depriving the expression-competent ones from nutrients > necessary to build your protein of interest. > > There is a good deal of precedent regarding lack of expression from > previously expressing cells (specifically E. coli, even more specifically - > the DE3 cryptic lysogens) after glycerol stocks are used to start cultures > rather than single colonies. There is a recent article in Microbial Cell > Factories journal that analyzes this phenomenon - and the authors insist > that the cells do not lose the plasmid, but rather mutate the lambda > polymerase to become inefficient. This obviously does not happen to ALL the > cells in the glycerol stock - the proportion of mutant must be relatively > low, but repeated re-culturing brings them to the forefront so to speak, by > allowing the mutants to win over their competitors. > > Therefore, in practical terms - if you just transform a bunch of cells with > a pure plasmid - it's perfectly OK to use the entire transformation for > growth. On the other hand, aged glycerol stocks made of such transformations > (i.e. without purifying single colonies) may pose a problem later. > > Artem CCP4bb navigationCCP4bb <-- 2008 <-- February 2008 <-- 02 February 2008 |
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