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 -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Andreas Förster Sent: Saturday, February 02, 2008 11:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Codon Optimized Expression There has been a somewhat related discussion in the lab the other day. If some colonies might express well and other not so well, why don't I just scoop up loads and start my culture from them? This way I'll be more certain to get the clones that express. The clones that don't express will dilute the yield, but I doubt they'd outcompete the rest, would they? For me, the start-with-plenty procedure has always worked well, but some insist on starting from single colonies. Andreas James Stroud wrote: > On Feb 2, 2008, at 2:15 AM, M T wrote: >> One classical way to optimize expression level is to screen culture >> conditions. >> >> For my proteins, I solved my expression problems by changing the >> expression vector to a pET or changing a pET 20 to a pET 30 (if the >> protein is toxic). >> >> But keep in mind that a low but folded expression is better than a >> high expression to inclusion body. > > Anecdotal evidence also suggests to try picking several colonies from > the original cloning procedure and doing test expressions with each. > Some clones express better than others even though they should in > principle express identically. I have no idea what the mechanism for > this differential expression could be. > > -- > James Stroud > UCLA-DOE Institute for Genomics and Proteomics > Box 951570 > Los Angeles, CA 90095 > > http://www.jamesstroud.com >
