I have a library with variants of the same gene, and after selection steps (antibiotic resistance) I have observed a large number of variants with deletions. I don't have these deletions in my naive library, they are found only in the selected clones. I use the SNO 301 strain (AMPD1, ampA1, ampC8, pyrB, recA, rpsL) for screenings. Anybody have any suggestions about this issue?
Just to clarify, is your strain recA, or ΔrecA? I don't believe recA deletion strains should recombine plasmids. From what you describe, it seems like you are probably selecting for deletion mutants in your selection. Any naive library will contain a certain level of indel mutants, but you may not be able to detect them in your initial library characterization if they are much less abundant than your point mutants (usually the case). The power and problem with selection experiments is that they can enrich for very rare variants, such as your deletion mutants.Without knowing more, it sounds like maybe that's what is happening. Alternatively, it might be that your other variants are all binding so well that your elution only releases the partial deletion mutants. Have you performed a mock selection with all appropriate controls with pre-defined pseudolibraries to ensure that your experimental procedure is indeed achieving what you think it's supposed to?
RecA is involved primarily in homologous recombination. Deletions are more likely to be caused by non-homologous processes that are independent of RecA. It sounds like your insert is intrinsically unstable in E. coli or alternatively, it, or the variants, are poisonous to the cells. If the latter is the case, you will notice big variations in colony size between whole and deleted inserts. It might help to clone in an expression vector and turn off the expression of the insert, or use a lambda vector that is going to kill the cell anyway. Another advantage of lambda is that it establishes lysogeny at single-copy, so you get less overexpression and less toxicity.
Good luck. Working with unstable inserts can get very complicated. In the worst case you may have to use a different host organism.
Prophages encode recombinases too. For exemple the defective prophage rac encode for RecT recombinase. Usually these prophage genes are shut down but sometimes the regulation is not active and these genes are expressed.
Furthermore, in bacteria these recombinases appear to recombine iverged sequences, and can make major deletions as presented here : http://www.ncbi.nlm.nih.gov/pubmed/?term=Vine%2C+CE%2C+MC+Justino%2C+LM+Saraiva%2C+J+Cole.+2010.+Detection+by+whole+genome+microarrays+of+a+spontaneous+126%E2%80%90gene+deletion+during+construction+of+a+ytfE+mutant%3A+confirmation+that+a+ytfE+mutation+results+in+loss+of+repair+of+iron%E2%80%90sulfur+centres+in+proteins+damaged+by+oxidative+or+nitrosative+stress.+J+Microbiol+Methods+81%3A77%E2%80%9079.
Are you strain prophage free?
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