Differentiating frequency of recombination from frequency of recombinants

In recombineering, the frequency of recombinant cells is often used as a measure of the frequency of recombination events. We used the “founder effect” (Lezin G. et al., in preparation) and PCR screening to study the origin and propagation of mini‐lambda‐mediated recombinants in liquid cultures. Specifically, we deleted a Neo marker from an mStrawberry cassette‐containing BAC, and then replaced mStrawberry with eYFP, ZsGreen or turboGFP. Upon deletion of Neo, some recombinants were PCR positive but Neo resistant, suggesting the formation of multiple targets during BAC recombinereing. Additionally, after replacement of mStrawberry with eYFP, there was evidence of partial or complete eYFP duplication at the first two enrichment cycles. However, at the third cycle, only one eYFP copy was detected. Both aberrations are similar to previously reported data (Poteete AR at al., BMC Mol Biol 2004). Upon replacement of Neo‐less mStrawberry or eYFP with ZsGreen or turboGFP, we observed similar frequency of recombination to the first eYFP enrichment cycle (estimated via the fraction of negative wells, Luria & Delbruck, Genetics 1943). However, we were unable to isolate ZsGreen and turboGFP recombinants because PCR products became progressively smeared at subsequent enrichment cycles, suggesting delayed DNA structure rearrangement. These data show that lower frequency of recombinant cells does not necessarily imply decreased frequency of recombination, and these two variables need to be independently assessed.