Here we have a problem with a multi-drug resistance bacteria (Enterobacter cloacae) in gene deletion by using pKD46. However, this strain is a clinical isolation, resistant for most antibiotics. We modified the pKD46 with a Kanamycin resistance gene, and successfully obtained the positive transconjugant.  In this situation, we are very difficult to find another marker for second round selection(homologous recombination). So we decide to reuse kanamycin for second round selection, however, it is very hard to eliminate the resistance from pKD46 by culture in high temperature(42 C). 

So my question is, is it possible to select recombinant by using single marker in red-lambda recombination system.

So, the most important question  is : Will the high temperature (42 C) kill the pKD46 at efficiency of 100%?

More Liang Huang's questions See All
Similar questions and discussions