i am in the middle of a very peculiar problem. I am the final stage of my experiment, which involve gene inactivation in pseudomonas. The gene will inactivated by incorporate an antibiotic cassette in the middle of the gene, hence hopefully will inactivate the gene. Due to problem that cannot be avoided, i only manage to have ~200bp of homology sequence as the downstream, but i able to have 900bp homology sequence as my upstream. Everything was going well, i manage to conjugate s17 harbouring my construct (pDM4 + insert+ antibiotic cassette) with the recipient, pseudomonas. selection was done one mineral salt medium with antbiotic. E.coli will die, as they are auxotrophic (cannot produce thiamine and proline) while, wild-type will die due to the antiobiotic. Control plates was done and give the right result and observations. Subsequently, i pick randomly 10 colonies from selective plate and grow in broth without any antibiotic, to promote second crossing over. Next, i did dilution and spread on 15% sucrose + antibiotic. I got few colonies, so i patch each of them on plate with chloramphenicol (merodiploid are resistant while mutants or wild type are sensitve), i got no growth even after 2 days. Finally i did colony pcr on them, and got 2 bands, with one of the band represent the size of a wild-type. i conclude that i might have isolated merodiploid strains in stead of mutants. i have done this whole process 2 times, with modification in incubation temperature and time, to promote homologous recombination.
So my question is, if the one i have now is merodiploid, will that strain can revert to mutant strains? because if i am not mistaken, merodiploid is unstable.
I am lost right now, any ideas or suggestions will be highly appreciated!