Hello everyone!

I am currently working on the genomic integration in E. coli MG1655.

I selected two attB sites to work with - attB lambda and attB 186. I have successfully integrated my gene via a plasmid carrying the attB lambda site and flipped the integration module using pE-FLP. However, in the case of attB 186, I have been successful in integrating the plasmid at primary 186 attB but unable to flip the integration module with Kanamycin resistance.

Initially, I allowed the 186 attB integrants to recover at 30 °C for 1 hour after pE-FLP transformation, plated 200 µl cells at 50 µg/ml LA-Ampicillin plates, and incubated them at 30 °C overnight. I picked a few colonies and streaked them at 15 µg/ml LA-Kanamycin and LA plates and incubated them at 30 °C overnight. This protocol is working fine for me with attB lambda integrants, but it failed with the attB 186 integrants, which already have integration at lambda attB.

I picked a few attB 186 integrants and repeated the flipping protocol but failed. Then, I increased the recovery time after the transformation of pE-FLP to 2.5 hours at 30 °C and plated 200 µl cells at both 50 and 100 µg/ml LA-Ampicillin plates. I also tried incubating the streaked colonies on LA-Kan and LA plates picked from pE-FLP transformed plates at 42 °C for 2 h and 37 °C overnight. I have been unable to flip the integration module as of now.

1 FRT scar remains at the attB lambda site due to flipping of the integrated plasmid. Does this cause problems with flipping at the other attB site?

Since flippase is under the control of a constitutive promoter in pE-FLP, I do not understand the logic of incubating the plates or allowing recovery at 42 °C.

Are there any other protocols available that can be used? Any help will be appreciated. Thank you!