I’m currently working with Acinetobacter baumannii ATCC 19606 and have been running into issues with transformation. Our aim is to integrate a disrupted gene into the genome through recombination.

• We have tried electroporation using the plasmid PCR-BLUNT II-TOPO carrying the disrupted gene. We tried different electroporation programs.
• We also attempted conjugation using E. coli S17 λpir as the donor strain, with the same plasmid modified to include an RSF1010 OriT sequence (inserted via restriction enzymes).
• So far, neither approach has been successful.

I would greatly appreciate advice from anyone who has worked with this strain:

• Have you found a reliable protocol for transformation (electroporation or conjugation) in ATCC 19606?
• Are there specific plasmids, selection markers, or growth conditions that improve transformation efficiency in this strain?
• Any troubleshooting tips you’ve found useful when working with A. baumannii?

Thank you in advance for any suggestions or references you can share!