Acinetobacter baylyi ADP1 Genome Manipulations

Genome manipulations in Acinetobacter baylyi ADP1 can be performed without the need for exogenous recombinase expression, plasmid electroporation, or other practices used in other canonical systems such as E. coli. With just the transformation of PCR products, one can make gene knockouts, insertions, allelic replacement, and deletions. Novel DNA sequences are inserted into the ADP1 genome by recombination between flanking DNA added through ligation or overlap extension PCR and a targeted homologous region of the genome. The listed protocols use the tdk-kan selection/counter selection cassette as retrieved from the ADP1 single gene knockout project, but other selection/counter-selection cassettes (such as kan-sacB) may also be used. The Barrick Lab primarily uses the Golden Transformation method linked below for constructing modified ADP1 strains which is fast and nearly scarless.

Protocols for ADP1 genome manipulations include:

• Golden Transformation: utilizing the Golden Gate Cloning methodology to join products by restriction digested ends
  
• Overlap Extension PCR Transformation: utilizing PCR products with annealing ends joined by an additional round of PCR
  

DNA for genome modifications can be transformed into ADP1 using either overnight culture transformations or "in plate" or "puddle" transformations.