Acinetobacter baylyi ADP1 Genome Manipulations - Note: Protocol Construction In Progress

Overview

Genome manipulations in Acinetobacter baylyi ADP1 (AB) 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. The listed protocols use the tdk-kan selection/counter selection cassette as retrieved from the AB single gene knockout project, but other selection/counter-selection cassettes (such as kan-sacB) may also be used. Our general strategy is to first construct our transforming constructs with targeted homology to the genome using a 3 piece overlap PCR and then (for all procedures minus simple knock outs) rescuing the insertion of that cassette into the genome with the desired construct using the tdk counter-selection.

Overlap PCR Construct Generation

The following is a standard procedure designing and constructing AB genome manipulation constructs using overlap PCR.

The Design of Overlap PCR Primers and Constructs

In order to ensure a successful assembly of transforming constructs, particular care should be made when designing the appropriate primers.

Notes/Considerations in AB transformations

• to avoid the use wet plates or plates with abberant surface texture. Wet plates will haze individual colonies and abberant surface texture produces irregular morphologies.
• Linearized plasmid will not recircularize in an AB transformation unless two separate digestions with different cut sites on the plasmid are used. It is best to just transform whole plasmid.

-- Main.BrianRenda - 13 Jun 2013