Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering
Local: Acinetobacter_ADP1.pdf

Abstract:
> Acinetobacter sp. strain ADP1 is a naturally transformable
> gram-negative bacterium with simple culture requirements, a
> prototrophic metabolism and a compact genome of 3.7 Mb which has
> recently been sequenced. Wild-type ADP1 can be genetically
> manipulated by the direct addition of linear DNA constructs to
> log-phase cultures. This makes it an ideal organism for the
> automation of complex strain construction. Here, we demonstrate the
> flexibility and versatility of ADP1 as a genetic model through the
> construction of a broad variety of mutants. These include marked and
> unmarked insertions and deletions, complementary replacements,
> chromosomal expression tags and complex combinations thereof. In the
> process of these constructions, we demonstrate that ADP1 can
> effectively express a wide variety of foreign genes including
> antibiotic resistance cassettes, essential metabolic genes,
> negatively selectable catabolic genes and even intact operons from
> highly divergent bacteria. All of the described mutations were
> achieved by the same process of splicing PCR, direct transformation
> of growing cultures and plating on selective media. The simplicity of
> these tools make genetic analysis and engineering with Acinetobacter
> ADP1 accessible to laboratories with minimal microbial genetics
> expertise and very little equipment. They are also compatible with
> complete automation of genetic analysis and engineering protocols.



Re: Alternatives to Gel Electrophoresis
From: "Mackenzie Cowell" <macowell@gmail.com>
To: diybio@googlegroups.com
CC: "Tom Knight" <tk@csail.mit.edu>
Date: 2008-10-21 05:41:46 pm
WOW! Acinetobacter ADP1 sounds amazing!

Quotes:

1. ADP1 is naturally competent: "...these properties allow genetic manipulation by simple addition of linear PCR products to small volumes of growing cell culture, followed by a few hours of incubation and plating on appropriate selective media"

2. ADP1 is similar to E. coli: "...the close relationship between E.coli and ADP1, combined with the newly available whole-genome sequence of ADP1, allows the tremendous amount of existing knowledge related to gene function and metabolism of E.coli to be applied directly to ADP1


How would biobrick alpha parts work with ADP1?:

1. Would one just linearize a biobrick part + plasmid and hope it self-ligated inside ADP1 after uptake?  Do circular vectors even exist in Acinetobacter?

2. Or would we be trying to integrate the biobrick part into the ADP1 genome with homologous recombination?  If so, what might a modified biobrick transformation strategy look like for ADP1

3. Lastly, do you think we would need to do any codon optimization when transferring E. coli biobricks (or any other construct) into ADP1?


Thanks a bunch TK!

Money quote:
In this work, splicing PCR (12) was used to create recombinant constructs for transformation of Acinetobacter ADP1 (13), and these constructs were used to generate a wide variety of mutants, including marked and unmarked gene deletions, chromosomal protein tags for purification, replacements of wild-type alleles with mutant alleles, interspecific gene complementations, recursive serial deletions, whole operon insertions and nonpolar deletions. These manipulations required only a PCR machine, shaking and stationary incubators, and oligonucleotide synthesis. The efficiency and broad applicability of this simple technique suggest that ADP1 is an ideal organism for genetic analysis and engineering. The tools needed to generate specific mutant strains of ADP1 are available to virtually any researcher, student or industrial worker in need of such a system. All manipulations in this work were performed in very small culture volumes with raw PCR products, straightforward selections and simple post-manipulation analytical methods, in order to demonstrate the possibility of automating these processes with existing robotic technologies.