Nucleic Acids Research, Vol 27, Issue 4 1056-1062, Copyright © 1999 by Oxford University Press
A Melnikov and PJ Youngman
We describe a general method for random mutagenesis of cloned genes by
error-prone PCR or DNA shuffling that eliminates the need for post-
amplification subcloning following each cycle of mutagenesis. This method
exploits the highly efficient and recombinogenic nature of DNA uptake
during natural transformation in the Gram-positive bacterium Bacillus
subtilis and the Gram-negative bacterium Acinetobacter calcoaceticus.
Plasmid systems were designed that allow capture of PCR- amplified DNA
fragments by marker-replacement recombination with a structurally similar
helper plasmid resident in the transformation recipient. This recombination
event simultaneously transfers the amplified sequences into the helper
plasmid and restores the integrity of a drug resistance gene, thereby
affording a direct selection for fragment capture. Although this strategy
was sufficiently effective to permit recovery in B. subtilis of up to 10(3)
transformants/microgram of PCR product, equivalent plasmid systems were
approximately 100 times more efficient in A.calcoaceticus. Acinetobacter
calcoaceticus also offers the advantage of essentially constitutive
transformation competence in ordinary complex broth, such as LB, in
contrast to two- step growth in semi-synthetic media required for optimal
transformation of B.subtilis.
ARTICLES
Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus
Department of Genetics, University of Georgia, Athens, GA 30602, USA.
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