J G Wallace1, R R Breaker. 1. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA.
Abstract
AIMS: Bacillus halodurans C-125 is a Gram-positive bacterium that was the first alkaliphilic species to have its genome completely sequenced. Despite its many years as a model for alkaliphily and source of industrially important enzymes, genetic manipulation of B. halodurans C-125 remains difficult, and therefore, we sought to develop a robust method to allow routine transformation of this organism. METHODS AND RESULTS: A plasmid artificial modification system (PAM system, Yasui et al. 2008) for B. halodurans C-125 was created that increases transformation efficiency by 10- to 1000-fold. Also, recovering transformed protoplasts on succinate nutrient agar (SNA) yields faster, more robust colony recovery than on the traditional recovery medium. Combining these two techniques often allows recovery of transformants in as little as 48 h. CONCLUSIONS: Use of the B. halodurans C-125 PAM system and SNA greatly improves the efficiency and speed of protoplast transformation of B. halodurans C-125. SIGNIFICANCE AND IMPACT OF THE STUDY: These techniques allow routine genetic manipulation of B. halodurans C-125, a model alkaliphilic bacterium with important industrial properties.
AIMS: Bacillus halodurans C-125 is a Gram-positive bacterium that was the first alkaliphilic species to have its genome completely sequenced. Despite its many years as a model for alkaliphily and source of industrially important enzymes, genetic manipulation of B. halodurans C-125 remains difficult, and therefore, we sought to develop a robust method to allow routine transformation of this organism. METHODS AND RESULTS: A plasmid artificial modification system (PAM system, Yasui et al. 2008) for B. halodurans C-125 was created that increases transformation efficiency by 10- to 1000-fold. Also, recovering transformed protoplasts on succinate nutrient agar (SNA) yields faster, more robust colony recovery than on the traditional recovery medium. Combining these two techniques often allows recovery of transformants in as little as 48 h. CONCLUSIONS: Use of the B. halodurans C-125 PAM system and SNA greatly improves the efficiency and speed of protoplast transformation of B. halodurans C-125. SIGNIFICANCE AND IMPACT OF THE STUDY: These techniques allow routine genetic manipulation of B. halodurans C-125, a model alkaliphilic bacterium with important industrial properties.
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