Integrant chromosome nonstarch hydrolyzing integrant

Figure 1. Double-crossover recombination shows the integration of a fragment of DNA from a linearized vector that is unable to replicate in Bacillus species. (a) Recombination event involving sequences homologous to the target DNA. (b) Recombination event involving heterologous DNA, in this case inserted between 5' (amyEA) and 3' (AamyE) fragments of the nonessential amyEgene encoding a-amylase. Integrants lose the ability to hydrolyze starch.

fragility of the wall-less protoplasts means that transformation needs to be carried out in an isosmotic medium; the cell walls are subsequently regenerated on complex regeneration media. Protoplast transformation was originally developed for B. subtilis by Chang and Cohen (30) but has subsequently been modified in many laboratories. A major advantage of protoplast transformation is that plasmid DNA remains double stranded throughout the procedure and consequently is less likely to undergo rearrangements. Despite its advantages, protoplast transformation methods are laborious and difficult to reproduce; transformation frequencies up to 10% per protoplast are claimed but in practice are usually a great deal lower. In addition, the complexity of the regeneration media precludes the selection for prototrophic markers. Protoplast transformation protocols have been developed for several other Bacillus species including B. amyloliquefaciens, B. licheniformis, B. stearothermophilus, B. anthracis, and B. firmus.

Genome Analysis and Manipulation

The genome of B. subtilis strain 168 is 4.2 Mbp in length, has a G + C content of 43.5%, and encodes slightly more

E. coli origin of replication

Resistance gene selectable in E. coli

E. coli origin of replication

Resistance gene selectable in E. coli

Controllable promoter

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