Abstract
A novel approach to the cloning large DNAs in theBacillus subtilis chromosome was examined. AnEscherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome ofB. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In theB. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kbB. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in theB. subtilis chromosome and possible applications of this technique are discussed.
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Itaya, M. Toward a bacterial genome technology: integration of theEscherichia coli prophage lambda genome into theBacillus subtilis 168 chromosome. Molec. Gen. Genet. 248, 9–16 (1995). https://doi.org/10.1007/BF02456608
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DOI: https://doi.org/10.1007/BF02456608