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Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon
Published on: November 16, 2012
Engineering the ribosomal DNA in a megabase synthetic chromosome
Weimin Zhang1,2, Guanghou Zhao1,3, Zhouqing Luo1
1Key Laboratory for Industrial Biocatalysis (Ministry of Education) and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
None:
We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.
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