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Schlagwörter:
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Zusammenfassung:
Recent developments in DNA-assembly methods make the synthesis of
synthetic chromosomes a reachable goal. However, the redesign of primary
chromosomes bears high risks and still requires enormous resources. An
alternative approach is the addition of synthetic chromosomes to the
cell. The natural secondary chromosome of Vibrio cholerae could
potentially serve as template for a synthetic secondary chromosome in
Escherichia coli. To test this assumption we constructed a replicon
named synVicII based on the replication module of V. cholerae chromosome
II (oriII). A new assay for the assessment of replicon stability was
developed based on flow-cytometric analysis of unstable GFP variants.
Application of this assay to cells carrying synVicII revealed an
improved stability compared to a secondary replicon based on E. coli
oriC. Cell cycle analysis and determination of cellular copy numbers of
synVicII indicate that replication timing of the synthetic replicon in
E. coli is comparable to the natural chromosome II (ChrII) in V.
cholerae. The presented synthetic biology work provides the basis to use
secondary chromosomes in E. coli to answer basic research questions as
well as for several biotechnological applications.
