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Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae
Author(s) -
Messerschmidt Sonja J.,
Kemter Franziska S.,
Schindler Daniel,
Waldminghaus Torsten
Publication year - 2015
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201400031
Subject(s) - replicon , vibrio cholerae , biology , chromosome , escherichia coli , synthetic biology , genetics , bacterial artificial chromosome , dna replication , dna , computational biology , gene , plasmid , genome , bacteria
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.