DNA Replication in Engineered Escherichia coli Genomes with Extra Replication Origins.
The standard outline of bacterial genomes is a single circular chromosome with a single replication origin. From the bioengineering perspective, it appears attractive to extend this basic setup. Bacteria with split chromosomes or multiple replication origins have been successfully constructed in the last few years. The characteristics of these engineered strains will largely depend on the respective DNA replication patterns. However, the DNA replication has not been investigated systematically in engineered bacteria with multiple origins or split replicons. Here we fill this gap by studying a set of strains consisting of (i) E. coli strains with an extra copy of the native replication origin (oriC), (ii) E. coli strains with an extra copy of the replication origin from the secondary chromosome of Vibrio cholerae (oriII), and (iii) a strain in which the E. coli chromosome is split into two linear replicons. A combination of flow cytometry, microarray-based comparative genomic hybridization (CGH), and modeling revealed silencing of extra oriC copies and differential timing of ectopic oriII copies compared to the native oriC. The results were used to derive construction rules for future multiorigin and multireplicon projects.
LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-University , Marburg, Hans-Meerwein-Strasse 6, D-35043 Marburg, Germany.
Sarah
S
Milbredt
LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-University , Marburg, Hans-Meerwein-Strasse 6, D-35043 Marburg, Germany.
Neda
N
Farmani
LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-University , Marburg, Hans-Meerwein-Strasse 6, D-35043 Marburg, Germany.
Patrick
P
Sobetzko
LOEWE Center for Synthetic Microbiology, SYNMIKRO, Philipps-University , Marburg, Hans-Meerwein-Strasse 6, D-35043 Marburg, Germany.
Torsten
T
Waldminghaus
D002876:Chromosomes, Bacterial / Q000235:genetics; D055028:Comparative Genomic Hybridization; D056915:DNA Copy Number Variations; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D023281:Genomics; D000074041:Microorganisms, Genetically-Modified; D051738:Origin Recognition Complex / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics*
D004269:DNA, Bacterial; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex
bacteria; genetic engineering; genome design; synthetic chromosomes; synthetic genomes