DNA Replication in Engineered Escherichia coli Genomes with Extra Replication Origins

Abstract

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.

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics

“…The integration of a plasmid-derived origin that could be induced with IPTG at a location ∼450 kb away from oriC was successful, but if this origin was active, it repressed activity of oriC (Kouzminova and Kuzminov, 2008). In another study, integration of a shorter oriC fragment in two chromosomal locations, one roughly equivalent to the oriZ position while the second was closer to the termination area (1.6 Mbp), did not result in any detectable initiation at the ectopic origins (Milbredt et al, 2016). The authors suggested that origin activity might be influenced by the presence of flanking genes (Milbredt et al, 2016), which would explain why the longer 5 kb oriC region stretch developed in the Sherratt lab (Wang et al, 2011) proved active.…”

“…In another study, integration of a shorter oriC fragment in two chromosomal locations, one roughly equivalent to the oriZ position while the second was closer to the termination area (1.6 Mbp), did not result in any detectable initiation at the ectopic origins (Milbredt et al, 2016). The authors suggested that origin activity might be influenced by the presence of flanking genes (Milbredt et al, 2016), which would explain why the longer 5 kb oriC region stretch developed in the Sherratt lab (Wang et al, 2011) proved active. However, our attempts to integrate the same 5 kb oriC fragment into the malT gene at 76.5 min, approximately 1/4 into the left-hand replichore, to generate oriC + oriY + cells ( Figure 2C), proved unsuccessful.…”

Optimization and Characterization of the Synthetic Secondary Chromosome synVicII in Escherichia coli

“…Spreading the genetic information to multiple replicons might actually have considerable benefits (Liang et al, 2013; Schindler and Waldminghaus, 2015; Milbredt et al, 2016). Since using the replication origin of the V. cholerae secondary chromosome as basis for a synthetic secondary chromosome in E. coli has proven a suitable approach, we set out to probe the origin diversity of the Vibrio genus for its potential as third chromosome.…”

“…One interesting alternative is the replication origin of the secondary chromosome of Vibrio cholerae . This origin has been shown to replicate in Escherichia coli and was used in several respective genome engineering projects (Egan and Waldor, 2003; Liang et al, 2013; Messerschmidt et al, 2015; Milbredt et al, 2016; Zhou et al, 2016). …”

BiFCROS: A Low-Background Fluorescence Repressor Operator System for Labeling of Genomic Loci

“…Measuring the whole-cell fluorescence can be performed precisely and in high throughput by flow cytometry on a single-cell level. This would be a great advance compared to conventional copy number measurements, such as quantitative PCR or marker frequency analysis by microarrays or next-generation sequencing (Milbredt et al 2016; Skovgaard et al 2011; Stokke et al 2011). In our experiments, fluorescence intensities of cells with two copies of the OL1/UAS hybrid FROS array were higher than cells with only a single copy (Figure 6).…”