Whole-Genome Sequencing of Syn3A-mCherry to Determine the Ori:Ter Ratio

We perform whole-genome sequencing of JCVI-syn3A (Syn3A-mCherry) in triplicate in order to measure the ori:ter ratio during exponential and stationary growth phases. Syn3A-mCherry cultures were grown in SP4 media, and genomic DNA was extracted from cell pellets collected at 5 hours (mid-exponential phase), 9 hours (late-exponential phase) and 21 hours (stationary phase). The shotgun genomic libraries were prepared using the Illumina DNA Prep kit and sequenced on an Illumina MiSeq platform with 251 bp paired-end reads. The measured ori:ter ratio of 1.21 in both the mid-exponential and late-exponential phase indicates that Syn3A undergoes roughly one replication initiation event per cell cycle.