Control of Gene Expression through Control of Plasmid Copy Number

As mobile genetic elements capable of self-replication, plasmids are perhaps the most versatile and widely used tool in synthetic biology. Though we have a clear picture of the elegantly evolved mechanisms governing the control of plasmid replication our understanding of the effects of plasmid copy number on gene expression and host physiology is vague. In this work we demonstrate robust control of plasmid copy number in ColE1 plasmids in several manners – using inducible promoters and massively parallelized assays to investigate the parameters governing plasmid copy number. We leverage this control to better understand the effect of plasmid copy number on host cell processes. The effects of copy number on gene expression and their entailed effects on host cell growth are investigated as are the effect of gene copy number on the efficiency of CRISPRi-based repression. We further use our control of plasmid copy number to investigate the dual perils that necessitate control of plasmid copy number in the first place - plasmid loss and metabolic burden, casting light onto the limits of plasmid replication.