Antibiotic Markers: An Overlooked Design Choice in Synthetic Biology?

Date

2021

Authors

Young, Eleanor

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Abstract

Synthetic biology seeks to engineer living systems to enhance human health, improve material synthesis and degradation, and simplify diagnostics. When researchers design synthetic constructs, they rely on predictable host organisms and predictable genetic parts to engineer them. Antibiotics and antibiotic resistance genes are essential to synthetic designs, but different antibiotics kill cells or inhibit their growth in different ways, and antibiotic resistance genes provide resistance through different mechanisms. In general, we do not know if and how these parts impose additional fitness costs or interfere with desired genetic functions in engineered cells. To test how antibiotic choice affected synthetic devices, I examined seven near-identical plasmids. Each plasmid expressed a fluorescent protein and included a different antibiotic resistance gene. When the strains were cultured with different antibiotic concentrations, some saw a decrease in final cellular density and fluorescence while others maintained normal performance at up to ten times the selection concentration of the antibiotic. When evolved for three weeks under normal selection conditions all strains lost RCP expression by the end of the time period as populations evolved to eliminate burden. The majority of strains began to lose expression around 11 or 12 days, some lost expression as early as day 5 or as late as day 16. Each population was sequenced to reveal that the strains had accrued mutations and adjusted their copy number in different manners over the course of the experiment. Four of the strains had accumulated mutations in their RCP gene and one had mutations in the antibiotic resistant gene, some had developed possible compensatory mutations in the genome, and one of the strains with a more stable expression profile over the course of the experiment had high IS element activity and a relatively high number of mutations possibly indicating activation of the SOS response. Overall, ampicillin (beta lactamase resistance gene) was the most stable in expression while tetracycline (efflux pump resistance gene) was the least stable. These experiments show that antibiotic resistance genes, a basic design choice in synthetic biology, alter the reliability of genetic constructs.

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