Precise Control of Lycopene Production to Enable a Fast-Responding, Minimal-Equipment Biosensor
Synthetic Biology Engineering Evolution Design SEED
2016
2016 Synthetic Biology: Engineering, Evolution & Design (SEED)
Poster Session
Accepted Posters
Zinc deficiency contributes to hundreds of thousands of childhood deaths annually, but current detection methods are cost-prohibitive for use in the regions most affected. Our group recently developed a low-cost bacterial biosensor that can produce different visible outputs based on the concentration of zinc in which the cells grow. However, these initial sensor cells always produce some pigment, which necessitates that they be grown from a very small inoculum so that initial coloration is minimal – thus requiring overnight assay times. To make an easy-to-use, field-deployable assay, we are engineering cells that completely repress pigmentation during growth and quickly produce the red pigment lycopene upon induction. This would allow sample addition to concentrated pre-cultured cells, decreasing assay time from about 24 hours (the time required for growth) to 3 hours (for visible pigment production without growth).
A sensor that produces a pigment instead of fluorescent proteins is advantageous in resource-limited settings, since sensor readout can be interpreted without advanced equipment. However, complete repression of pigment production requires more precise control than what is necessary for most synthetic biology applications, since small amounts of enzyme can produce visible amounts of pigment. Inducing high levels of pigmentation poses an additional challenge, as overexpression of crtEBI, the genes that produce lycopene, can cause cell toxicity and prevent accumulation of lycopene. Since maximum pigment production does not correspond with maximum protein production, precise control of the induced expression level is also necessary to express the appropriate amount of pigment-producing proteins.
Three commonly used inducible promoter systems—the lac, ara, and T7 systems—were tested for precise control of lycopene production. The arabinose-inducible promoter pBAD proved most repressible but still could not fully repress lycopene production during uninduced growth, and in all systems lycopene production decreased upon induction because of crtEBI-related toxicity issues. Modification of polymerase binding sites in the promoters significantly decreased induced expression level (enabling overexpression of lycopene upon induction), but only slightly decreased the repressed level (which was insufficient to repress lycopene production during uninduced growth). Translational modifications proved much more effective in controlling lycopene production. When the original ribosomal binding sites for the crtE, crtB, and crtI genes were replaced with weak RBSs, the lac and ara regulatory systems completely repressed pigmentation during uninduced growth and produced visible amounts of lycopene at full induction. Upon induction, the lac and ara systems produced visible pigment in about 3 and 6 hours, respectively – a large improvement over the 18-24 hours required in the original assay. Zinc-responsive elements and other pigments can be added to these systems to create circuits that produce different pigments in response to different zinc concentrations.
As the applications of synthetic biology expand to systems that require precise control of metabolic state, transcriptional control alone will likely be insufficient. Even the most repressible promoters could not fully repress lycopene production, suggesting that post-transcriptional modifications may be a better starting point in combatting unwanted protein expression.