(423f) Variants of the Quorum-Sensing Repressor Modulated Promoter, Pesar, Alter Amplitude, Signal Sensitivity and Switch-Like Behavior

Synthetic biology has advanced to but is also challenged by engineering microbial communities that exhibit coordinated, population-dependent behaviors. Studies of multicellular systems have been conducted incorporating acyl-homoserine lactone (AHL)-based quorum-sensing (QS) systems. To date, the most employed QS regulator has been a transcriptional activator, LuxR, but not limited. Expanding the use of QS components, we focus on a transcriptional repressor, EsaR from Pantoea stewartii, identified to repress its cognate DNA binding site, the esa box. In addition to protein engineering, promoter engineering also provides an alternative to fine-tune the regulatory systems. We have observed that placing the wild-type Pesa promoter in a low copy-number plasmid, repression was detected in the absence of AHL. However, when Pesa promoter is harbored by a high copy-number plasmid, it shows no detectable repression by EsaR. To address this issue, we developed a set of mutant promoters that include additional copies of the esa box. We hypothesized that providing two esa boxes in one promoter could increase EsaR affinity for the promoter and lead to switch-like behavior in high-copy plasmids. We will show that altering the position of a second esa box within Pesa affects promoter behavior in a manner that depends on its position. In addition to identifying a novel promoter that shows AHL-inducible gene expression in our high copy plasmid, our new promoters show a range of AHL-dependencies and amplitudes. These promoters, in combination with our previously evolved EsaR variants with varying signal sensitivities, represent a new set of tools for enabling cell-cell communication.