Expanding the quorum sensing toolbox: Promoter libraries and hybrid promoter for dynamic genetic circuits

The interesting features of quorum sensing systems make them very appealing for synthetic biology applications. The first steps have already been taken in characterizing these systems to accelerate their successful implementation. In this work, we explore the next step, tuning. Multiple strategies exist for tuning genetic circuits and quorum sensing systems in general, where the most straightforward solution is to vary the expression level of the synthase or transcription factor. However, these tuning possibilities can be broadened by expanding the variety of quorum sensing-regulated promoters. Here, we expand the range of tuning possibilities by constructing promoter libraries for P_lasI (LasI/LasR system) and P_esaR/PesaS (EsaI/EsaR system), enabling direct modulation of downstream gene expression levels. Next, the quorum sensing synthetic biology toolbox was further expanded with the creation of a new-to-nature genetic part, the hybrid promoter, which requires two autoinducers for activation, namely 3-oxo-hexanoyl- and 3-oxo-dodecanoyl-homoserine lactone. This promoter was optimized and applied to design a multistage genetic switch capable of creating a growth stage followed by two sequential production stages. Additionally, the circuit was optimized by incorporating the new promoter variants created in this study. This complex genetic circuit demonstrates the true potential of these quorum sensing systems for achieving various cell-density related dynamic circuits.