Construction of a Synthetic Promoter Library Employing Well-Studied Regulatory Components

Even though the evolution of synthetic biology over the last decade has been signicant, the number of molecular parts with well-defined functions still remains small.The need for developing libraries of well-characterized molecular components has been discussed before [1]. Libraries of well-defined molecular elements will allow to program biological function easier and in a more predictable manner. To this end, we develop a library of synthetic inducible promoters employing regulatory elements of the well-studied tetracycline, lactose and lambda-phage operons. We demonstrate that the dierential positioning of operator sites yields a range of expression and repression proles.We tested the promoters strength and leakiness using flow cytometry and monitoring the output signal for a wide range of inputs. We next explored the modularity of the designed promoters. Lastly, we introduced point mutations to two of the synthetic promoters and analyzed the role of individual nucleotides on the overall promoter behavior. In conjunction with the experiments, we developed mathematical models that capture the behavior of the synthetic promoters. This study provides a dozen varying strength synthetic promoters that can be readily utilized as stand-alone molecular systems or integrated into higher level synthetic devices.

References

1) T. Ellis, X. Wang, and J.J. Collins. Diversity-based, model-guided construction of synthetic gene  networks with predicted functions. Nature biotechnology, 27(5):465-471, 2009.