Design of a transcriptional repressor-based biosensor in E. coli

Open Access
Author:
Frei, Christopher Scott
Graduate Program:
Chemical Engineering
Degree:
Master of Engineering
Document Type:
Master Thesis
Date of Defense:
September 23, 2010
Committee Members:
  • Patrick Cirino, Thesis Advisor
  • Patrick C Cirino, Thesis Advisor
Keywords:
  • polyketide
  • ActR
  • transcriptional repressor
  • Biosensor
Abstract:
The development of in vivo high-throughput screening techniques for the detection of polyketides is essential to alleviate our dependence of expensive synthetic and time consuming methods for the production these industrial bioactive molecules. Nature provides a vast array of polyketides in a myriad of organisms, produced from highly malleable biosynthetic pathways. Protein transcriptional regulators offer a gateway to mitigate the current processes of polyketide extraction and/or chemical synthesis. By naturally having an affinity for molecules such as polyketides and with the genetic tools available today, transcriptional regulators can be evolved to detect other industrially significant ligands and novel bioactive compounds through directed evolution. This will allow for the design of inexpensive high yield processes when expressed in a genetically manipulative host such as Escherchia coli. In this study, we describe the design of transcriptional repressor-based biosensor for the in vivo detection of polyketides in E. coli. The biosensor was engineered with ActR, a TetR-like transcriptional repressor protein, controlling expression of the gfp reporter gene which produces a green fluorescent protein (GFP). The incorporation of a GFP reporter allows for high-throughput screening of ActR and polyketide biosynthetic pathway combinatorial libraries using fluorescence-activated cell sorting (FACS). The ActR mutants will be screened based on their affinity and selectivity for a specific product produced through an engineered polyketide biosynthetic pathway, which the pathway can then be screened for increased yields. The broad range of genetic techniques available for manipulation of E. coli combined with the ActR-based biosensor engineered in E. coli, a new window of opportunity has been presented for engineering more efficient microbial production of polyketide compounds.