(125c) Novel, Synthetic, Tetracycline-Inducible Transcription Regulators for E. Coli

摘要

We are designing, constructing and characterizing novel synthetic gene regulators. These regulators range from individual inducible fusion protein and promoter pairs to complex nonlinear regulatory networks. All of the regulators are designed to be compatible in E. coli and to control gene expression by direct transcription activation. Our investigations have complementary experimental and computer modeling components which allow us to build and characterize these complex and robust synthetic systems while reducing the required expense and effort to construct future networks. Our initial focus has been on designing, constructing and characterizing two-part synthetic regulatory systems. They are composed of an inducible fusion regulatory protein and a complementary fusion promoter. The fusion proteins are novel combinations of the inducible DNA binding tetracycline represser (TetR) or its derivative reverse-TetR (rTetR), and the transcription activation domain LuxRAN(2-162) (LuxRAN). The fusion promoter contains the complementary operator sequences, tetO and luxbox, for TetR/rTetR and LuxRAN respectively. We are experimentally characterizing the systems' gene expression regulation using real-time PCR, immunoblotting, culture fluorescence and flow cytometry while making small adjustments for optimization. In parallel, we are building and testing stochastic models with the aim of accurately capturing the systems' behavior, independent of context. Real-time PCR results indicate that the rTetR-LuxRAN facilitates significant transcription upregulation upon induction by anhydrous tetracycline (aTc). We also observe a parallel increase in protein synthesis, monitoring mean culture fluorescence and individual cell analysis by flow cytometry. After induction with aTc, the transcription activator upregulates target gene expression, initiating the emergence of a cell population with GFP expression 2 orders of magnitude above the uninduced population.