The goal of this study was to improve upon the widely used T7 expression system in E. coli by significantly reducing basal levels of gene expression. Two plasmids, pZE21_A12C_T7RNAPol and pZA21_T7sfGFP are the products of this effort. The former plasmid incorporates a cis-repressing RNA element into the 5’ UTR of the gene for T7 RNA Polymerase. The second plasmid provides a multiple cloning site driven by a T7 promoter. The plasmids have different resistance markers and antibiotic resistance markers and can be transformed into one cell at the same time. The improved T7 Riboregulation System is a foundational advance in synthetic biology. PCR Screening Results Confirm Presence of T7 RNA Polymerase and T7 Artificial Riboregulation System

Expression of Construct with GFP

Expression of Anti-biofouling Peptide

Assessing Adhesion of Peptide

Biofilm Assay Results

We simulated a 24 hour period and determined the optimal time to induce the cells is around mid-log (~7.5 hours). Inducing at this time maximizes production of the peptide. The graph below shows E. coli growth with induction at different times. They follow a logistic growth model until the inducer is added and then there is an exponential decay.

Overlayed with this graph is a plot of total production of of the peptide vs. time of induction, (with induction at every 6 minutes over a 24 hour period). The highest production of peptide over the lifespan of these bacteria is represented by the peak of this plot, which corresponds to induction at mid-log, as we previously hypothesized.
The MATLAB code for our model can be found Here.