Difference between revisions of "Part:BBa K2825002"

Revision as of 02:51, 18 October 2018

PcaU inducible promoter with high sensitivity

Inducible promoter device for protocatechuic acid (PCA). The PcaU transcription factor represses its own transcription as well as a target transcript when PCA is not present by binding to the operator region. When PCA is present, PcaU releases the region and transcription of the target transcript occurs.

This version of PcaU is derived from evolutionary engineering courtesy of Jha et. al. See design section for details

Usage and Biology

PcaU is an IclR transcription factor originally obtained from a PCA catabolism operon in Acinetobacter baylyi. (Jha et al., 2018). PcaU was then optimized for sensitivity and specificity in response to PCA in Pseudomonas putida through directed evolution.

Characterization

PcaU has been inserted into the pGLO plasmid, in which it expresses GFP when induced by PCA. Fluorescent intensity scales hyperbolically with PCA concentration. Above 10mM concentration, PCA was toxic to BL21 E. coli containing the PcaU reporter. We used a logarithmic scale in the chart below to clearly indicate the relationship between PCA concentration and fluorescence.

The lower range of PcaU sensitivity is in single micromolar concentrations. When measuring PCA concentrations this dilute in a multi well plate, wells on the edge of the plate show increased error. For accurate results, fill outer wells with water and use inner wells only. The figure below used 32 wells in the center of a 48 well plate (n=8). Washing wells in PBS will also reduce error. We used a logarithmic scale in the chart below to clearly indicate the relationship between PCA concentration and fluorescence.

PcaU is functional in DH5a E. coli, but is more sensitive in BL21 DE3 E. coli. We used a linear scale in the chart below to show the equal uninduced fluorescence of these cells.

For parts submission, we mutated an EcoR1 site in the noncoding region from GAATTC to GAAATC. The chart below shows that the mutation did not affect sensitivity. We used a linear scale in the chart below to show the equal uninduced fluorescence of these cells.

Sequence and Features

References

1. Jha, R. K., Kern, T. L., Fox, D. T., & M. Strauss, C. E. (2014). Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry. Nucleic Acids Research, 42(12), 8150–8160. http://doi.org/10.1093/nar/gku444