Device
PcaU

Part:BBa_K2825002:Design

Designed by: Chris Kuffner   Group: iGEM18_UMaryland   (2018-10-10)


PcaU inducible promoter with high sensitivity


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 171


Design Notes

optimized from acinobacter to work in E. coli BL21 DE3 by Jha et. al. 2014. Jha codon optimized PcaU, added E. coli RBS and promoter regions, then used directed evolution to select for mutants with maximum sensitivity. On top of Jha's work, we have codon optimized PCAU open reading frame using the IDT codon optimizer. The noncoding regulatory region is identical to the one used in Jha's paper, apart from the exchange of a GAATTC EcoR1 site for GAAATC, which did not hinder functionality.

Source

See first reference

References

---The 2014 paper in which the PcaU for E. coli originated:

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



Extra sources:

---2017 paper in which PcaU was optimized in Putida and used to detect enzymatic activity

Ramesh K. Jha, Jeremy M. Bingen, Christopher W. Johnson, Theresa L. Kern, Payal Khanna, Daniel S. Trettel, Charlie E.M. Strauss, Gregg T. Beckham, Taraka Dale (2018). A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution. Metabolic Engineering Communications, 6, 33-38,

---Paper on PcaK, a PCA active transporter that increases PCA concentration in E. coli roughly 5x. PcaK could be used to improve this part in the future.

Nichols, N. N., & Harwood, C. S. (1997). PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida. Journal of Bacteriology, 179(16), 5056–5061.

---More details on the mechanism of PcaU

Gerischer, U., Segura, A., & Ornston, L. N. (1998). PcaU, a Transcriptional Activator of Genes for Protocatechuate Utilization in Acinetobacter. Journal of Bacteriology, 180(6), 1512–1524. Kok,R.G., D’Argenio,D.A. and Ornston,L.N. (1998) Mutation analysis of PobR and PcaU, closely related transcriptional activators in Acinetobacter. J. Bacteriol., 180, 5058–5069.