Part:BBa_K5178012

Coding sequence for human pancreatic lipase enzyme with a signal peptide and histidine purification tag. This part is under the control of the constitutive P43 promoter (Part:BBa_K143013). The part sequence is codon optimized for expression in B. subtilis. Addition of a signal peptide allows the enzyme to be more efficiently secreted by transformed B. subtilis. A His6 tag was added for easy enzyme detection and quantification during experimental assays. This part also includes coding sequences for RiboJ—a ribozyme that increases access to the adjacent RBS, a generic RBS: MF001, and a generic terminator: B0015.

This part is a modified version of a part designed previously which used the inducible PT7lac promoter. After a re-design of our parts, this part was developed. We chose to list this part on the registry as it is more accessible and usable by other teams, as it is not dependent on the T7RNAP gene.

Coding sequences were gathered from the following sources.

PNLIP: Winkler, F., D'Arcy, A. & Hunziker, W. Structure of human pancreatic lipase. Nature 343, 771–774 (1990). https://doi.org/10.1038/343771a0

P43: Part:BBa_K143013

RiboJ: Lou, C., Stanton, B., Chen, Y. J., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature biotechnology, 30(11), 1137–1142. https://doi.org/10.1038/nbt.2401

MF001: Castillo-Hair, S. M., Fujita, M., Igoshin, O. A., & Tabor, J. J. (2019). An Engineered B. subtilis Inducible Promoter System with over 10 000-Fold Dynamic Range. ACS synthetic biology, 8(7), 1673–1678. https://doi.org/10.1021/acssynbio.8b00469

B0015: Castillo-Hair, S. M., Fujita, M., Igoshin, O. A., & Tabor, J. J. (2019). An Engineered B. subtilis Inducible Promoter System with over 10 000-Fold Dynamic Range. ACS synthetic biology, 8(7), 1673–1678. https://doi.org/10.1021/acssynbio.8b00469

Design considerations: This part was codon optimized for expression in Bacillus subtilis. Coenzyme, procolipase is necessary for lipase to activate in the human body.