Part:BBa_K3016030

Vibrio natriegens’ TatAB

This part contains the Vibrio natriegens’ native tatAB operon. It encodes the TatA and TatB proteins of the twin-arginine translocation pathway. The proteins also need TatC (link to part) to operate normally. The twin-arginine translocation pathway is capable of translocating fully folded proteins up to 150 kDa. It also contains a quality control feature of rejecting misfolded proteins. In some cases, disulfide bridge formation is not required for successful translocation. (Alanen et al., 2015)

Comparision to E. coli

Unlike in E. coli, Vibrio natriegens’ tatAB and tatC are located in separate operons. V. natriegens is also missing the tatD and tatE components present in E. coli. The ratio of TatA:TatB:TatC is about 50:25:1 in E. coli (Jack et al., 2001). This ratio is unconfirmed in V. natriegens and the separation into two operons with different sigma-factor binding sites possibly implies that the ratio is not static.

Use

This part can be used to overexpress the twin-arginine translocation pathway components to facilitate increased transport of proteins into the periplasm. Note that chromosomal overexpression might be better than expression from a plasmid. (Browning et al., 2017)

Translocation using the tat-pathway requires the protein to contain a signal peptide with a twin-arginine (RR) motif. The signal peptide is cleaved during the translocation process. A collection of V. natriegens’ native twin-arginine signal peptides identified by Aalto-Helsinki can be found here (link to peptides)

Aalto-Helsinki 2019 set out to overexpress the tatAB and tatC operons by adding an inducible promoter upstream of both operons. We chose to use the IPTG-inducible ptac and plac promoters. Further insights into our design and decisions can be found in our wiki (link to wiki/design)

References:

Alanen, H. I., Walker, K. L., Suberbie, M. L. V., Matos, C. F., Bönisch, S., Freedman, R. B., ... & Robinson, C. (2015). Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1853(3), 756-763.

Browning, D. F., Richards, K. L., Peswani, A. R., Roobol, J., Busby, S. J., & Robinson, C. (2017). Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway. Biotechnology and bioengineering, 114(12), 2828-2836.

Jack, R. L., Sargent, F., Berks, B. C., Sawers, G., & Palmer, T. (2001). Constitutive expression of Escherichia coli tat genes indicates an important role for the twin-arginine translocase during aerobic and anaerobic growth. Journal of bacteriology, 183(5), 1801-1804.