Part:BBa_K3802001
pTac Promoter
Sequence and Features
The ptac promoter is a hybrid of trp and lac UV5 promoters. This part is IPTG inducible and is also regulated by the lacI repressor [1].
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage: MC-LR Degradation
Most methods of microcystin-LR (MC-LR) degradation involve the enzyme microcystinase or MlrA. A problem that has consistently occurred with MlrA expression in E. coli has been that it goes directly to the cytoplasm, which reduces the efficiency of its capability of MC-LR degradation. This problem was solved two ways, as listed below:
Membrane anchoring of MlrA
The first solution addressed this issue by expressing MlrA on the outer membrane of E. coli [1]. The advantage of this system is that microcystin (MC-LR) does not have to be transported into the cytosol of E. coli for degradation, but rather can be directly broken down if MC-LR is within proximity of E. coli . Thus we expressed MlrA on the outer membrane via a fusion protein. This fusion protein consists of the MlrA gene linked with the gene for PgsA (poly-γ-glutamate synthetase A), a protein natively found in Bacillus subtilis [2].
This MlrA-PgsA fusion protein was also recorded as a composite part: (BBa_K3802000).
TatExpress: Upregulated MlrA export
The bacterial twin arginine translocase (Tat) pathway transports fully folded proteins across the inner membrane to the periplasmic space, thereby offering a promising solution to the problems posed by heterologous expression of MlrA in E. coli [3]. Despite the potential of the Tat secretion system, one drawback that has limited industrial applications is the relatively low yield of exported heterologous proteins. This low yield is primarily due to the small amount of Tat secretion machinery naturally produced in E. coli , as relatively few native proteins proteins utilize the pathway [4]. Our team hoped to address this anticipated limitation by reproducing the super-secreting “TatExpress” strain of E. coli engineered by Browning et al. The strong IPTG inducible promoter ptac was introduced upstream of the TatABCD operon on the chromosome, producing a strain with enhanced secretion capacity while avoiding the negative selective pressures introduced by the use of multiple plasmids bearing an extra copy of the gene with the strong promoter [3][1].
References
[1] Dziga, D., Wladyka, B., Zielińska, G., Meriluoto, J., & Wasylewski, M. (2012). Heterologous expression and characterisation Of microcystinase. Toxicon, 59(5), 578–586. https://doi.org/10.1016/j.toxicon.2012.01.001
[2] Narita, J., Okano, K., Tateno, T., Tanino, T., Sewaki, T., Sung, M.-H., Fukuda, H., & Kondo, A. (2005). Display of active enzymes on the cell surface of Escherichia COLI using PgsA ANCHOR protein and their application to bioconversion. Applied Microbiology and Biotechnology, 70(5), 564–572. https://doi.org/10.1007/s00253-005-0111-x
[3] Lee, P. A., Tullman-Ercek, D., & Georgiou, G. (2006). The Bacterial Twin-Arginine Translocation Pathway. Annual Review of Microbiology, 60(1), 373–395. https://doi.org/doi.org/10.1146/annurev.micro.60.080805.142212
[4] Barrett, C. M. L., Ray, N., Thomas, J. D., Robinson, C., & Bolhuis, A. (2003). Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli. Biochemical and Biophysical Research Communications, 304(2), 279–284. https://doi.org/doi.org/10.1016/s0006-291x(03)00583-7
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