Part:BBa_K4359000:Design
E.coli K-12 Cytolysin A (ClyA)
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1003
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1003
Illegal NheI site found at 82 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1003
Illegal BglII site found at 159
Illegal BamHI site found at 997 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1003
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1003
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
ClyA was amplified from the E.coli K-12 w3110 genome with an EcoN1 site added upstream and BamH1 site added downstream to enable ligation into the plasmid backbone pGEX-4T1. An N-terminal 6X His tag and thrombin recognition site were included. The thrombin site was optimized to remove the BamH1 site
Source
This part is derived from composite part (BBa_K4359006) which was provided by Dr. Vipul Gujrati, TUM Munich.
References
[1] Gujrati, V., Kim, S., Kim, S.-H., Min, J. J., Choy, H. E., Kim, S. C., & Jon, S. (2014). Bioengineered Bacterial Outer Membrane Vesicles as Cell-Specific Drug-Delivery Vehicles for Cancer Therapy. ACS Nano, 8(2), 1525–1537. https://doi.org/10.1021/nn405724x
[2] Kim, J.-Y., Doody, A. M., Chen, D. J., Cremona, G. H., Shuler, M. L., Putnam, D., & DeLisa, M. P. (2008). Engineered Bacterial Outer Membrane Vesicles with Enhanced Functionality. Journal of Molecular Biology, 380(1), 51–66. https://doi.org/10.1016/j.jmb.2008.03.076
[3] Kulshrestha, A., Maurya, S., Gupta, T., Roy, R., Punnathanam, S., & Ayappa, K. G. (2021). Conformational flexibility is a key determinant of the lytic activity of the pore forming protein, Cytolysin A [Preprint]. Biophysics. https://doi.org/10.1101/2021.10.23.465544
[4] Murase, K. (2022). Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins, 14(2), 78. https://doi.org/10.3390/toxins14020078