Part:BBa_K3581017
Hagfish intermediate filaments alpha subunit with T7 parts and TorA signal peptide
This composite part includes T7 promoter BBa_I712074, RBS BBa_B0030, the alpha subunit BBa_K3581000, His tag BBa_K3581002, TorA signal peptide BBa_K3581014 and T7 terminator BBa_B0016. The aim of this part is to replicate results of a previous experimental data which also used T7 parts by secreting alpha subunit of hagfish intermediate filaments. The His tag will be used for purifying the protein and TEV protease cut site can cleave out the tag once the protein is purified. The signal peptide is flanked by HindIII cut sites which will allow us to secrete the protein extracellularly, when we can also cut out the signal peptide using HindIII to secrete them as inclusion bodies to determine the best method with highest yield. The terminator is a T7 terminator which is commonly paired with T7 promoters.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2242
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2091
Illegal BsaI.rc site found at 2223
References:
1. Jing, F., A. Guerette, P., Pavesi, A., Horbelt, N., Chwee Teck, L., J. Harrington, M., & Miserez, A. (2017). Artificial hagfish protein fibers with ultra-high and tunable stiffness, 9(35), 12908–12915. The Royal Society of Chemistry.
2. England, N. (n.d.). TEV Protease | NEB. Retrieved October 13, 2020, from https://international.neb.com/products/p8112-tev-protease#Product%20Information
3. Wikipedia. (2020, October 14). TEV Protease. Wikipedia. Retrieved October 13, 2020, from https://en.wikipedia.org/wiki/TEV_protease
4. Palmer, I., & Wingfield, P. T. (2004). Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Current protocols in protein science, Chapter 6, Unit–6.3. https://doi.org/10.1002/0471140864.ps0603s38
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