Composite
AraC_TEV-F
Part:BBa_K627009:Design
Designed by: iGEM11 Potsdam_Bioware Group: iGEM11_Potsdam_Bioware (2011-09-21)
Arabinose inducible TEV protease
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1239
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Illegal SapI.rc site found at 1563
Design Notes
This biobrick was built by PCR using the following PCR primers:
Primer used for site directed mutagenesis:
Fragment 1:
- r_TEV_ACCAGC: GGAATGTGCAGCTGGTGTCTGACACC
- f_TEV_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGAGAAAGCTTGTTTAAGGGA
Fragment 2:
- r_TEV_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTATTGCGAGTACACCAATTCATTCAT
- f_TEV_ACCAGC: GGTGTCAGACACCAGCTGCACATTCC
Primer used for assembly PCR of mutated fragments:
- f_TEV_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGAGAAAGCTTGTTTAAGGGA
- r_TEV_iGEM_BamHI: TATAGGATCCACTGCAGCGGCCGCTACTAGTTTATTGCGAGTACACCAATTCATTCAT
Primers used for amplification of pBAD arabinose-inducible induction system:
- f_AraC_iGEM_HindIII: TATAAGCTTGAATTCGCGGCCGCTTCTAGATTATGACAACTTGACGGCTACATCATT
- r_AraC_NgoMIV: ATAGCCGGCCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGCCC
Primers used for amplification and modification of TEV protease:
- f_TEV_AraFusion_NgoMIV: ATATTGCCGGCATGGGAGAAAGCCTGTTTAAGGGA
- r_TEV_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTATTGCGAGTACACCAATTCATTCAT
Source
Arabinose inducible operon form pBAD_iGEM_express, TEV protease from Gunther Stier et. al.
References
- Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. and Bottomley, S. P. (2007). Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-2367
- Kapust, R. B., Tözsér, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., and Waugh, D. S. (2001). Tobacco etch virus protease: Mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Prot. Eng. 14: 993-1000.
- Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30: 544-550.