Difference between revisions of "Part:BBa K627008:Design"

(→‎Source)
(→‎References)
 
Line 32: Line 32:
  
 
===References===
 
===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.

Latest revision as of 15:48, 21 September 2011

Fusion part of arabinose-inducible induction system and the TEV protease


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1239
  • 1000
    INCOMPATIBLE 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.