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

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K627008 short</partinfo>
 
<partinfo>BBa_K627008 short</partinfo>
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===Design Notes===
 
===Design Notes===
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This biobrick was built by PCR using the following PCR primers:<br>
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<br>
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<b>Primer used for site directed mutagenesis:</b><br>
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<i>Fragment 1:</i><br>
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*r_TEV_ACCAGC: GGAATGTGCAGCTGGTGTCTGACACC<br>
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*f_TEV_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGAGAAAGCTTGTTTAAGGGA<br>
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<i>Fragment 2:</i><br>
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*r_TEV_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTATTGCGAGTACACCAATTCATTCAT<br>
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*f_TEV_ACCAGC: GGTGTCAGACACCAGCTGCACATTCC<br>
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<br>
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<b>Primer used for assembly PCR of mutated fragments:</b><br>
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*f_TEV_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGAGAAAGCTTGTTTAAGGGA<br>
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*r_TEV_iGEM_BamHI: TATAGGATCCACTGCAGCGGCCGCTACTAGTTTATTGCGAGTACACCAATTCATTCAT<br>
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<br>
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<b>Primers used for amplification of pBAD arabinose-inducible induction system:</b><br>
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*f_AraC_iGEM_HindIII: TATAAGCTTGAATTCGCGGCCGCTTCTAGATTATGACAACTTGACGGCTACATCATT<br>
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*r_AraC_NgoMIV: ATAGCCGGCCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGCCC<br>
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<br>
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<b>Primers used for amplification and modification of TEV protease:</b><br>
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*f_TEV_AraFusion_NgoMIV: ATATTGCCGGCATGGGAGAAAGCCTGTTTAAGGGA<br>
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*r_TEV_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTATTGCGAGTACACCAATTCATTCAT<br>
  
 
===Source===
 
===Source===
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Arabinose inducible operon form pBAD_iGEM_express, TEV protease from Gunther Stier <i>et. al</i>.
  
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===References===
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*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
  
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*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.
  
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===References===
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*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.