Difference between revisions of "Part:BBa K627011:Design"

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===Design Notes===
 
===Design Notes===
 
This biobrick was built by PCR using the following PCR primers:<br>
 
This biobrick was built by PCR using the following PCR primers:<br>
−
Forward primer (p_TorA-f): CTTCTAGATGAACAATAACGATCTCTTTCAGGCATC <br>
+
<b>Site directed mutagenesis of HRV 14 3C protease:</b><br>
−
Reverse primer (o_bla_igem_r): CTACTAGTATTAACCGGTCCAATGCTTAATCAGTGAGGCAC
+
<i>Fragment 1:</i>
 +
* f_14_3C_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGACCAAACACAGAATTTGCACTATCC
 +
* r_14_3C_ACCAGC: ACCATTTGCTGGTACATCATCACCAGG
 +
<i>Fragment 2:</i>
 +
* f_14_3C_ACCAGC: CCTGGTGATGATGTACCAGCAAATGGT
 +
* r_14_3C_tm_XbaI208_A-T: CACTGTAAGCTCAAGATTAATGTTCTC
 +
<i>Fragment 3:</i>
 +
* f_14_3C_tm_Xba208_A-T: GAGAACATTAATCTTGAGCTTACAGTG
 +
* r_14_3C_tm_XbaI280_A-T: ATCCACACCTTCGAGATCTTCTGATAT
 +
<i>Fragment 4:</i>
 +
* f_14_3C_tm_Xba280_A-T: ATATCAGAAGATCTCGAAGGTGTGGAT
 +
* r_14_3C_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTTGTTTCTCTACAAAATATTGTTTTTTAAGTTGAGCTGA
 +
<b>Primers used for first assembly PCR of mutated fragments:</b><br>
 +
<i>Fragment 5, containing Fragment 1 and 2:</i>
 +
* f_14_3C_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGACCAAACACAGAATTTGCACTATCC
 +
* r_14_3C_tm_Xba208_A-T: CACTGTAAGCTCAAGATTAATGTTCTC
 +
<i>Fragment 6, containing Fragment 3 and 4:</i>
 +
* f_14_3C_tm_Xba208_A-T: GAGAACATTAATCTTGAGCTTACAGTG
 +
* r_14_3C_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTTGTTTCTCTACAAAATATTGTTTTTTAAGTTGAGCTGA
 +
<b>Primer used for final assembly PCR of mutated fragments:</b><br>
 +
<i>Complete mutated 14_3C protease, containing Fragment 5 and 6:</i>
 +
* f_14_3C_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGACCAAACACAGAATTTGCACTATCC
 +
* r_14_3C_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTTGTTTCTCTACAAAATATTGTTTTTTAAGTTGAGCTGA
 +
<i>Primers used for amplification of the pBAD arabinose-inducible induction system:</i>
 +
* f_AraC_iGEM_HindIII: TATAAGCTTGAATTCGCGGCCGCTTCTAGATTATGACAACTTGACGGCTACATCATT
 +
* r_AraC_NgoMIV: ATAGCCGGCCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGCCC
 +
<i>Primers used for amplification and modification of HRV 14 3C protease:</i>
 +
* f_14_3C_AraFusion_NgoMIV: ATATTGCCGGCATGGGACCAAACACAGAATTTGCACTATCC
 +
* r_14_3C_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTTGTTTCTCTACAAAATATTGTTTTTTAAGTTGAGCTGA
  
 
===Source===
 
===Source===
−
TorA signal sequence and the lactamase was amplified via PCR from pJC354, which originates from two iGEM compatible BioBricks: BBa_K208005 and BBa_I757010. The cleavage site was created, based on the information of the Brenda enzymes database, via 2 oligonucleotides ordered from Sigma-Aldrich.
+
Arabinose inducible operon form pBAD_iGEM_express was ampilied via PCR, 14_3C protease was isolated from the genome of human rhinovirus 14_3C and mutated with primers listed in part description BBa: to remove all iGEM restriction sites inside the protease gene.
  
 
===References===
 
===References===
 +
* Combination of Two Separate Binding Domains Defines Stoichiometry between Type III Secretion System Chaperone IpgC and Translocator Protein IpaB, Ravi Kumar Lokareddy, Michele Lunelli, Björn Eilers, Vivien Wolter, and Michael Kolbe, December 17, 2010 The Journal of Biological Chemistry, 285, 39965-39975.
 +
 +
* Cleavage of Small Peptides In Vitro by Human Rhinovirus 14 3C Protease  expressed in Escherichia coli, MICHAEL G. CORDINGLEY,* R. BRUCE REGISTER, PIA L. CALLAHAN, VICTOR M. GARSKY,AND RICHARD J. COLONNO JOURNAL OF VIROLOGY, Dec. 1989, p. 5037-5045

Latest revision as of 16:58, 21 September 2011

Fusion part of pBAD arabinose-inducible induction system and the HRV 14_3C protease


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1523
    Illegal BglII site found at 1711
    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


Design Notes

This biobrick was built by PCR using the following PCR primers:
Site directed mutagenesis of HRV 14 3C protease:
Fragment 1:

  • f_14_3C_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGACCAAACACAGAATTTGCACTATCC
  • r_14_3C_ACCAGC: ACCATTTGCTGGTACATCATCACCAGG

Fragment 2:

  • f_14_3C_ACCAGC: CCTGGTGATGATGTACCAGCAAATGGT
  • r_14_3C_tm_XbaI208_A-T: CACTGTAAGCTCAAGATTAATGTTCTC

Fragment 3:

  • f_14_3C_tm_Xba208_A-T: GAGAACATTAATCTTGAGCTTACAGTG
  • r_14_3C_tm_XbaI280_A-T: ATCCACACCTTCGAGATCTTCTGATAT

Fragment 4:

  • f_14_3C_tm_Xba280_A-T: ATATCAGAAGATCTCGAAGGTGTGGAT
  • r_14_3C_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTTGTTTCTCTACAAAATATTGTTTTTTAAGTTGAGCTGA

Primers used for first assembly PCR of mutated fragments:
Fragment 5, containing Fragment 1 and 2:

  • f_14_3C_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGACCAAACACAGAATTTGCACTATCC
  • r_14_3C_tm_Xba208_A-T: CACTGTAAGCTCAAGATTAATGTTCTC

Fragment 6, containing Fragment 3 and 4:

  • f_14_3C_tm_Xba208_A-T: GAGAACATTAATCTTGAGCTTACAGTG
  • r_14_3C_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTTGTTTCTCTACAAAATATTGTTTTTTAAGTTGAGCTGA

Primer used for final assembly PCR of mutated fragments:
Complete mutated 14_3C protease, containing Fragment 5 and 6:

  • f_14_3C_iGEM: ATATAGAATTCGCGGCCGCTTCTAGATGGGACCAAACACAGAATTTGCACTATCC
  • r_14_3C_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTTGTTTCTCTACAAAATATTGTTTTTTAAGTTGAGCTGA

Primers used for amplification of the pBAD arabinose-inducible induction system:

  • f_AraC_iGEM_HindIII: TATAAGCTTGAATTCGCGGCCGCTTCTAGATTATGACAACTTGACGGCTACATCATT
  • r_AraC_NgoMIV: ATAGCCGGCCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGCCC

Primers used for amplification and modification of HRV 14 3C protease:

  • f_14_3C_AraFusion_NgoMIV: ATATTGCCGGCATGGGACCAAACACAGAATTTGCACTATCC
  • r_14_3C_iGEM_BamHI: ATATAGGATCCACTGCAGCGGCCGCTACTAGTTTTGTTTCTCTACAAAATATTGTTTTTTAAGTTGAGCTGA

Source

Arabinose inducible operon form pBAD_iGEM_express was ampilied via PCR, 14_3C protease was isolated from the genome of human rhinovirus 14_3C and mutated with primers listed in part description BBa: to remove all iGEM restriction sites inside the protease gene.

References

  • Combination of Two Separate Binding Domains Defines Stoichiometry between Type III Secretion System Chaperone IpgC and Translocator Protein IpaB, Ravi Kumar Lokareddy, Michele Lunelli, Björn Eilers, Vivien Wolter, and Michael Kolbe, December 17, 2010 The Journal of Biological Chemistry, 285, 39965-39975.
  • Cleavage of Small Peptides In Vitro by Human Rhinovirus 14 3C Protease expressed in Escherichia coli, MICHAEL G. CORDINGLEY,* R. BRUCE REGISTER, PIA L. CALLAHAN, VICTOR M. GARSKY,AND RICHARD J. COLONNO JOURNAL OF VIROLOGY, Dec. 1989, p. 5037-5045