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===

Revision as of 16:57, 21 September 2011

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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 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

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.

References

Used parts from the Registry of Standard Biological Parts:

  • BBa_K208005
  • BBa_I757010