Difference between revisions of "Part:BBa K1603000:Design"

 
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===Design Notes===
 
===Design Notes===
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20 base pair overlapping sequence between the signal peptide from STE2 and MAM2 had to be constructed through PCR to allow Gibson assembly of the two gene fragments. Added Prefix and Suffix through PCR.
+
Added 20 overlapping base pairs between the two gene fragments through PCR with primers with overhangs. This allowed the two gene fragments to be combined through Gibson assembly. Added Prefix and Suffix through PCR with primers with overhangs.
  
 +
Primers for isolation of ''SPSTE2'' with Prefix and overlap to ''MAM2-SP'' from genomic DNA:
  
 +
FW:GCTTCTAGATGTCTGATGCGGCTCC
  
 +
RV:AGTTGGGCAGACAAAGTCATCTGAGTAACAGTACTGTTAACTAAACCTTGC
 +
 +
 +
Primers for isolation of ''MAM2-SP'' with Suffix from genomic DNA:
 +
 +
FW:ATGACTTTGTCTGCCCAACT
 +
 +
RV:AGCCTGCAGCGGCCGCTACTAGTATTACGTCCACTTTTTAGTTTCAGATTC
 +
 +
 +
The primers were designed to remove the EcoRI site from the insert when constructing the biobrick to reduce the amount of base pairs in the primers. The primers also add 3 extra protective basepairs to the PstI site in the insert.
 +
 +
 +
The construction of the final biobrick was initiated by cutting [https://parts.igem.org/Part:BBa_J04450 BBa_J04450] with XbaI, PstI, KnpI and FastAP and the insert with XbaI and PstI.
  
 
===Source===
 
===Source===
  
−
The non-cytoplasmic N-terminal signal peptide from STE2 is a genomic sequence from Saccharomyces cerevisiae CEN.PK2 and the Pheromone P-factor receptor MAM2, without its signaling peptide, is a genomic sequence from Schizosaccharomyces pombe Δ8 h-.
+
The non-cytoplasmic N-terminal signal peptide from ''STE2'' is a genomic sequence from ''Saccharomyces cerevisiae'' CEN.PK2 and the Pheromone P-factor receptor MAM2, without its signaling peptide, is a genomic sequence from ''Schizosaccharomyces pombe'' Cut Δ8 -h.
  
 
===References===
 
===References===

Latest revision as of 14:36, 18 September 2015


Fusion GPCR STE2MAM2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 408
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Added 20 overlapping base pairs between the two gene fragments through PCR with primers with overhangs. This allowed the two gene fragments to be combined through Gibson assembly. Added Prefix and Suffix through PCR with primers with overhangs.

Primers for isolation of SPSTE2 with Prefix and overlap to MAM2-SP from genomic DNA:

FW:GCTTCTAGATGTCTGATGCGGCTCC

RV:AGTTGGGCAGACAAAGTCATCTGAGTAACAGTACTGTTAACTAAACCTTGC


Primers for isolation of MAM2-SP with Suffix from genomic DNA:

FW:ATGACTTTGTCTGCCCAACT

RV:AGCCTGCAGCGGCCGCTACTAGTATTACGTCCACTTTTTAGTTTCAGATTC


The primers were designed to remove the EcoRI site from the insert when constructing the biobrick to reduce the amount of base pairs in the primers. The primers also add 3 extra protective basepairs to the PstI site in the insert.


The construction of the final biobrick was initiated by cutting BBa_J04450 with XbaI, PstI, KnpI and FastAP and the insert with XbaI and PstI.

Source

The non-cytoplasmic N-terminal signal peptide from STE2 is a genomic sequence from Saccharomyces cerevisiae CEN.PK2 and the Pheromone P-factor receptor MAM2, without its signaling peptide, is a genomic sequence from Schizosaccharomyces pombe Cut Δ8 -h.

References