Difference between revisions of "Part:BBa K1783000:Design"

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Restriction Enzyme cloning:
 
Restriction Enzyme cloning:
 
Double digest of pSB1C3 backbone + K1783000 of EcoRI-HF and PstI-HF (1 unit each) for 1 hr.
 
Double digest of pSB1C3 backbone + K1783000 of EcoRI-HF and PstI-HF (1 unit each) for 1 hr.
−
Ligation with T4 DNA Ligase overnight
+
Ligation of an equimolar amount of both inserts + backbone with T4 DNA Ligase overnight.
 +
Transformation into DH5-alpha chemically competent cells.
  
−
DH5α was used as the cell strain for transformations.
+
DH5α was used as the cell strain for transformations due to the absence of recombinases.
  
 
===Source===
 
===Source===
  
−
All parts are from previous BioBricks. pBAD promoter is from E. coli, while Miraculin gene comes from S. dulcificum.
+
All parts are from previous BioBricks. pBAD promoter is from ''E. coli'', while Miraculin gene comes from ''S. dulcificum''.
  
 
===References===
 
===References===
 +
Matsuyama T, Satoh M, Nakata R, Aoyama T, Inoue H. Functional expression of miraculin, a taste-modifying protein in Escherichia coli. J Biochem. 2009;145(4):445-50.

Latest revision as of 21:05, 17 September 2015


Miraculin Generator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Parts were assembled using 3A assembly of K206000 (pBAD promoter) and K1033120 (RBS-Miraculin), following the iGEM protocol. Since both parts were in the pSB1C3 backbone, we initially assembled the final construct in the pSB1A3 backbone, then moved the composite part into the pSB1C3 backbone using RE cloning.

Restriction Enzyme cloning: Double digest of pSB1C3 backbone + K1783000 of EcoRI-HF and PstI-HF (1 unit each) for 1 hr. Ligation of an equimolar amount of both inserts + backbone with T4 DNA Ligase overnight. Transformation into DH5-alpha chemically competent cells.

DH5α was used as the cell strain for transformations due to the absence of recombinases.

Source

All parts are from previous BioBricks. pBAD promoter is from E. coli, while Miraculin gene comes from S. dulcificum.

References

Matsuyama T, Satoh M, Nakata R, Aoyama T, Inoue H. Functional expression of miraculin, a taste-modifying protein in Escherichia coli. J Biochem. 2009;145(4):445-50.