Part:BBa_K1465102:Design

Fumarate reductase (frd) from E. coli under the control of the T7 promoter

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1352
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 666
  Illegal AgeI site found at 741
  Illegal AgeI site found at 3023
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

• Amplification of the pSB1C3-Backbone (BBa_K1172502) using the specific Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_frd_pre pSB1C3_pre_frd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3__suf_frd pSB1C3_frd_suf]:
  

pSB1C3_pre_frd (43 bp)

5'-GCAAGATCGGCTTGAAAGGTTTGCATCTAGTATTTCTCCTCTTTC-3'

pSB1C3_suf_frd (47 bp)

5'-CTGATTGGTGTCGTTACAATCTAATACTAGTAGCGGCCGCTGCAGTC-3'

• Amplification of the fumerate reductase (frd) from Escherichia coli KRX containing the T7 promotor and the RBS BBa_B0034 in the specific overhang of the Gibson-Primer [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#T7_frd_Ec_fwd T7_frd_Ec_fwd] and [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#T7_frd_Ec_rev T7_frd_Ec_rev]:
  

T7_frd_Ec_fwd (64 bp)

5'-TAATACGACTCACTATAGGGAAAGAGGAGAAATACTAGATGCAAACCTTTCAAGCCGATCTTGC-3'

T7_frd_Ec_rev (44 bp)

5'-TGCAGCGGCCGCTACTAGTATTAGATTGTAACGACACCAATCAG-3'

• [http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson Gibson-Assembly] of the pSB1C3-Backbone containg also the T7 promoter and the RBS BBa_B0034 and the coding sequence of the fumerate reductase (frd) from E. coli.
  

Source

[http://www.promega.de/resources/protocols/technical-bulletins/101/single-step-krx-competent-cells-protocol/ Escherichia coli KRX (Promega)]:

• Fumarate reductase (anaerobic), catalytic and NAD/flavoprotein subunit: [http://www.ncbi.nlm.nih.gov/gene/948667 frdA]
  
• Fumarate reductase (anaerobic), Fe-S subunit: [http://www.ncbi.nlm.nih.gov/gene/948666 frdB]
  
• Fumarate reductase (anaerobic), membrane anchor subunit: [http://www.ncbi.nlm.nih.gov/gene/948680 frdC]
  
• Fumarate reductase (anaerobic), membrane anchor subunit: [http://www.ncbi.nlm.nih.gov/gene/948668 frdD]