Device

Part:BBa_K1465102:Design

Designed by: iGEM-Team Bielefeld 2014   Group: iGEM14_Bielefeld-CeBiTec   (2014-10-03)
Revision as of 17:42, 7 October 2014 by Tore (Talk | contribs) (→‎Design Notes)

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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]

References