Composite

Part:BBa_K1796015

Designed by: Nannan Xie   Group: iGEM15_SCU_China   (2015-09-18)


complete line of nif cluster

whole line of nif cluster,contain Pnif,nifBHDKENXV,hesA

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2163
    Illegal EcoRI site found at 6951
    Illegal PstI site found at 2689
    Illegal PstI site found at 6597
    Illegal PstI site found at 7295
    Illegal PstI site found at 7866
    Illegal PstI site found at 7937
    Illegal PstI site found at 8743
    Illegal PstI site found at 8983
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2163
    Illegal EcoRI site found at 6951
    Illegal PstI site found at 2689
    Illegal PstI site found at 6597
    Illegal PstI site found at 7295
    Illegal PstI site found at 7866
    Illegal PstI site found at 7937
    Illegal PstI site found at 8743
    Illegal PstI site found at 8983
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2163
    Illegal EcoRI site found at 6951
    Illegal BglII site found at 3655
    Illegal BglII site found at 10747
    Illegal BamHI site found at 10570
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2163
    Illegal EcoRI site found at 6951
    Illegal PstI site found at 2689
    Illegal PstI site found at 6597
    Illegal PstI site found at 7295
    Illegal PstI site found at 7866
    Illegal PstI site found at 7937
    Illegal PstI site found at 8743
    Illegal PstI site found at 8983
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2163
    Illegal EcoRI site found at 6951
    Illegal PstI site found at 2689
    Illegal PstI site found at 6597
    Illegal PstI site found at 7295
    Illegal PstI site found at 7866
    Illegal PstI site found at 7937
    Illegal PstI site found at 8743
    Illegal PstI site found at 8983
    Illegal NgoMIV site found at 5942
    Illegal NgoMIV site found at 6189
    Illegal NgoMIV site found at 7624
    Illegal AgeI site found at 1136
    Illegal AgeI site found at 2096
    Illegal AgeI site found at 2451
    Illegal AgeI site found at 3680
    Illegal AgeI site found at 4362
    Illegal AgeI site found at 4735
    Illegal AgeI site found at 5202
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2268


iGEM2018_Nanjing China Experiment

This year our team used the nitrogen fixation gene cluster from Paenibacillus polymyxa CR1, which shares a close biological relationship with Paenibacillus sp.WLY78, so the data can provide some reference to this part.

To test whether the nitrogen fixation gene cluster could express in gram-negative E. coli JM109 , pUC57-nif was inreoduced into JM109 via electroporation (Figure 1a). But before qRT-PCR determination, the function and strength of the native promoter in nif cluster (Pnif) were firstly tested in JM109 by fusing Dronpa as the reporter. T5 promoter (BBa_M50075) severed as control. As shown in Figure 1b, compared with T5 promoter, Pnif was much stronger in driving the expression of RFP and its expression pattern was constitutive. Transcriptional analysis was carried out afterward. As shown in Figure 2, Pnif was strong enough to drive the expression of each structure gene in the nif cluster including nifB though with different relative expression level.

Figure 1a）Engineered E. coli cells with nitrogenase
1b）Fluorescence intensity detemination

Figure 2. Expression profiles of each structure gene in the nif cluster that overexpressed in engineered E.coli JM109 (EJNC). E.coli JM109 (EJ) severs as control and relative expression compared to the housekeeping gene 16S rRNA is shown. N.D. represent not ditected.

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