Composite

Part:BBa_K1796015

Designed by: Nannan Xie   Group: iGEM15_SCU_China   (2015-09-18)
Revision as of 06:56, 13 October 2018 by Manbu (Talk | contribs)


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

In order to test the expression efficiency of the nif cluster,firstly we measured the transcriptional activity of nif promoter by combining it with the gene of fluorescent protein Dronpa,with T5 (IPTG Inducible) Promoter BBa_M50075 as a positive control(Fig 2).

Fig 2：Expression efficiency of Pnif

Comparison of the expression efficiency of Pnif and T5 (IPTG Inducible) Promoter.
T5 (IPTG Inducible) Promoter BBa_M50075; Pnif: nif promoter BBa_K1796001.

As demonstrated above, nif promoter is quite strong,however, how capable it is in our nitrogen fixation system remains an unclear question. So we also detected the expression level of the essential components in our system by conducting Real-time Quantitative PCR(QPCR),using 16S DNA as an internal reference.The results are shown in Fig3.
After we compare the result with the ideal expression ratio in Paenibacillus CR1 and model the transcription, we plan to optimize the nif gene cluster by adding promoters or altering the position of genes.

T--Nanjing-China--QPCR1.jpg

Fig 3. The qPCR results for components of nitrogen fixation system

Nitrogenase can not only reduce dinitrogen to ammonia but also reduce ethylene to acetylene. Therefore, we use gas chromatography to detect the amount of acetylene reduced, and indirectly detect its nitrogen fixation activity.

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