Difference between revisions of "Part:BBa K1796015"
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<h2>iGEM2018_Nanjing China Experiment</h2>
 
<h2>iGEM2018_Nanjing China Experiment</h2>
  
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<p>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(<strong>Fig 2</strong>).</p>
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<p>This year our team use the nitrogen fixation  gene cluster from <em>Paenibacillus polymyxa</em> CR1, which shares a close  biological relationship with <em>Paenibacillus sp.</em> WLY78, so the data can  provide some reference to this part.<br>
 +
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, and use T5 (IPTG Inducible) Promoter, BBa_M50075 as a positive control(<strong>Fig 1</strong>).</p>
 +
 
 
[[File:T--Nanjing-China--11part.png|800px|thumb|center|Fig 2：Expression efficiency of Pnif]]
 
[[File:T--Nanjing-China--11part.png|800px|thumb|center|Fig 2：Expression efficiency of Pnif]]
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<p>Comparison of the expression efficiency of  Pnif and T5 (IPTG Inducible) Promoter. <br />
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<p>Comparison of the expression efficiency of  Pnif and T5 (IPTG Inducible) Promoter. <br>
 
T5 (IPTG Inducible) Promoter BBa_M50075;  Pnif: nif promoter BBa_K1796001.</p>
 
T5 (IPTG Inducible) Promoter BBa_M50075;  Pnif: nif promoter BBa_K1796001.</p>
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<p>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 <strong>Fig3</strong>.<br />
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<p>As demonstrated above, nif promoter is  quite strong,however, how capable it is in our nitrogen fixation system remains  unclear. So we test expression profiles of each structure gene in the nif cluster that overexpressed in EJNC by conducting Real-time Quantitative PCR(qPCR). Relative expression compared to the housekeeping gene 16S rRNA is  shown(<strong>Fig2)</strong>.</p>
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  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.</p>
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   [[File:T--Nanjing-China--QPCR1.jpg|800px|thumb|center]]<br />
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   [[File:T--Nanjing-China--qRT-PCR.jpg|800px|thumb|center|Fig 2. The qPCR results for components of nitrogen fixation system]]
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  [[File:T--Nanjing-China--QPCR2.jpg|800px|thumb|center|Fig 3. The qPCR results for components of nitrogen fixation system]]<p>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. </p>
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  <p>qRT-PCR analysis demonstrates that all the component genes of the nif cluster are significantly over expressed in EJNC whereas the transcription of these genes are no detected (N.D.) in nondiazotrophic E.coli JM109.  </p>

Revision as of 11:08, 15 October 2018


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 use 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.
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, and use T5 (IPTG Inducible) Promoter, BBa_M50075 as a positive control(Fig 1).

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 unclear. So we test expression profiles of each structure gene in the nif cluster that overexpressed in EJNC by conducting Real-time Quantitative PCR(qPCR). Relative expression compared to the housekeeping gene 16S rRNA is shown(Fig2).

Fig 2. The qPCR results for components of nitrogen fixation system

qRT-PCR analysis demonstrates that all the component genes of the nif cluster are significantly over expressed in EJNC whereas the transcription of these genes are no detected (N.D.) in nondiazotrophic E.coli JM109.