Difference between revisions of "Part:BBa K1796015"
 
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<partinfo>BBa_K1796015 parameters</partinfo>
 
<partinfo>BBa_K1796015 parameters</partinfo>
 
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<h2>Nanjing-China2018</h2>
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<h2>iGEM2018_Nanjing China Experiment</h2>
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<p>Based on the existing part complete line of  nif cluster, BBa_K1796015, which contains essential components for nitrogen  fixation: nif Promoter, nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV  from the <em>Paenibacillus sp.</em> WLY78. We choose a new nitrogen fixation gene cluster from more common strain <em>Paenibacillus polymyxa</em> CR1, to comprise the nitrogen fixation system in our project.</p>
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<p>This year our team used the nitrogen fixation gene cluster from&nbsp;Paenibacillus polymyxa&nbsp;CR1, which shares a close biological relationship  with&nbsp;Paenibacillus sp.WLY78, so the data can provide some reference to  this part.</p>
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<p>In our this year&rsquo;s project, we intends to  establish a sound and ideal whole-cell photocatalytic nitrogen fixation system. We use the engineered <em>E. coli</em> cells to express nitrogenase(<strong>Fig 1</strong>) and in-situ synthesize of CdS semiconductors in the biohybrid system. Instead  of ATP-hydrolysis, such system is able to photocatalytic N2(nitrogen) to  NH3(ammonia). The biohybrid system based on engineered E. coli cells with  biosynthesis inorganic materials will likely become an alternative approach for  the convenient utilization of solar energy.</p>
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<p>To test whether the nitrogen fixation gene cluster could express in gram-negative <em>E. coli</em> JM109 , pUC57-<em>nif </em>was inreoduced into JM109 via electroporation (Figure 1a). But before qRT-PCR determination, the function and strength of the native promoter in <em>nif</em> cluster (P<em>nif</em>) 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, P<em>nif </em>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, P<em>nif</em> was strong enough to drive the expression of each structure gene in the <em>nif</em> cluster including <em>nif</em>B though with different relative expression level.</p>
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  <p>[[File:T--Nanjing-China--011part-design.png|800px|thumb|center|Fig 1. Design of our project: Engineered E. coli cells with nitrogenase]] </p>
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<p>[[File:T--Nanjing-China--1%2B2.jpg|800px|thumb|center|Figure 1a）Engineered E. coli cells with nitrogenase<br />
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<p>So, certainly we need not only a powerful solar energy transition system but also a strong nitrogen  fixation system to improve the efficiency of our whole-cell photocatalytic  nitrogen fixation system. According to the above requirements, we choose a  different nif gene cluster from <em>Paenibacillus polymyxa</em> CR1 to test its  expression level compared with the BBa_K1796015 from <em>Paenibacillus sp.</em> WLY78.</p>
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1b）Fluorescence intensity detemination]] </p>
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<p>&nbsp;</p>
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<p> [[File:T--Nanjing-China--qRT-PCR.jpg|800px|thumb|center|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.]]</p>
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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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[[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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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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  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--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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Latest revision as of 10:45, 16 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 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.