View source for Part:BBa K1796015


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<partinfo>BBa_K1796015 short</partinfo>

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

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===Usage and Biology===

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K1796015 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K1796015 parameters</partinfo>
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<h2>iGEM2018_Nanjing China Experiment</h2>
<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>
<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>
<p>[[File:T--Nanjing-China--1%2B2.jpg|800px|thumb|center|Figure 1a）Engineered E. coli cells with nitrogenase<br />
1b）Fluorescence intensity detemination]] </p>
 <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>