Difference between revisions of "Team:Nanjing-China/Improve"

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     <div style=" position:absolute; top:-55px;left:-10px; z-index:3;"><img src="https://static.igem.org/mediawiki/2018/f/fb/T--Nanjing-China--part-i.png" width="45%"; ></div>
 
     <div style=" position:absolute; top:-55px;left:-10px; z-index:3;"><img src="https://static.igem.org/mediawiki/2018/f/fb/T--Nanjing-China--part-i.png" width="45%"; ></div>
 
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    <p>The part number for the existing part we are improving in the box below:<a href="http://parts.igem.org/Part:BBa_K1796007"><strong>BBa_K1796007</strong></a> </p>
 
<p>The part number of our new part in the box below:
 
<a href="http://parts.igem.org/Part:BBa_K2740012 "><strong>BBa_K2740012 </strong></a>
 
 
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<p>Based on the existing part, BBa_K1796007, which is an essential component from <em>Paenibacillus sp.</em> WLY78&rsquo;s nitrogen fixation gene cluster: <em>nif</em> Promoter,<em> nifB, nifH, nifD, nifK, nifE, nifN, nifX,  hesA, nifV,</em> We choose a new nitrogen fixation gene cluster from a more common  strain <em>Paenibacillus polymyxa</em> CR1 and make some improvements, to  comprise the nitrogen fixation system in our project. </p>
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<p>he existing part, <a href="http://parts.igem.org/Part:BBa_K1796007">BBa_K1796007</a>, is an essential component of the <em>Paenibacillus sp.</em> WLY78&rsquo;s nitrogen fixation gene (<em>nif</em>)  cluster arranged in the order of <em>nif</em>B, <em>nif</em>H, <em>nif</em>D, <em>nif</em>K, <em>nif</em>E, <em>nif</em>N, <em>nif</em>X, <em>hes</em>A, <em>nif</em>V. Instead of directly cloning WLY78 <em>nif</em>B using BBa_K1796007 as the template, a minimal <em>nif</em> cluster from <em>Paenibacillus  polymyxa</em> CR1 that also contained <em>nif</em>B  (nucleoid acid sequence similarity 96% as compared to WLY78 <em>nif</em>B) was chemically synthesized and  incorporated into commercially available cloning vector pUC57. After that, CR1 <em>nif</em>B was obtained by PCR amplification using pUC57-<em>nif</em> as the template and subsequently introduced into pSB1C3 backbone through restriction enzyme digestion. Below, we discuss why we made such an improvement:<br />
<p>Firstly, Because of existence of the  illegal <em>PstI</em> sites and <em>EcoRI</em> sites, the original gene sequence from <em>Paenibacillus  polymyxa</em> CR1 and the existing part, BBa_K1796007 is not RFC10 compatible, which is not convenient for us and other teams to use this part. So to make the  part easier to operate, we make some synonymous mutations to reform the gene sequence and chemically synthesize the entire nitrogen fixation gene cluster,  then we can PCR then isolated gene gene or basic part like <em>nifB</em> to get them. The new part is RFC10 compatible which ensures a greater diversity when designing synthetic biology projects.</p>
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(1) Both <em>nif</em>B genes from WLY78  and CR1 contain unwanted restriction sites that can not meet the compatibility  requirements of the iGEM Parts Guidelines. Therefore, elimination of these site  through chemical synthesis is necessary.<br />
<p>Secondly, in our this year’s project, we intends to establish a sound and ideal whole-cell photocatalytic nitrogen fixation system. And we use the engineered E. coli cells to express nitrogenases(Fig 1) and in-situ synthesize of CdS semiconductors in the biohybrid system. Instead of ATP-hydrolysis, such system is able to photocatalytic N<sub>2</sub>(nitrogen) to NH<sub>3</sub>(ammonia). So certainly we need to test the nitrogen fixation’s heterologous expression level in <em>E.coli</em> to make sure the efficiency of photocatalytic nitrogen fixation.</p>
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(2) The complete genome of <em>Paenibacillus polymyxa</em> CR1 has  been thoroughly sequenced and deposited in NCBI with the accession number  CP006941.2. Considering that there exist some other genes possessing regulatory  function for the <em>nif</em> cluster, <em>nif</em>B of a bacterium with clear genetic  background, such as<em> Paenibacillus polymyxa</em> CR1, may be more valuable for  researchers of relevant field. </p>
<div class="word-note"><img src="https://static.igem.org/mediawiki/parts/b/b5/T--Nanjing-China--011part-design.png" width="70%"  />
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    <p>In addition, to test whether the <em>nif</em>B  could express in gram-negative <em>E. coli</em> JM109 as a part of the <em>nif</em> cluster,  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><font size="-1">Fig 1. Design of our project: Engineered <em>E. coli</em> cells with nitrogenase</font></p></div>
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<div class="word-note" align="center"><img src="https://static.igem.org/mediawiki/2018/b/b6/T--Nanjing-China--1%2B2.jpg" width="55%" >
<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><font size="-1">Figure 1a)Engineered E. coli cells with nitrogenase<br />
  <div class="word-note"><img src="https://static.igem.org/mediawiki/parts/f/f9/T--Nanjing-China--11part.png" width="70%" >
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1b)Fluorescence intensity detemination
   <p><font size="-1">Fig 2:Expression efficiency of Pnif</font></p></div>
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</font></p></div>
<p>Comparison of the expression efficiency of  Pnif and T5 (IPTG Inducible) Promoter. <br />
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    </div>
T5 (IPTG Inducible) Promoter BBa_M50075;  Pnif: nif promoter BBa_K1796001.</p>
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<div class="word-note" align="center"><img src="https://static.igem.org/mediawiki/2018/4/47/T--Nanjing-China--qRT-PCR.png" width="55%" >
<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>.</p>
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  <p><font size="-1">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.</font></p></div>
  <div class="word-note" align="center"><img src="https://static.igem.org/mediawiki/2018/4/47/T--Nanjing-China--qRT-PCR.png" width="55%" >
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   <p>Hopefully, the aforementioned improvements and relevant testing results can facilitate the utilization of the improved part for other iGEM team.   </p>
    <p><font size="-1">Fig 3. The qPCR results for components of nitrogen fixation system</font></p></div>
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  <p align="left">From the results of qPCR we have known that not only the nitrogen gene cluster can  successfully heterologously expressed in the engineered <em>E. coli </em>and but  also the relative transcriptional level of each component of nitrogen gene  cluster is different. Based on these analysis, our team created a mathematical  model to optimize the arrangement of the <em>nif</em> gene cluster. This model helped we  optimized our design and provided some new perspectives of our  nitrogen-fixation system in transcriptional level. And you can see the detailed  model by clicking the following link.<br />
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    <a href="https://2018.igem.org/Team:Nanjing-China/Model">https://2018.igem.org/Team:Nanjing-China/Model</a> </p>
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   <p align="left">The  improvements above have facilitate our team to accomplish our project and we  sincerely wish it can help other use the gene cluster. </p>
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Revision as of 11:39, 16 October 2018

Nanjing-China2018

he existing part, BBa_K1796007, is an essential component of the Paenibacillus sp. WLY78’s nitrogen fixation gene (nif) cluster arranged in the order of nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV. Instead of directly cloning WLY78 nifB using BBa_K1796007 as the template, a minimal nif cluster from Paenibacillus polymyxa CR1 that also contained nifB (nucleoid acid sequence similarity 96% as compared to WLY78 nifB) was chemically synthesized and incorporated into commercially available cloning vector pUC57. After that, CR1 nifB was obtained by PCR amplification using pUC57-nif as the template and subsequently introduced into pSB1C3 backbone through restriction enzyme digestion. Below, we discuss why we made such an improvement:
(1) Both nifB genes from WLY78 and CR1 contain unwanted restriction sites that can not meet the compatibility requirements of the iGEM Parts Guidelines. Therefore, elimination of these site through chemical synthesis is necessary.
(2) The complete genome of Paenibacillus polymyxa CR1 has been thoroughly sequenced and deposited in NCBI with the accession number CP006941.2. Considering that there exist some other genes possessing regulatory function for the nif cluster, nifB of a bacterium with clear genetic background, such as Paenibacillus polymyxa CR1, may be more valuable for researchers of relevant field.

In addition, to test whether the nifB could express in gram-negative E. coli JM109 as a part of the nif cluster, 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.

Hopefully, the aforementioned improvements and relevant testing results can facilitate the utilization of the improved part for other iGEM team.