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

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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 />
 
(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 />
 
(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>
 
(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>
     <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>
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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) served 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>
 
<div class="word-note" align="center"><img src="https://static.igem.org/mediawiki/2018/b/b6/T--Nanjing-China--1%2B2.jpg" width="95%" >
 
<div class="word-note" align="center"><img src="https://static.igem.org/mediawiki/2018/b/b6/T--Nanjing-China--1%2B2.jpg" width="95%" >
 
   <p><font size="-1">Figure 1a)Engineered E. coli cells with nitrogenase<br />
 
   <p><font size="-1">Figure 1a)Engineered E. coli cells with nitrogenase<br />
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     </div>
 
     </div>
 
<div class="word-note" align="center"><img src="https://static.igem.org/mediawiki/2018/4/47/T--Nanjing-China--qRT-PCR.png" width="60%" >
 
<div class="word-note" align="center"><img src="https://static.igem.org/mediawiki/2018/4/47/T--Nanjing-China--qRT-PCR.png" width="60%" >
   <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>
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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) serves as control and relative expression compared to the housekeeping gene 16S rRNA is shown. N.D. represent not ditected.</font></p></div>
 
   <p>Hopefully, the aforementioned improvements and relevant testing results can facilitate the utilization of the improved part for other iGEM team.  </p>
 
   <p>Hopefully, the aforementioned improvements and relevant testing results can facilitate the utilization of the improved part for other iGEM team.  </p>
 
     </div>
 
     </div>

Revision as of 07:34, 17 October 2018

Nanjing-China2018

The 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) served 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) serves 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.