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

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         <div class="word" id="journal"  align="center">
 
         <div class="word" id="journal"  align="center">
<div class="bottom" onclick="MM_effectBlind('may', 1000, '0%', '100%', true);" align="center">MAY</div>
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<div class="bottom" onclick="MM_effectBlind('March', 1000, '0%', '100%', true);" align="center">MARCH</div>
 
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<div class="word-1" id="may" style="display:none; overflow:hidden;">
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<div class="word-1" id="March" style="display:none; overflow:hidden;">
 
<div class="bottom-2">1</div>
 
<div class="bottom-2">1</div>
<div class="word-2" style=" border-right:2px #999999 solid;"><div style=" padding:20px;"><p>asdjfha ebfaiup bwfjbsan ;fkjabs jdkf aj;sbdf jnajb fwnlfna;j asdfabf iubwfiub ajsfbsjadaf njaj enfjnas;jfndjs nfj;nfajsn fja;fnwjf ndnf;</p></div></div>
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<div class="word-1" ><div style=" padding:20px;"><p>Our team was founded this week! We met and communicated with each other. Through our team leader’s presentation, we knew IGEM a lot. Each member defined his/her mission in the team.
<div class="word-2" style=" border-left:2px #999999 solid; width:49%;"><div style=" padding:20px;"><p>asdjfha ebfaiup bwfjbsan ;fkjabs jdkf aj;sbdf jnajb fwnlfna;j asdfabf iubwfiub ajsfbsjadaf njaj enfjnas;jfndjs nfj;nfajsn fja;fnwjf ndnf;</p></div></div>
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We broadly read literature and brainstormed this year’s project. At first we came up with several different ideas, but later we reached an agreement that the most interesting and meaningful one was about nitrogen fixation.
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<div class="bottom" onclick="MM_effectBlind('April', 1000, '0%', '100%', true);" align="center">April</div>
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<div class="word-1" id="April: " style="display:none; overflow:hidden;">
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<div class="bottom-2">1</div>
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<div class="word-2" style=" border-right:2px #999999 solid;"><div style=" padding:20px;">
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  <p><strong>Human Practice &amp;Society:</strong><br />
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    We learned about nitrogenous fertilizer  production status in quo and the advancement of China&rsquo;s nitrogenous fertilizer  industry. In order to better understand the actual demand of nitrogenous  fertilizer in agriculture, we decided to visit farmers in Xiaohe Bei Village.</p>
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Having learned of the dearth of efficient and  affordable fertilizer, we spared no effort to seek a cost effective nitrogen  fixation method. Inspired by our previous work(Nanjing-China 2016), we  creatively proposed an idea of &ldquo;whole-cell  photocatalytic nitrogen fixation&rdquo;</div></div>
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<div class="word-2" style=" border-left:2px #999999 solid; width:49%;"><div style=" padding:20px;">
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  <p><strong>Technical works Wet&amp;Dry labs:</strong> <br />
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    Wet Lab:Having confirmed the theme of our  project, we began to work on our design. We read latest papers about biological  nitrogen fixation, focused on the method sections and discussed what we didn&rsquo;t  understand in details. During the last week of this month, we worked out the  first version of our design.<br />
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    Dry lab:We communicated and exchanged ideas  frequently in order to identify possible modeling directions which could  provide useful guidance to our wet experiments. Later we proposed a few  directions. The idea of developing homologous modeling of nitrogenase didn&rsquo;t  work successfully because we couldn&rsquo;t get access to relevant software.</p>
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</div></div>
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</div><div class="bottom" onclick="MM_effectBlind('May', 1000, '0%', '100%', true);" align="center">MAY</div>
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<div class="word-1" id="May" style="display:none; overflow:hidden;">
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<div class="bottom-2">1</div>
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  <p><strong>Human  Practice&amp;Society:</strong><br />
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    We planned to investigate the current  production of nitrogenous fertilizer so we prepared interview questions and  contacted Yantai Wuzhou Feng Fertilizer Plant. Then we went there, met the  manager and were shown around the factories. We communicated with the technical  R&amp;D personnel and realized the big challenge we had to overcome before  putting our project into practical application.</p>
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</div></div>
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<div class="word-2" style=" border-left:2px #999999 solid; width:49%;"><div style=" padding:20px;">
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  <p><strong>Technical  works Wet&amp;Dry Labs:</strong></p>
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  <p>Wet lab:We measured the transcriptional  activity of nif promoter and constructed the complete line of nif cluster,  BBa_K1796015. Then we transformed the plasmid Pcb1C3 containing the nif cluster  and the fusion protein  expression plasmid including <em>E. coli</em> outer membrane protein OmpA and the  PbrR protein into <em>E. coli</em> strain pUC57. Besides,based  on our Human Practice, we modified our design by adding Cd2+  toxicity test to it.</p>
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</div></div>
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</div>
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<div class="bottom" onclick="MM_effectBlind('March', 1000, '0%', '100%', true);" align="center">MARCH</div>
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<div class="word-background-block" style=" height:10px;"></div>
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<div class="word-1" id="March" style="display:none; overflow:hidden;">
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<div class="bottom-2">1</div>
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<div class="word-1" >
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  <div style=" padding:20px;">
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    <p><strong>Exam Break</strong></p>
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  </div></div>
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<div class="bottom" onclick="MM_effectBlind('July', 1000, '0%', '100%', true);" align="center">July</div>
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<div class="word-1" id="July" style="display:none; overflow:hidden;">
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<div class="bottom-2">1</div>
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  <p><strong>Technical  works Wet&amp;Dry Labs:</strong><br />
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    Wet  lab:We finished the last step in construction of whole-cell  photocatalytic nitrogen fixation system----biosynthesis of CdS semiconductor. Then we performed gas chromatography to detect the amount of  acetylene reduced to indirectly test the nitrogen fixation activity of our  system. <br />
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    Meanwhile,we conducted Real-time  Quantitative PCR(QPCR).<br />
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    Dry lab:Enlightened by the different  relative transcriptional level of each nitrogenase component which was shown in  the result of QPCR, we turned our attention to the  extreme complexity of nitrogenase system. We perused literature on the stoichiometry  of nitrogenase components.</p>
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</div></div>
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<div class="word-2" style=" border-left:2px #999999 solid; width:49%;"><div style=" padding:20px;">
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  <p><strong>Human practice&amp;Society:</strong><br />
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    We invited Professor Haoqian Zhang and held  a meet up with iGEM teams in Nanjing. <br />
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    We were interviewed by Nanjing University  Student Career Guidance Center. The Wechat Push introducing our team was issued  on the public account &ldquo;NJU Employment&rdquo;. </p>
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</div></div>
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</div>
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<div class="bottom" onclick="MM_effectBlind('August', 1000, '0%', '100%', true);" align="center">August</div>
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<div class="word-background-block" style=" height:10px;"></div>
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<div class="word-1" id="August" style="display:none; overflow:hidden;">
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<div class="bottom-2">1</div>
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<div class="word-2" style=" border-right:2px #999999 solid;"><div style=" padding:20px;">
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  <p><strong>Technical  works Wet&amp;Dry Labs:</strong><br />
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    Wet  lab:We conducted ICP-MS measurement of Cd2+ adsorption, Cd2+  toxicity test and TEM-EDX analysis. In addition,we  improved our part to make it easier to operate.<strong> </strong><br />
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    Dry lab:We finally decided to model on the  best stoichiometry of nif gene cluster. We looked through many common algorithm  and figured out two modeling methods. After further comparation, we finally  chose a method similar to greedy algorithm. We drew a flow diagram to describe  the core idea of our method and as a reference for programming. Then we  programmed with python, debugged our code and received the result.</p>
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</div></div>
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<div class="word-2" style=" border-left:2px #999999 solid; width:49%;"><div style=" padding:20px;">
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  <p><strong>Human practice&amp;Society:</strong><br />
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    We distributed brochures about our project at  NJU.<br />
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    We performed Language project with IIT  Madras and received the finished video a few days later. We attended the 5th  Conference of China iGEMer Community at ShanghaiTech University to  demonstrate our project to all teams in China and learn from others.</p>
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</div></div>
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</div>
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<div class="bottom" onclick="MM_effectBlind('September', 1000, '0%', '100%', true);" align="center">September</div>
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<div class="word-background-block" style=" height:10px;"></div>
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<div class="word-1" id="September" style="display:none; overflow:hidden;">
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<div class="bottom-2">1</div>
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  <p><strong>Technical  works Wet&amp;Dry Labs:</strong><br />
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    Hardware:  Inspired by our Human Practice, we formed an idea of designing a device for the  growth of engineered E.coli strain<em>. </em>First we drew a draft on paper and  then used the software solidworks to draw a 3D version draft. Eventually, a  real device came out. The device provided a great help to our further  experiments.</p>
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Dry lab:We refined our model</div></div>
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<div class="word-2" style=" border-left:2px #999999 solid; width:49%;"><div style=" padding:20px;">
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  <p><strong>Human practice&amp;Society:</strong><br />
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    We helped another IGEM team, CSU-China to  establish their team. We issued our Emoji chanllenge on the  official website and received some interesting feedback shortly after that.</p>
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</div></div>
 
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Revision as of 14:43, 13 October 2018

Nanjing-China2018

MARCH
April
MAY
MARCH
July
August
September

Protocol

ICP-MS(Inductively Coupled Plasma Mass Spectrometry) measurement of Cd2+ adsorption

Escherichia coli BL21 containing OmpA-PbrR-PJQ200SK (pBAD33) plasmid was cultured in LB medium to an OD600 of 0.4-0.6. Arabinose and CdCl2 were added to the medium to a final arabinose concentration of 40 μM and a final Cd2+ concentration of 100 μM, to induce the formation of CdS nano semiconductors.
From the start of the induction, 5 ml of the bacterial solution was taken from the culture every 6 hours (sampling to 24 hours), centrifuged at 4000 rpm for 2 minutes, and washed three times with water to remove the medium involved in the bacterial surface.
The washed bacteria were resuspended in 5 ml of water. OD600 was measured, and the bacteria were collected by centrifugation.
3 ml of concentrated nitric acid was added and the mixture was digested overnight at 90 °C.
The Cd2+ content in the sample was measured using ICP-MS.

Cd2+ toxicity test

Multiple groups of LB medium were prepared, and arabinose with a final concentration of 40 μM and different amounts of CdCl2 were added to the medium to form a Cd2+ gradient of 0,150 μM, 300 μM, 600 μM, and 1000 μM.
E. coli BL21 containing the OmpA-PbrR-PJQ200SK (pBAD33) plasmid and plasmid-free E. coli BL21 (control) were cultured in different media.
The OD600 value was measured every 2 hours and measured for 12 hours.

Transmission electron microscopy with energy-dispersive x-ray spectroscopy (TEM-EDX)

After the Cd2+ adsorption induction was completed, the bacteria were collected by centrifugation and resuspended in ultrapure water. Samples were sent for TEM image acquisition.
The thick carbon film (20 to 30 nm) on the copper grid was immersed in the bacteria solution for 1 second before imaging, dried under atmospheric conditions, and then imaged using TEM. At the same time, the EDX system (EDAX, AMETEK) was attached to the microscope for elemental analysis. All TEM images were imaged using a JEOL JEM-2100 electron microscope at an acceleration bias of 200 kV.

Reference

  1. Kathryn RF,Yanning Z,et.al.(2016)Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium PNAS
  2. Wang L,Zhang L,Liu Z,Zhao D,Liu X et.al(2013) A minimal Nitrogen Expression of Active Nitrogenase in Escherichia coli PLOS Genetics9(10):e1003865
  3. Katherine AB,Derek FH,Molly BW et.al(2016) Light-driven nitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid Science352,448
  4. Wei W,Sun PQ,Li Z,Song KS,Su WY,Wang B,Liu YZ,Zhao J et.al (2018) A surface display biohybrid approach to light-driven hydrogen production in air Science eaap9253
  5. Wei W,Zhu T,Wang Y et.al(2012) Engineering a gold-specific regulon for cell-based visual detection and recovery of gold Chem.Sci,3,1780-1784
  6. James BH&Douglas CR(1996) Structural Basis of Biological Nitrogen Fixation Chem.Rev.96,2965-2982