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| | <title>Nanjing-China2018</title> | | <title>Nanjing-China2018</title> |
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| | <div id="HOME"> | | <div id="HOME"> |
| | <div class="sub"> | | <div class="sub"> |
| − | <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Notebook">Notebook</a></ul></li></div> | + | <ul><li><a href="https://2018.igem.org/Team:Nanjing-China/Design">Design</a></li></ul></div> |
| − | | + | |
| − | <li><a href="# journal"> Journal</a></li> | + | <li><a href="# cds"> CdS</a></li> |
| − | <li><a href="# protocol"> Protocol</a></li> | + | <li><a href="# nitrogen"> <font size="-1">Nitrogen fixation</font></a></li> |
| − | <li><a href="# reference"> Reference</a></li> | + | <li><a href="# device"> Device</a></li> |
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| − | <li><a href="https://2018.igem.org/Team:Nanjing-China">N<font size="-1"><sub>2</sub></font> CHASER</a> | + | <li><a href="https://2018.igem.org/Team:Nanjing-China">N<font size="-1"><sub>2</sub></font>CHASER</a> |
| | <ul> | | <ul> |
| | <li><a href="https://2018.igem.org/Team:Nanjing-China/Team">Team</a></li> | | <li><a href="https://2018.igem.org/Team:Nanjing-China/Team">Team</a></li> |
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| | </ul> | | </ul> |
| | </div> | | </div> |
| − | <div class="header"><img src="https://static.igem.org/mediawiki/2018/b/bf/T--Nanjing-China--title-NOTEBOOK.png" width="100%" onload="MM_effectAppearFade(this, 1000, 0, 100, false);MM_effectBlind('HOME', 1000, '0%', '100%', true)" > | + | <div class="header"><img src="https://static.igem.org/mediawiki/2018/4/40/T--Nanjing-China--title-4.png" width="100%" onload="MM_effectAppearFade(this, 1000, 0, 100, false);MM_effectBlind('HOME', 1000, '0%', '100%', true)"> |
| | </div> | | </div> |
| − | <div class="contain" > | + | <div class="contain"> |
| − | <div class="word " id="journal" align="center"> | + | |
| − | | + | <div style=" position:absolute; top:-90px; z-index:3; left:-10px;"> |
| − | <div class="bottom" onclick="MM_effectBlind('March', 1000, '0%', '100%', true)">March</div></div> | + | <img src="https://static.igem.org/mediawiki/2018/8/8a/T--Nanjing-China--PROJECT-d.jpg" width="50%" /></div> |
| − | <div class="word-background-block" style=" height:10px;"></div> | + | <div class="word- 1" style="height: 20px;"></div> |
| − | <div class="word-1" id="March" style="display:none;"> | + | </div> |
| − | <div class="bottom-2">3</div> | + | <div class="word" id=" overview"> |
| − | <div class="word-1" style="border-top:rgba(153,153,153,0.6) 4px dotted;" ><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 was assigned his/her mission in the team.</p> | + | <h2>Overview</h2> |
| − | <p>We broadly read paper and brainstormed this year’s project. At first we came up with several different ideas, later we reached an agreement that the most interesting and meaningful one was about nitrogen fixation. | + | <p>Our design is composed of three parts: biosynthesis of CdS semiconductor, light-driven nitrogen fixation and a light-driven biohybrid reaction device. This system is the expansion of our previous project of hydrogen production (Nanjing-China 2016), and it proves that surface display mechanism is capable of being expanded to a general principle for light-driven biohybrid reactions.</p> |
| − | </p></div> | + | </div> |
| − | </div></div> | + | <div class="word" id="cds"> |
| − | <div class="word-background-block" style=" height:10px;"></div> | + | <h2>Biosynthesis of CdS semiconductor on cell surface</h2> |
| − | | + | <p align="left">To construct our light-driven system, we induce the in situ synthesis of CdS semiconductor on the <em>E. coli</em> cell surface. One key element of our system is fused protein OmpA-PbrR. OmpA (Outer membrane protein A) fix the protein complex on outer cell membrane while PbrR (lead-specific binding protein) adsorb Cd<sup>2+</sup> in the environment and further form CdS semiconductor on cell surface.</p> |
| − | <div class="bottom" onclick="MM_effectBlind('April', 1000, '0%', '100%', true);">April</div></div>
| + | <p> After Cd<sup>2+</sup> ions are added into the culture, the ions specifically bind to PbrR protein contributing to the aggregation of Cd<sup>2+</sup>. Combining with S<sup>2-</sup> ions in the media, CdS semiconductors are therefore formed on the outer membrane of cells.</p></div> |
| − | <div class="word-background-block" style=" height:10px;"></div> | + | <div class="word"> |
| − | <div class="word-1" id="April" style="display:none; overflow:hidden;"> | + | <div id="design2" style="left:10%; width:80%;"> |
| − | <div class="bottom-2">4</div> | + | <div id="Layer11" style="visibility: hidden"><img src="https://static.igem.org/mediawiki/2018/2/27/T--Nanjing-China--d-1-8.png" width="150" /></div> |
| − | <div class="word-2" style=" border-right:3px #999999 solid;"><div style=" padding:20px;"> | + | <div id="Layer12" style="height: 61px; visibility: hidden; left: 26px; top: 8px;"><img src="https://static.igem.org/mediawiki/2018/8/85/T--Nanjing-China--d-1-7.png" width="60" /></div> |
| − | <h4>Human Practices, Collaboration&Society:</h4>
| + | <div id="Layer13" style="visibility: hidden; left: 103px; top: 7px;"><img src="https://static.igem.org/mediawiki/2018/a/a7/T--Nanjing-China--d-2-2.png" width="100" /></div> |
| − | <p>We learned about the advancement of nitrogenous fertilizer production in China. In order to better understand the actual demand of nitrogenous fertilizer in agriculture, we decided to visit farmers in Xiaohe Bei Village.<br />
| + | <div id="Layer14" style="visibility: hidden"><img src="https://static.igem.org/mediawiki/2018/0/0d/T--Nanjing-China--d-2-1.png" width="200" /></div> |
| − | We distributed brochures about our project at NJU.<br/>
| + | <div id="Layer15" style="visibility: hidden; left: 247px; top: 5px;"><img src="https://static.igem.org/mediawiki/2018/1/1d/T--Nanjing-China--d-2-3.png" width="130" /></div> |
| − | 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 “whole-cell photocatalytic nitrogen fixation”.<br />
| + | <div id="Layer16" style="visibility: hidden"><img src="https://static.igem.org/mediawiki/2018/2/26/T--Nanjing-China--d-2-4.png" name="Image1" width="202" /></div> |
| − | We helped Nanjing Forestry University build their team.<br />
| + | <div id="Layer17" style="visibility: hidden; left: 245px; top: 196px;"><img src="https://static.igem.org/mediawiki/2018/0/03/T--Nanjing-China--d-2-5.png" width="130" /></div> |
| − | We held conferences with Nanjing Agricultural University and China Pharmaceutical University to share experiences of being iGEMers.
| + | <div id="Layer18" style="visibility: hidden"><img src="https://static.igem.org/mediawiki/2018/8/8a/T--Nanjing-China--d-2-6.png" width="200" /></div> |
| − | </p>
| + | <div id="Layer-all2" style="visibility: visible"><img src="https://static.igem.org/mediawiki/2018/2/29/T--Nanjing-China--d-2-all.png" width="500" /></div> |
| − | </div></div>
| + | <div id="b"> |
| − | <div class="word-2" style=" border-left:0px #999999 solid; width:49%;"><div style=" padding:20px;"> | + | <div class="play" class="button" onclick="MM_timelinePlay('Timeline2')">play</div> |
| − | <h4>Technical works Wet& Dry labs:</h4>
| + | <div class="stop" class="button" onclick="MM_timelineStop('Timeline2')">stop</div></div> |
| − | <p>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’t understand in details. During the last week of this month, we worked out the first version of our design.</p>
| + | |
| − | <p>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’t work successfully because we couldn’t get access to relevant software. | + | |
| − | </p> | + | |
| − | </div></div> | + | |
| | </div> | | </div> |
| − | <div class="word-background-block" style=" height:10px;"></div>
| |
| − | <div class="month_w"><div class="bottom" onclick="MM_effectBlind('May', 1000, '0%', '100%', true);">May</div>
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| − | <div class="word-background-block" style=" height:10px;"></div>
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| − | <div class="word-1" id="May" style="display:none; overflow:hidden;">
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| − | <div class="bottom-2">5</div>
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| − | <div class="word-2" style=" border-right:0px #999999 solid;"><div style=" padding:20px;">
| |
| − | <h4>Human Practices, Collaboration& Society:</h4>
| |
| − | <p>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&D personnel and realized the big challenge we had to overcome before putting our project into practical application.</p>
| |
| − | </div></div>
| |
| − | <div class="word-2" style=" border-left:3px #999999 solid; width:49%;"><div style=" padding:20px;">
| |
| − | <h4>Technical works Wet& Dry labs:</h4>
| |
| − | <p>Wet lab: We measured the transcriptional activity of <em>nif</em> promoter. Then we transformed the plasmid pUC57 containing the <em>nif</em> cluster and the fusion protein expression plasmid including <em>E. coli</em> outer membrane protein <em>OmpA</em> and the <em>PbrR</em> protein into <em>E. coli</em> strain JM109. Besides, based on our Human Practice, we modified our design by adding Cd<sup>2+</sup> toxicity test to it.</p>
| |
| − | </div></div>
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| − | </div></div>
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| − | <div class="word-background-block" style=" height:10px;"></div>
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| − | <div class="month_w">
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| − | <div class="bottom" onclick="MM_effectBlind('June', 1000, '0%', '100%', true);" align="center">June</div>
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| − | <div class="word-background-block" style=" height:10px;"></div>
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| − | <div class="word-1" id="June" style="display:none; overflow:hidden;">
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| − | <div class="bottom-2">6</div>
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| − | <div class="word-1" style="border-top:rgba(153,153,153,0.6) 4px dotted;" >
| |
| − | <div style=" padding:20px;" align="center">
| |
| − | <h4>Exam Break</h4>
| |
| − | </div></div>
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| − | </div></div>
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| − | <div class="word-background-block" style=" height:10px;"></div>
| |
| − | <div class="month_w">
| |
| − | <div class="bottom" onclick="MM_effectBlind('July', 1000, '0%', '100%', true);" align="center">July</div>
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| − | <div class="word-background-block" style=" height:10px;"></div>
| |
| − | <div class="word-1" id="July" style="display:none; overflow:hidden;">
| |
| − | <div class="bottom-2">7</div>
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| − | <div class="word-2" style=" border-right:0px #999999 solid;">
| |
| − | <div style=" padding:20px;">
| |
| − | <h4>Human Practices, Collaboration& Society:</h4>
| |
| − | <p>We invited Professor Haoqian Zhang and held a meet up with iGEM teams in Nanjing. <br />
| |
| − | We were interviewed by Nanjing University Student Career Guidance Center. The Wechat Push introducing our team was issued on the public account “ NJU Employment”.<br />
| |
| − | We borrowed the No.5 plasmid in the InterLab kit to team AHUT.</p>
| |
| − | </div></div>
| |
| − | <div class="word-2" style=" border-left:3px #999999 solid; width:49%;"><div style=" padding:20px;">
| |
| − | <h4>Technical works Wet& Dry labs:</h4>
| |
| − | <p>Wet lab: We conducted Real-time Quantitative PCR(qPCR). Meanwhile, we conducted Cd<sup>2+</sup> toxicity test and ICP-MS measurement of Cd<sup>2+</sup> adsorption. <br/>
| |
| − | We improved our part to make it easier to operate.</p>
| |
| − | <p> Dry lab: Enlightened by the different relative transcriptional levels of each nitrogenase component which was shown in the result of qPCR, we turned our attention to the complexity of nitrogenase system. We perused literature on the stoichiometry of nitrogenase components.</p>
| |
| − | </div></div>
| |
| | </div> | | </div> |
| | + | <div class="word" id="nitrogen"> |
| | + | <h2>Light-driven nitrogen fixation in <em>E. coli</em> cells</h2> |
| | + | <p>When the system is exposed in light, electrons of the CdS semiconductor conduct transit, and CdS provide these excited electrons to the electrons to Mo-Fe protein subunit of nitrogenase. Subsequently, the Mo-Fe protein utilizes the energy from these electrons to reduce N<sub>2</sub>(dinitrogen) to NH<sub>3</sub>(ammonia). Finally, the semiconductor regains its lost electron from sacrificial electron donors.</p> |
| | + | <p>This design is of general applications as OmpA protein is merely a surface display mechanism for <em>E. coli</em>, and PbrR can be replaced with other proteins with different specificity. </p> |
| | </div> | | </div> |
| − | <div class="word-background-block" style=" height:10px;"></div> | + | <div class="word"> |
| − | | + | <div id="design3" style=" left:10%; width:80%;"> |
| − | <div class="bottom" onclick="MM_effectBlind('August', 1000, '0%', '100%', true);" align="center">August</div>
| + | <div id="Layer21" style="visibility: hidden; left: 0px;"><img src="https://static.igem.org/mediawiki/2018/c/c4/T--Nanjing-China--d-3-8.png" width="240" /></div> |
| − | <div class="word-background-block" style=" height:10px;"></div> | + | <div id="Layer22" style="visibility: hidden; top: 19px; left: 30px;"><img src="https://static.igem.org/mediawiki/2018/9/9e/T--Nanjing-China--d-3-7.png" width="170" /></div> |
| − | <div class="word-1" id="August" style="display:none; overflow:hidden;"> | + | <div id="Layer23" style="visibility: hidden"><img src="https://static.igem.org/mediawiki/2018/2/21/T--Nanjing-China--d-3-1.png" width="250" /></div> |
| − | <div class="bottom-2">8</div> | + | <div id="Layer24" style="visibility: hidden; left: 314px; top: 14px;"><img src="https://static.igem.org/mediawiki/2018/f/fd/T--Nanjing-China--d-3-2.png" width="60" /></div> |
| − | <div class="word-2" style=" border-right:0px #999999 solid;"><div style=" padding:20px;"> | + | <div id="Layer25" style="visibility: hidden"><img src="https://static.igem.org/mediawiki/2018/a/a8/T--Nanjing-China--d-3-3.png" width="260" /></div> |
| − | <h4>Human Practices, Collaboration& Society:</h4>
| + | <div id="Layer26" style="visibility: hidden"><img src="https://static.igem.org/mediawiki/2018/9/9d/T--Nanjing-China--d-3-4.png" width="211" /></div> |
| − | <p>We attended the 5th Conference of China iGEMer Community at Shanghai Tech University to demonstrate our project to all teams in China and learn from each other.<br />
| + | <div id="Layer27" style="visibility: hidden; left: 370px; top: 97px; height: 103px;"><img src="https://static.igem.org/mediawiki/2018/2/28/T--Nanjing-China--d-3-6.png" width="100" /></div> |
| − | We helped Central South University found team.<br />
| + | <div id="Layer28" style="visibility: hidden"><img src="https://static.igem.org/mediawiki/2018/1/1d/T--Nanjing-China--d-3-5.png" width="150" /></div> |
| − | We communicated with two numbers of iGEM USTC.</p>
| + | <div id="b2"> |
| − | </div></div>
| + | <div class="play" class="button" onclick="MM_timelinePlay('Timeline3')">play</div> |
| − | <div class="word-2" style=" border-left:3px #999999 solid; width:49%;"><div style=" padding:20px;"> | + | <div class="stop" class="button" onclick="MM_timelineStop('Timeline3')">stop</div></div> |
| − | <h4>Technical works Wet& Dry labs:</h4>
| + | |
| − | <p>Wet lab: We conducted TEM-EDX analysis and UV-vis scanning. Then we used methyl viologen to verify the generation of electron. Finally, we successfully biosynthesized CdS semiconductor which could be excited by visible light to generate electrons.</p>
| + | |
| − | <p>Dry lab: We finally decided to model on the best stoichiometry of <em>nif</em> gene cluster. We looked through many common algorithms and figured out two modeling methods. After further comparison, 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>
| + | |
| − | </div></div> | + | |
| | </div> | | </div> |
| | </div> | | </div> |
| − | <div class="word-background-block" style=" height:10px;"></div> | + | <div class="word" id="device"> |
| − | <div class="month_w">
| + | <h2>Reaction device</h2> |
| − | <div class="bottom" onclick="MM_effectBlind('September', 1000, '0%', '100%', true);" align="center">September</div>
| + | <p>We also designed a light- driven biohybrid reaction device to apply our system to practical use. After a few test, we proved our device to be quite practical. <a href=" https://2018. igem. org/ Team: Nanjing- China/ Hardware">( see hardware for more details)</ a> </p></div> |
| − | <div class="word-background-block" style=" height:10px;"></div>
| + | |
| − | <div class="word-1" id="September" style="display:none; overflow:hidden;">
| + | |
| − | <div class="bottom-2">9</div> | + | |
| − | <div class="word-2" style=" border-right:0px #999999 solid;"><div style=" padding:20px;">
| + | |
| − | <h4>Human Practices, Collaboration& Society:</h4>
| + | |
| − | <p>We performed Language project with IIT Madras and received the finished video a few days later. <br /> | + | |
| − | 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>
| + | |
| − | </div></div>
| + | |
| − | <div class="word-2" style=" border-left:3px #999999 solid; width:49%;"><div style=" padding:20px;">
| + | |
| − | <h4>Technical works Wet& Dry labs:</h4>
| + | |
| − | <p>Wet lab: Inspired by our Human Practice, we raised an idea of designing a device for the growth of engineered <em>E.coli</em> strain<em>. </em>First we drew a draft on paper and then used the software Solid Works to draw a 3D version draft. Eventually, a real device came out. The device provided a great help to our further experiments because it provided a suitable place for the engineered strain to grow.<br/>
| + | |
| − | Near the end of this month, we performed gas chromatography to detect the amount of acetylene reduced to indirectly test the nitrogen fixation activity of our system. Up to wiki freeze, samples have been sent out and we will receive the result about five days later.
| + | |
| − | </p>
| + | |
| − | <p>Dry lab: We refined our model by further reading literature and verifying one of our assumptions. In that way, we got a more accurate result. This result provided useful guidance to our further experiments. </p>
| + | |
| − | </div></div>
| + | |
| − | </div></div>
| + | |
| − | </div>
| + | |
| − | <div class="word" id="protocol">
| + | |
| − | <h2>Protocol</h2>
| + | |
| − | <h3>Plasmids and Bacterial Strains.</h3><p> The bacterial strains, plasmids and primers used in this study are all listed in Table 1. <em>Escherichia coli</em>JM109 was purchased from Takara and designated EJ. A high-copy plasmid, pUC57-<em>nif</em> (pMB1 <em>ori</em>), harboring the minimal nitrogen fixation gene cluster (<em>nif</em>) of <em>Paenibacillus polymyxa</em> CR1 was chemically synthesized and then transformed into <em>E. coli</em> JM109, and the resulting recombinant was designated EJN. For construction of the second plasmid, pJQ200SK <em>OmpA/PbrR</em> (with a compatible p15A <em>ori</em>), a lab store plasmid pBAD24-<em>OmpA/PbrR</em> was used as the template to PCR-amplify <em>OmpA/PbrR</em> with P200F and P200R primers. After confirmation by sequencing, the PCR product was digested with <em>Kpn</em> I and <em>Hind</em> III and then insert into pJQ200SK to yield pJQ200SK-<em>OmpA/PbrR</em>. EJN transformed with pJQ200SK-<em>OmpA/PbrR</em> was selected from LB agar plates containing appropriate antibiotics, and the resulting strain was designated EJNC.</p>
| + | |
| − | <h3>Culture Conditions.</h3>
| + | |
| − | <p>LB broth for <em>E. coli</em> JM109 growth contained 10g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract. KPM minimal medium was adopted for all nitrogen fixation assays and contained per liter 1040 mg Na<sub>2</sub>HPO<sub>4</sub>, 3400 mg KH<sub>2</sub>PO<sub>4</sub>, 26 mg CaCl<sub>2</sub>·2H<sub>2</sub>O, 30 mg MgSO<sub>4</sub>, 7.5 mg Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O, 0.3mg MnSO<sub>4</sub>, 8000 mg glucose, 500 mg casein hydrolysate, 36 mg ferric citrate, 10 mg para-aminobenzoic acid, 5 mg biotin, and 1 mg vitamin B<sub>1</sub>, supplied with 10 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> (KPM-HN) for pregrowth or 10 mM glutamate (KPM-LN) for nitrogenase activity assays. Antibiotics were supplemented as required at the following concentrations: 100 μg/mL of ampicillin, and 20 μg/mL of gentamycin. </p>
| + | |
| − | <h3>Quantitative Real-time PCR. </h3>
| + | |
| − | <p>After harvesting bacteria from LB medium, purification of total RNA was performed using RNAiso Plus reagent (TaKaRa, Japan) following the protocol described by the manufacturer. One microgram of qualified total RNA was subjected to reverse transcription with a PrimeScript RT reagent Kit with gDNA Eraser per the manufacturer’s instructions (TaKaRa, Japan). qRT-PCR of the resulting cDNA was performed with gene-specific primers (Table 1) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA) with a SYBR Premix Ex Taq (Tli RNaseH Plus) Kit (TaKaRa, Japan). Standard curves of cDNA dilutions were used to determine the PCR efficiency. An expression data analysis was performed by the Pfaffl method of relative quantification using CFX Manager 3.1 software (Bio-Rad, USA).</p>
| + | |
| − | <h3>Nitrogenase Activity Assay.</h3>
| + | |
| − | <p>The C<sub>2</sub>H<sub>2</sub> reduction method was used to assay nitrogenase activity. EJNC was initially grown overnight in KPM-HN medium and then diluted in 2 mL KPM-LN medium in 20 mL sealed tube to a final OD600 of about 0.3. Air in the tubes was repeatedly evacuated and replaced with argon. After incubation at 37 °C for 6 to 8 h, 2 mL C<sub>2</sub>H<sub>2</sub> was injected. 1 mL of gas was sampled from the gas phase 16 h later and analyzed with a GC-7890B (Agilent, USA) gas chromatograph after appropriate 10-fold serially dilution with nitrogen. Both EJ and EJN severed as controls. </p>
| + | |
| − | <h3>ICP-MS(Inductively Coupled Plasma Mass Spectrometry) measurement of Cd<sup>2+</sup> adsorption.</h3>
| + | |
| − | <p><em>Escherichia coli</em> BL21 containing <em>OmpA-PbrR</em>-PJQ200SK (pBAD33) plasmid was cultured in LB medium to an OD<sub>600</sub> of 0.4-0.6. Arabinose and CdCl<sub>2</sub> were added to the medium to a final arabinose concentration of 40 μM and a final Cd<sup>2+</sup> 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. OD<sub>600</sub> 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 Cd<sup>2+</sup> content in the sample was measured using ICP-MS.</p>
| + | |
| − | <h3>Cd<sup>2+</sup> toxicity test.</h3><p>Multiple groups of LB medium were prepared, and arabinose with a final concentration of 40 μM and different amounts of CdCl<sub>2</sub> were added to the medium to form a Cd<sup>2+</sup> gradient of 0,150 μM, 300 μM, 600 μM, and 1000 μM.<em>E. coli</em> BL21 containing the <em>OmpA-PbrR</em>-PJQ200SK (pBAD33) plasmid and plasmid-free <em>E. coli</em> BL21 (control) were cultured in different media.The OD<sub>600</sub> value was measured every 2 hours and measured for 12 hours.</p>
| + | |
| − | <h3>Transmission electron microscopy with energy-dispersive x-ray spectroscopy (TEM-EDX).</h3><p>After the Cd<sup>2+</sup> 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.</p>
| + | |
| − | <h3>Characterization of biologically precipitated CdS nanoparticles</h3>
| + | |
| − | <p>The photocatalytic MV<sup>2+</sup> reduction assay was performed using a 10-mm quartz cuvette with a cap and a light source(350-W Xe lamp).<em> E.coli </em>cells containing biosynthesized CdS nanoparticles were harvested from LB medium by centrifugation (4000 rpm for 10 min). The reaction system consisted of the same amounts of different semiconductors [TiO<sub>2</sub> anatase (<em>10</em>) and synthesized free CdS nanoparticles (<em>29</em>)] and 3ml of 100 mM tris-HCl(PH 7), 150mM NaCl, 5% glycerol, 100mM ascorbic acid, and 5mM MV<sup>2+</sup> in the quartz cuvette. O<sub>2</sub> was removed by bubbling N<sub>2</sub> into the solution for 30 min. The reaction was initiated by light irradiation and stopped by centrifugation and separation of <em>E.coli</em>-CdS nanoparticles from the MV buffer. The absorption spectra were immediately measured after centrifugation (1000<em>g</em> for 1 min). The amount of reduced MV<sup>2+</sup>(MV<sup>+</sup>) that formed was calculated by monitoring the OD<sub>605</sub> using the molar conversion coefficient ɛ=1.3 × 10<sup>4</sup> M<sup>-1</sup> cm<sup>-1</sup>.</p>
| + | |
| − | <table border="0" cellspacing="0" cellpadding="0" width="0">
| + | |
| − | <tr class="t">
| + | |
| − | <td class="t" width="123"><p align="left"><strong>Strains</strong></p></td>
| + | |
| − | <td class="t" width="406"><p align="center"><strong><em>E. coli</em></strong></p></td>
| + | |
| − | <td class="t" width="84"><p align="center"><strong>Source</strong></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">EJ</p></td>
| + | |
| − | <td width="406"><p align="center"><em>E. coli</em> JM109 </p></td>
| + | |
| − | <td width="84"><p align="center">TaKaRa</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">EJN</p></td>
| + | |
| − | <td width="406"><p align="center"><em>E. coli</em> JM109 harboring plasmid pUC57-<em>nif</em></p></td>
| + | |
| − | <td width="84"><p align="center">This study</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">EJNC</p></td>
| + | |
| − | <td width="406"><p align="center"><em>E. coli</em> JM109 harboring plasmids pUC57-<em>nif and </em>pJQ200SK-OmpA/PbrR</p></td>
| + | |
| − | <td width="84"><p align="center">This study</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="t">
| + | |
| − | <td class="t" width="123"><p align="left"><strong>Plasmids</strong></p></td>
| + | |
| − | <td class="t" width="406"><p align="center"><strong>characteristic</strong><strong> </strong></p></td>
| + | |
| − | <td class="t" width="84"><p align="center"><strong>Source</strong></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">pUC57</p></td>
| + | |
| − | <td width="406"><p align="center">Cloning vector; pMB1 <em>ori</em>; Ampr</p></td>
| + | |
| − | <td width="84"><p align="center">Lab store</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">pUC57-<em>nif</em></p></td>
| + | |
| − | <td width="406"><p align="center">pUC57 with <em>nif</em>; pMB1 <em>ori</em>; Ampr</p></td>
| + | |
| − | <td width="84"><p align="center">Chemically synthesized</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">pJQ200SK</p></td>
| + | |
| − | <td width="406"><p align="center">Cloning vector;p15A <em>ori</em>; Gmr</p></td>
| + | |
| − | <td width="84"><p align="center">Lab store</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">pJQ200SK-<em>OmpA/PbrR</em></p></td>
| + | |
| − | <td width="406"><p align="center">pJQ200SK with<em> OmpA/PbrR</em>; p15A <em>ori</em>; Gmr</p></td>
| + | |
| − | <td width="84"><p align="center">This study</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="t">
| + | |
| − | <td class="t" width="123"><p align="left"><strong>PCR Primers</strong></p></td>
| + | |
| − | <td class="t" width="406"><p align="center"><strong>sequence</strong><strong> </td>
| + | |
| − | <td class="t" width="84"><p align="center"><strong>Amplicon</strong></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">P200F</p></td>
| + | |
| − | <td width="406"><p align="center">5’-GCTCTAGACATGAAAAAGACAGCTATCGCGA</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center"><em>OmpA/PbrR </em></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">P200R</p></td>
| + | |
| − | <td width="406"><p align="center">5’-TCCCCCGGGTCAGATCTTATCGTCGTCATC</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr class="t">
| + | |
| − | <td class="t" width="123"><p align="left"><strong>qRT-PCR </strong><strong>Primers</strong></p></td>
| + | |
| − | <td class="t" width="406"><p align="center"><strong>sequence</strong><strong> </strong></p></td>
| + | |
| − | <td class="t" width="84"><p align="center"><strong>Amplicon</strong></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifBF </p></td>
| + | |
| − | <td width="406"><p align="center">5’-TCGGCCGTGCCAAGGAATTT</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center"><em>nifB</em>for qRT-PCR</p></td>
| + | |
| − | </tr>
| + | |
| − | | + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifBR</p></td>
| + | |
| − | <td width="406"><p align="center">5’-CCTATGCCGGACGACAGCAG</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifHF</p></td>
| + | |
| − | <td width="406"><p align="center">5’-TGCGCCGTATGACCGTTACC</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center"><em>nifH</em> for qRT-PCR</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifHR</p></td>
| + | |
| − | <td width="406"><p align="center">5’-CCGGACGCCTCAGCTTTGTT</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifDF</p></td>
| + | |
| − | <td width="406"><p align="center">5’-GCCCGACCAAGACGATGGAG</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center"><em>nifD</em> for qRT-PCR</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifDR</p></td>
| + | |
| − | <td width="406"><p align="center">5’-CCGCAGTCCGCCAATCAGAA</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifKF</p></td>
| + | |
| − | <td width="406"><p align="center">5’-ACCTGAAGTTCGCGGCCAAA</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center"><em>nifK</em> for qRT-PCR</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifKR</p></td>
| + | |
| − | <td width="406"><p align="center">5’-ATCCGGAGCCTGCTCTTCCA</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifEF</p></td>
| + | |
| − | <td width="406"><p align="center">5’-TGCGGCAGATGGCTTACCTG</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center"><em>nifE</em> for qRT-PCR</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifER</p></td>
| + | |
| − | <td width="406"><p align="center">5’-AGCACTGCCCGCTTTCCTTT</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifNF</p></td>
| + | |
| − | <td width="406"><p align="center">5’-TCGAGAGCCGATTGCCGTTC</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center"><em>nifN</em> for qRT-PCR</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifNR</p></td>
| + | |
| − | <td width="406"><p align="center">5’-ATCCAGCGCCTCCTCCAGAT</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifXF</p></td>
| + | |
| − | <td width="406"><p align="center">5’-CGACGGAAGACGGTGTGCAT</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center"><em>nifX</em> for qRT-PCR</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifXR</p></td>
| + | |
| − | <td width="406"><p align="center">5’-TCCAGGAACTGGACGCCTGA</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifVF</p></td>
| + | |
| − | <td width="406"><p align="center">5’-TGGGCGCTGACCATTCGTTT</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center"><em>nifV</em> for qRT-PCR</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">QnifVR</p></td>
| + | |
| − | <td width="406"><p align="center">5’-ACTGCAGCCAGCGCCTTAAA</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">Q16SF</p></td>
| + | |
| − | <td width="406"><p align="center">5’-ACTCCTACGGGAGGCAGCAG</p></td>
| + | |
| − | <td width="84" rowspan="2"><p align="center">16S rRNAfor qRT-PCR</p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="123"><p align="left">Q16SR</p></td>
| + | |
| − | <td width="406"><p align="center">5’-ATTACCGCGGCTGCTGG</p></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | <div class="word" align="left" id="reference">
| + | |
| − | <h2>Reference</h2>
| + | |
| − | <ol><li>Wang, L., et al., <em>A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli.</em> PLoS Genet, 2013. <strong>9</strong>(10): p. e1003865.</li>
| + | |
| − | <li>Fixen, K.R., et al., <em>Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium.</em> Proc Natl Acad Sci U S A, 2016. <strong>113</strong>(36): p. 10163-7.</li>
| + | |
| − | <li>Brown, K.A., et al., <em>Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid.</em> Science, 2016. <strong>352</strong>(6284): p. 448-50.</li>
| + | |
| − | <li>Kuypers, M.M.M., H.K. Marchant, and B. Kartal, <em>The microbial nitrogen-cycling network.</em> Nat Rev Microbiol, 2018. <strong>16</strong>(5): p. 263-276.</li>
| + | |
| − | <li>Wei, W., et al., <em>A surface-display biohybrid approach to light-driven hydrogen production in air.</em> Sci Adv, 2018. <strong>4</strong>(2): p. eaap9253.</li>
| + | |
| − | <li>Wang, X., et al., <em>Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation.</em> PLoS One, 2013. <strong>8</strong>(7): p. e68677.</li>
| + | |
| − | <li>Yang, J., et al., <em>Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity.</em> Proc Natl Acad Sci U S A, 2017. <strong>114</strong>(12): p. E2460-E2465.</li>
| + | |
| − | <li>Yang, J., et al., <em>Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology.</em> Proc Natl Acad Sci U S A, 2018. <strong>115</strong>(36): p. E8509-E8517.</li>
| + | |
| − | <li>Yang, J.G., et al., <em>Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli.</em> Proceedings of the National Academy of Sciences of the United States of America, 2014. <strong>111</strong>(35): p. E3718-E3725.</li>
| + | |
| − | <li>Howard, J.B. and D.C. Rees, <em>Structural basis of biological nitrogen fixation.</em> Chemical Reviews, 1996. <strong>96</strong>(7): p. 2965-2982.</li>
| + | |
| − | </ol>
| + | |
| − | </div>
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