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| | + | <!-- start of content --> |
| | + | <body> |
| | + | <div class = "igem_2017_content_wrapper"> |
| | + | <h3></h3> |
| | + | <center> |
| | + | <img src="https://static.igem.org/mediawiki/2017/7/71/T--NYMU-Taipei--safety_title.jpg" |
| | + | style="width:45%;border-radius:50%;border:8px solid #412a07;"> |
| | + | </center> |
| | + | <h3></h3> |
| | + | <p></p> |
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| − | .Nitrogen_Starvation h4{ | + | <h1>Overview</h1> |
| − | padding:10px 0px 11px 20px;
| + | <blockquote> |
| − | color: #3dba32; | + | <p style='margin-top:9.6px'> Our team is deeply convinced that all biological experiments carry some risk to the environment and experimenter. Under the guideline of IGEM Headquarter, we strictly follow a high standard of biosafety and responsible biological engineering. We also take note of the Laboratory biosafety manual published by WHO to ensure our lab work conducting in compliance with safety regulation. Moreover, before conducting experiments in the lab, all team members have undergone lab training by our PI and team members in NYMU Taipei 2016. The training included laboratory emergency response procedures, operation protocols for all kinds of machines, and biological waste decontamination and disposal protocols. As one of the members of the synthetic biology community, we are responsible for abiding by the common rule.</p> |
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| − | .Nitrogen_Starvation p { | + | <h1>Biosafety</h1> |
| − | padding:0px 0px 0px 20px; | + | <blockquote> |
| − | font-size: 20px; | + | <h4>Organisms We Used</h4> |
| − | color: #0f2306;
| + | <p> We used (1) <font style='color: #3dba32;font-family:"Acme",sans-serif'>cyanobacteria <i>Synechococcus</i> sp. PCC 6803</font>, (2) <font style='color: #3dba32;font-family:"Acme",sans-serif'>cyanobacteria <i>Synechococcus</i> sp. PCC 7942</font> and (3) <font style='color: #3dba32;font-family:"Acme",sans-serif'><i>E.coli DH5α</i></font> in our project.</p> |
| − | } | + | <p> We extract NrtA gene from cyanobacteria <i>Synechocystis</i> sp. PCC 6803, which will then be transformed into <i>E.coli DH5α</i>. We co-culture the engineered <i>E.coli</i> with microalgae to induce nitrogen starvation – a pathway that will increase oil production of algae. After extracting oil from microalgae, we constructed a suicide mechanism to kill <i>E.coli</i> prevent contamination and potential danger of spreading engineered <i>E.coli</i>. To deal with the safety of <i>E.coli</i>, we construct a suicide mechanism.</p> |
| | + | <p> In pigment project, we transform pigment genes into PCC 7942, which express gene and produce pigments.</p> |
| | + | <h4></h4> |
| | + | <h4>Strain information of <i>E.coli</i></h4> |
| | + | <p> According to the Globally Harmonized System (GHS), <i>E.coli DH5α</i> is not a dangerous substance. As stated in Centers for Disease Control and Prevention (CDC) Guidelines (Biosafety in Microbiological and Biomedical Laboratories, 5th Edition<sup>1</sup>), <i>E.coli DH5α</i> is Biological Safety Level One organism.</p> |
| | + | <h4></h4> |
| | + | <h4>Strain information of i>Synechocystis sp.</i> PCC 6803 and PCC 7942</h4> |
| | + | <p> <i>Synechocystis sp.</i> PCC 6803 and PCC 7942 are the most widely used model organisms for photosynthesis research.<sup>2,3</sup> Both are the biosafety level one organism.</p> |
| | + | <h4></h4> |
| | + | <h4>Suicide Mechanism</h4> |
| | + | <p> To prevent potential environmental danger, we create a suicide mechanism to kill <i>E.coli</i>. We design suicide genes, endolysin and holin, to clear away all of engineered <i>E.coli</i>, ensuring the safety of <i>E. coli</i>. Endolysin and holin are similar to the mechanism used by team Pecking (2014 iGEM Beijing). Holin can induce holes on cell membrane, allowing Endolysin to pass through the membrane and decompose peptidoglycan. After the cell membrane and cell wall are destroyed, <i>E.coli</i> will be lysed.</p> |
| | + | </blockquote> |
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| − | .mark_backbone { | + | <h1>Lab safety</h1> |
| − | font-family: 'Acme', sans-serif; | + | <blockquote> |
| − | font-weight: bold; | + | <p> All experiments are done in Biosafety Level 1 (BSL1) laboratory. Our lab is equipped with safety facilities, such as emergency shower and fire extinguishers, and we conduct experiments under Laboratory Safe Hygiene Precautions of our school.<sup>4</sup> In addition, there are safety insert and postcards all around the lab as reminders. Lastly, all experiment protocols were checked and guided by our PI, Dr. Chou before we conducted.</p> |
| − | } | + | <h4></h4> |
| | + | <h4>Lab training</h4> |
| | + | <p> Every members of wet lab in NYMU iGEM team have finished lab safety lesson and training twice, which are directed by professors who instruct us step by step. Thus, members can conduct experiments properly and safely.</p> |
| | + | <h4></h4> |
| | + | <h4>Protection</h4> |
| | + | <p> In the laboratory, each member wears laboratory coat and gloves with long pants and shoes of full coverage. When we leave the lab, we make sure to remove our coat/gloves and wash our hands with antibacterial hand wash. We strictly ensured that all of these are done in addition to avoid touching door knobs with gloves to prevent carrying bacteria beyond the lab.</p> |
| | + | <h4></h4> |
| | + | <h4>Waste</h4> |
| | + | <p> We gather chemical and bacteria mixture into a bottle containing bleach after conducting the experiments. Besides, we sterilize all trash with autoclave and put it at a specific area of contaminative trash. All the wastes in our lab are handled seriously and strictly to prevent contamination.</p> |
| | + | </blockquote> |
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| − | .half_column { | + | <h1 style='font-size:36px;'>Reference</h1> |
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| + | <li>U.S. Department of Health and Human Services. (2009). <i>Biosafety in Microbiological and Biomedical Laboratories</i> (5th ed.). HHS Publication No. (CDC) 21-1112 |
| − | } | + | <li>Bertram M. Berla, Rajib Saha, Cheryl M Immethun, Costas D. Maranas, Tae Seok Moon, and Himadri B. Pakrasi. (2013). Synthetic biology of cyanobacteria: unique challenges and opportunities. <i>Front Microbiol, 13(4), 246.</i> |
| − |
| + | <li>Raul Muñoz, Cristina Gonzalez-Fernandez. (2017). <i>Microalgae-Based Biofuels and Bioproducts: From Feedstock Cultivation to End-Products.</i> Woodhead Publishing. |
| − | .half_column img {
| + | <li>Taiwan National Yang Ming University. (2010). Laboratory Safe Hygiene Precautions. (<a href='http://ces.web.ym.edu.tw/ezfiles/151/1151/img/183/laboratorysafehygieneprecautions-991001.pdf'>http://ces.web.ym.edu.tw/ezfiles/151/1151/img/183/laboratorysafehygieneprecautions-991001.pdf</a>) |
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| − | <div class="Nitrogen_Starvation_top">
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| − | <img src="https://static.igem.org/mediawiki/2017/2/20/T--NYMU-Taipei--nitrogen_starvation.jpg" width="100%">
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| − | </div>
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| − | <div class="Nitrogen_Starvation">
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| − | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;">
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| − | Background
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| − | </a>
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| − | <p style="padding-top:10px;"></p>
| + | |
| − | <p> With the development of global economy in the latest demands of energy in world, an average Taiwanese produces around 2.58 metric tons of carbon emission a year.
| + | |
| − | This number far surpasses those of China, Japan and South Korea.
| + | |
| − | Our project, as a result, works to use biofuel as an alternative fossil fuel to reduce the current energy crisis.
| + | |
| − | In our lab, we <font class='mark_backbone'>use microalgae as the source of biofuel</font> since they have the greatest capability of producing large amount of oil. </p>
| + | |
| − | <p> Our project aims to increase oil accumulation through <font class='mark_backbone'>nitrogen starvation</font>, which was brought up in a research we came upon.
| + | |
| − | Under nitrogen starvation, de novo synthesis of triacylglycerol from acyl-CoA increases.
| + | |
| − | Acyl moieties derived from the degradation of membrane lipids then recycle into triacylglycerol, increasing carbon flux towards glycerol-3 -phosphate and acyl-CoA for fatty acid synthesis.
| + | |
| − | As such, <font class='mark_backbone'>the oil accumulation under nitrogen starvation will increase</font>.</p>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2017/8/85/T--NYMU-Taipei--NS_pathway.jpg" style="width:70%;"></center>
| + | |
| − | <br>
| + | |
| − | <p> There are currently two types of cultivated systems: <font class='mark_backbone'>open-pond</font> and <font class='mark_backbone'>closed bioreactors</font>.
| + | |
| − | While open-pond costs way lower than closed bioreactors, open-pond cultivate microalgae is with significantly lower oil contents.
| + | |
| − | Many microalgae farms today cultivate microalgae in closed ponds, where regulations are made to keep the nitrogen level low.
| + | |
| − | This method, though, consumes lots of energy due to the need to maintain proper temperature, nutrition, light, and other growing factors.
| + | |
| − | We want to develop a new method that allows microalgae to reach nitrogen starvation in open-pond, and thus reaching <font class='mark_backbone'>the same effectiveness of closed bioreactors with the affordable price of open-pond</font>. </p>
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| − | <a href="#!" onclick="toggleHeight1(this, 1100);" style='color:#56914a'>
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| − | click to close
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| − | </center>
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| − | <div id="s2" class="expandable" style='height: 30px;padding-top:15px;'>
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| − | <a href="#!" onclick="toggleHeight2(this, 920); return false"
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| − | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;">
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| − | Design
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| − | </a>
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| − | <p style="padding-top:10px;"></p>
| + | |
| − | <p> NrtA protein sticks to the periplasmic membrane through a flexible linker to capture nitrite or nitrate in the periplasm.
| + | |
| − | Then delivery to the transmembrane complex that made by NrtB.
| + | |
| − | In our project, we try to <font class='mark_backbone'>transform NrtA gene from cyanobacteria <i>Synechocystis</i> sp. PCC 6803 to<i> E.coli</i>, and then co-culture the engineered <i>E.coli</i> with microalgae</font>.
| + | |
| − | Engineered <i>E.coli</i> will be capable of clutching nitrite or nitrate present in the environment.
| + | |
| − | They will not intake nitrate or nitrite since the gas accumulation may be lethal to cells.
| + | |
| − | But the amount of cells that contain nitrite will decrease.
| + | |
| − | Therefore, the microalgae will undergo <font class='mark_backbone'>nitrogen starvation</font> and produce oil more efficiently.</p>
| + | |
| − | <p> After building up the nitrogen starvation and extracting oil from microalgae, we need to kill <i>E.coli</i> to prevent contamination.
| + | |
| − | So we plan to <font class='mark_backbone'>use endolysin and holin for cell lysis</font>, which is similar to the mechanism used by team PeKing (2014 iGEM Beijing).
| + | |
| − | Holin can trigger the formation of holes on cell membrane.
| + | |
| − | When holin successfully forms holes on cell membrane, endolysin can pass through the membrane to decompose peptidoglycan.
| + | |
| − | <i>E.coli</i> is lysed after the cell membrane and cell wall are destroyed.
| + | |
| − | To control the suicide timing, we designed an inducible promoter for holin, so that we can induce <i>E.coli</i> suicide at the exact time we want.</p>
| + | |
| − | <center><img src='https://static.igem.org/mediawiki/2017/7/75/T--NYMU-Taipei--nitrogen_starvation_animation.gif' style='width:70%'></center>
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| − | <br>
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| − | click to close
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| − | </center>
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| − | <!--experiments-->
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| − | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;">
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| − | Experiments
| + | |
| − | </a>
| + | |
| − | <p style="padding-top:10px;"></p>
| + | |
| − | <h4>NrtA Expression</h4>
| + | |
| − | <p> We successfully transformed NrtA gene from cyanobacteria <i>Synechocystis</i> sp. PCC 6803 to <i>E.coli</i>.</p>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2017/d/da/T--NYMU-Taipei--NS_NrtA_RE.png" style="width:60%;"></center>
| + | |
| − | <p> This figure is restriction enzyme check electrophoresis result of NrtA construct.
| + | |
| − | We use XhoI and HindIII to digest the plasmid, and the expected length is 892bp, 1169bp, 1540bp.
| + | |
| − | M represents 1kb marker, and the result shows that number 2, 4, 6, 8, 10, 12 are right.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h4></h4>
| + | |
| − | <h4>Endolysin Construct</h4>
| + | |
| − | <div class="full_column">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/c/c7/T--NYMU-Taipei--NS_endolysin_snapgene.png" style="width:40%;float:left;margin:0 3vw 0 3vw;">
| + | |
| − | <p> We successfully constructed <font class='mark_backbone'>J23106-B0034-Endolysin-B0010-B0012</font>. The endolysin in this part is from iGEM released part, BBa_K112806, which is endolysin from enterobacteria phage T4. Besides endolysin, in this composite part, we choose BBa_J23106 as a constitutive promoter, BBa_B0034 as ribosome binding site, BBa_B0010 and BBa_B0012 as double terminator, all of which are widely used parts in iGEM.</p>
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| − | </div>
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| − | <div class="half_column">
| + | |
| − |
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/c/c4/T--NYMU-Taipei--NS_endolysin_PCR.png">
| + | |
| − | <p>This figure is electrophoresis result of endolysin PCR product.
| + | |
| − | The marker is 100bp. The length is 514bp as expected.
| + | |
| − | </p>
| + | |
| − | </div >
| + | |
| − | <div class="half_column">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/2/22/T--NYMU-Taipei--NS_endolysin_BB.png" >
| + | |
| − | <p>This figure is electrophoresis result of endolysin backbone PCR product.
| + | |
| − | The marker is 100bp. The length is 2268bp as expected.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <div class="clear"></div>
| + | |
| − |
| + | |
| − | <h4></h4>
| + | |
| − | <h4>Holin Construct</h4>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/6/62/T--NYMU-Taipei--NS_holin_snapgene.png" style="width:35%;float:left;margin:0 3vw 0 3vw;">
| + | |
| − | <p> We successfully constructed <font class='mark_backbone'>R0010-B0034-Holin-B0010-B0012</font>. To control the precise suicide timing, we choose a lactose-induced promoter, BBa_R0010, to regulate this suicide mechanism. Besides holin and inducible promoter, in this composite part, we also choose BBa_B0034 as ribosome binding site, and BBa_B0010 and BBa_B0012 as double terminator.</p>
| + | |
| − |
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| − | <div class="clear"></div>
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| − |
| + | |
| − | <div class="half_column">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/b/be/T--NYMU-Taipei--NS_holin_BB.png" style="width:50%">
| + | |
| − | <p>This figure is electrophoresis result of holin PCR product.
| + | |
| − | The marker is 100bp. The length is 657bp as expected.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="half_column">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/9/9a/T--NYMU-Taipei--NS_holin_PCR.png" >
| + | |
| − | <p>This figure is electrophoresis result of holin backbone PCR product.
| + | |
| − | The marker is 1kb. The length is 705bp as expected.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="clear"></div>
| + | |
| − |
| + | |
| − | <h4></h4>
| + | |
| − | <h4>Endolysin-Holin Construct</h4>
| + | |
| − | <div class="half_column">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/e/e9/T--NYMU-Taipei--NS_endolysin_holin_snapgene.png" >
| + | |
| − | <p> We successfully constructed <font class='mark_backbone'>R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012</font>. We combined holin and endolysin for suicide mechanism. In this composite part, holin functions as an important regulation role.</p>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <div class="half_column">
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| − | <img src="https://static.igem.org/mediawiki/parts/e/e2/T--NYMU-Taipei%E2%80%94Holin-Endolysin-REcheck.png" style="width:50%;">
| + | |
| − | <p>This is restriction enzyme check electrophoresis result of holin-endolysin construct.
| + | |
| − | The marker is 1kb. We use XmaI to digest the sample. The total length is 3843bp as expected.
| + | |
| − | </p>
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| − | </div>
| + | |
| − | <div class="clear"></div>
| + | |
| − |
| + | |
| − | <h4></h4>
| + | |
| − | <h4>Endolysin-Holin-NrtA Construct</h4>
| + | |
| − | <div class="half_column">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/c/ca/T--NYMU-Taipei--endolysin_holin_NrtA_snapgene.png">
| + | |
| − | <p> We successfully constructed <font class='mark_backbone'>R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012-J23118-B0034-NrtA-B0015</font>. This part combined holin, endolysin, and NrtA and had <font class='mark_backbone'>nitrate-capturing</font> function and <font class='mark_backbone'>suicide</font> function.</p>
| + | |
| − | </div>
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| − |
| + | |
| − | <div class="half_column">
| + | |
| − | <img src="https://static.igem.org/mediawiki/parts/b/b0/Holin-Endolysin-NrtA_REcheck.png" style="width:50%;">
| + | |
| − | <p>This figure is restriction enzyme check electrophoresis result of endolysin-holin-NrtA construct.
| + | |
| − | We use XmaI to digest sample 1~3. The result shows that 2 and 3 are right. M represents 1 kb marker.
| + | |
| − | </p>
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| − | </div>
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| − |
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| − | <div class="clear"></div>
| + | |
| − | <h4></h4>
| + | |
| − | <h4 style="color:#000000;">*See our parts: <a href="https://2017.igem.org/Team:NYMU-Taipei/Parts" target="_blank">click</a></h4>
| + | |
| − | <h4 style="color:#000000;">*See our experiments protocols: <a href="https://2017.igem.org/Team:NYMU-Taipei/Notebook" target="_blank">click</a></h4>
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| − |
| + | |
| − | <h4></h4>
| + | |
| − | <h4></h4>
| + | |
| − | <h4>Growth Curve Measurement</h4>
| + | |
| − | <p> In order to investigate <font class='mark_backbone'>the optimal time to co-culture our modified <i>E.coli</i> and <i>Chlorella vulgaris</i></font>, evaluate growth of <i>Chlorella vulgaris</i> before Nile Red staining for oil determination in microalgal cells, we will measure the OD value at 680nm by using the spectrophotometer and analyze the growth curve in R.
| + | |
| − | <br> However, we encountered some difficulties such as irregular measurement time and personal errors. Thus, we decided to search for better measurement method. Later, we borrowed a photo-bioreactor from Professor Ya Tang Yang, Department of Electrical Engineering, National Tsing Hua University, and used it to measure OD value for more precise bacterial growth curves.
| + | |
| − | </p>
| + | |
| − | <center><img src='https://static.igem.org/mediawiki/2017/4/48/T--NYMU-Taipei--NS_growth_curve_2.png' style='width:60%'></center>
| + | |
| − | <ol>
| + | |
| − | <li>The above line chart presents growth curve of <i>Chlorella vulgaris</i>. This measurement lasted more than 216 hours and it roughly meets our expectation.
| + | |
| − | <li>Although the later part of growth curve shows violent fluctuating range, it may be affected by environmental nitrogen metabolites from <i>Chlorella vulgaris</i>.
| + | |
| − | <li>The measurement result helps us determine lipid production of <i>Chlorella vulgaris</i>, and the time we add NrtA-transformed <i>E. coli</i> into the medium to establish a co-culture system.
| + | |
| − | </ol>
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| − |
| + | |
| − |
| + | |
| − | <h4></h4>
| + | |
| − | <h4>Automatic Measurement</h4>
| + | |
| − | <p> In the <font class='mark_backbone'>photo-bioreactor</font>, there are four light-emitting diode sources and two photodetectors. Once calibrated, the device can <font class='mark_backbone'>cultivate microbial cells and record their growth expression without human intervention</font>. We measure two kinds of microalgae during cell growth in same culture medium, BG-11.
| + | |
| − | </p>
| + | |
| − | <center><img src='https://static.igem.org/mediawiki/2017/7/76/T--NYMU-Taipei--NS_growth_curve_1.jpg' style='width:60%'></center>
| + | |
| − | <p> The photo-bioreactor was designed by Professor Yang. It was made with Arduino and some circuit components. Yang’s students also helped us assemble and teach us how to use this device. The photo-bioreactor itself can detect multiple units of organisms at same time, has pumps, fans, stir bars and some light bars. In Yang’s laboratory, we created a calibration curve for correcting and confirmed that there was a very high degree of correlation between voltage and OD value. This can be observed in the charts below.
| + | |
| − | </p>
| + | |
| − | <center><img src='https://static.igem.org/mediawiki/2017/1/13/T--NYMU-Taipei--NS_growth_curve_3.png' style='width:60%'></center>
| + | |
| − | <br>
| + | |
| − | <center><img src='https://static.igem.org/mediawiki/2017/2/2f/T--NYMU-Taipei--NS_growth_curve_4.png' style='width:60%'></center>
| + | |
| − | <br>
| + | |
| − | <p>And here are our results.</p>
| + | |
| − | <center><img src='https://static.igem.org/mediawiki/2017/6/6d/T--NYMU-Taipei--NS_growth_curve_5.png' style='width:60%'></center>
| + | |
| − | <ol>
| + | |
| − | <li>The above line chart presents growth curve of <i>Chlorella vulgaris</i>, under measurement near 10000 minutes made by the photo-bioreactor.
| + | |
| − | <li>In comparison to the stimulation <font class='mark_backbone'>model</font> we made, the result meets our expectation.
| + | |
| − | </ol>
| + | |
| − | <center><img src='https://static.igem.org/mediawiki/2017/6/67/T--NYMU-Taipei--NS_growth_curve_6.png' style='width:60%'></center>
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| − | <a href="#!" onclick="toggleHeight3(this, 7000);" style='color:#56914a'>
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| − | click to close
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| − | </a>
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| − | </center>
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| − | </div>
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| − | </div>
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| − |
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| − | <!--functional test NrtA-->
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| − | <div id="s4" class="expandable" style='height: 30px;padding-top:15px;'>
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| − | <a href="#!" onclick="toggleHeight4(this, 2900); return false"
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| − | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;">
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| − | NrtA Functional Test
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| − | </a>
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| − | <p style="padding-top:10px;"></p>
| + | |
| − | <p> We expected the engineered <i>E.coli</i> with NrtA gene could express NrtA protein. When we co-culture it with microalgae, the NrtA protein would capture nitrate and nitrite, and the microalgae would undergo nitrogen starvation and produce more oil. To test our hypothesis, first, we should verify NrtA protein could capture nitrate and nitrite, and then second, test whether the engineered <i>E.coli</i> could decrease nitrate and nitrite in the environment. </p>
| + | |
| − | <h4></h4>
| + | |
| − | <h4>NrtA Protein</h4>
| + | |
| − | <p> To test whether NrtA protein could capture nitrate and nitrite, we used <font class='mark_backbone'>French Pressure Press</font> to isolate NrtA protein, and used <font class='mark_backbone'>Cayman Nitrate/Nitrite Colorimetric Assay Kit</font> to measure the nitrate and nitrite concentration in the medium with NrtA protein. (You can see the detail in our Notebook page: <a href="https://2017.igem.org/Team:NYMU-Taipei/Notebook">click</a>.)</p>
| + | |
| − | <p> Here are our results.</p>
| + | |
| − | <div style="width:60%;margin:auto">
| + | |
| − | <img src="https://static.igem.org/mediawiki/parts/8/8c/T--NYMU-Taipei--NS_NrtA_func_1.png" style="width:95%">
| + | |
| − | <p style="font-size:16px;">Figure1. Nitrate concentration of cell lysate.
| + | |
| − | <br>Blank: nitrate concentration assay kit assay buffer.
| + | |
| − | <br>BG11: microalgae culture medium buffer.
| + | |
| − | <br>CC: competent cell.
| + | |
| − | <br>NrtA: isolated NrtA protein.
| + | |
| − | </p>
| + | |
| − | <br>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/e/ec/NYMU_2017_awards_basicpart_3.jpg" style="width:95%">
| + | |
| − | <p style="font-size:16px;">Table1. Dunnett’s T3 test of nitrate concentration of cell lysate.
| + | |
| − | <br>Blank: nitrate concentration assay kit assay buffer.
| + | |
| − | <br>BG11: microalgae culture medium buffer.
| + | |
| − | <br>CC: competent cell.
| + | |
| − | <br>NrtA: isolated NrtA protein.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <br>
| + | |
| − | <p> The nitrate and nitrite concentration of competent cell and NrtA was significant different. The results indicated that <font class='mark_backbone'>NrtA protein can capture nitrite and nitrate</font>! This is a milestone of our project!</p>
| + | |
| − |
| + | |
| − |
| + | |
| − | <h4></h4>
| + | |
| − | <h4>The Engineered <i>E.coli</i> with NrtA Gene</h4>
| + | |
| − | <p> Then we wanted to know whether the engineered <i>E.coli</i> could decrease nitrate and nitrite in the environment. We used Cayman Nitrate/Nitrite Colorimetric Assay Kit to measure the nitrate and nitrite concentration of the supernatant of the engineered <i>E.coli</i> liquid culture.</p>
| + | |
| − | <p>Here are our results.</p>
| + | |
| − | <div style="width:60%;margin:auto">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/0/0d/NYMU_2017_awards_basicpart_2.jpg" style="width:95%">
| + | |
| − | <p style="font-size:16px;">Figure 2. Nitrate concentration of cell.
| + | |
| − | <br>Blank: nitrate concentration assay kit assay buffer.
| + | |
| − | <br>BG11: microalgae culture medium buffer.
| + | |
| − | <br>CC: competent cell.
| + | |
| − | <br>NrtA: the supernatant of the engineered <i>E.coli</i> liquid culture.
| + | |
| − | </p>
| + | |
| − | <br>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/e/ec/NYMU_2017_awards_basicpart_3.jpg" style="width:95%">
| + | |
| − | <p style="font-size:16px;">Table 2. Dunnett’s T3 test of nitrate concentration of cell.
| + | |
| − | <br>Blank: nitrate concentration assay kit assay buffer.
| + | |
| − | <br>BG11: microalgae culture medium buffer.
| + | |
| − | <br>CC: competent cell.
| + | |
| − | <br>NrtA: the supernatant of the engineered <i>E.coli</i> liquid culture.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <br>
| + | |
| − | <p> The nitrate and nitrite concentration of NrtA and competent cell had slight but not significant difference. The result implied that NrtA protein could capture nitrate and nitrite while the engineered <i>E.coli</i> with NrtA gene couldn’t. The engineered <i>E.coli</i> with NrtA gene could express NrtA protein, while NrtA protein might be inside the cell, so nitrate and nitrite concentration outside the cell didn’t change a lot. To make NrtA protein become a secreted protein, we are trying to construct <font class='mark_backbone'>signal peptide sequence</font>, so that NrtA protein can be released to the medium.</p>
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| − | <a href="#!" onclick="toggleHeight4(this, 2900);" style='color:#56914a'>
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| − | click to close
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| − | </a>
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| − | </center>
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| − | </div>
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| − | <!--functional test suicide-->
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| − | <div class='panel'>
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| − | <div id="s6" class="expandable" style='height: 30px;padding-top:15px;'>
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| − | <a href="#!" onclick="toggleHeight6(this, 1950); return false"
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| − | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;">
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| − | Suicide Mechanism Functional Test
| + | |
| − | </a>
| + | |
| − | <p style="padding-top:10px;"></p>
| + | |
| − |
| + | |
| − | <p> Before we construct our suicide mechanism with NrtA, we want to <font class='mark_backbone'>make sure that our suicide mechanism, endolysin and holin, can be induced by lactose efficiently</font>. Therefore, we test our suicide mechanism by adding different concentration of lactose in order to find out the minimum effective concentration. Furthermore, to induce <i>E.coli</i> suicide at the time we want, we also want to know the changing degree of the OD value of bacterium at different time after the lactose is added.</p>
| + | |
| − | <p> We measure the OD<sub>600</sub> value of the sample, and then aliquot liquid culture into several cuvettes. Next, we add different amount of 0.5mM lactose into cuvettes to form different concentration of lactose, and measure the OD<sub>600</sub> value per hour.</p>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2017/4/4d/T--NYMU-Taipei--NS_suicide_func.png" style="width:95%"></center>
| + | |
| − | <p> As the figure shows, <font class='mark_backbone'>the suicide mechanism is induced immediately when lactose is added into the samples</font>. Besides, we can see that the lactose concentration and the degree of decline of OD value are positively correlated.</p>
| + | |
| − | <p> We also collaborate with team <font class='mark_backbone'>TAS Taipei</font>. They help us by testing our holin-endolysin-NrtA construct.</p>
| + | |
| − | <center><img src="https://static.igem.org/mediawiki/2017/8/8b/NYMU_2017_collaboration_TFG_.jpg" style="width:70%"></center>
| + | |
| − | <p> As the figure shows, the trend of the relative absorbance is downward as the lactose is added to induce the suicide mechanism. The concentration of lactose is also positively correlated with the declining degree of relative absorbance.</p>
| + | |
| − | <p> We need to mention that there are some differences in the process of team TAS Taipei’s and ours functional tests. We use different medium to culture the bacterium. For the bacteria with holin-endolysin construct, we use Luria-Bertani (LB) broth with 0.9% glucose to culture; team TAS Taipei cultures the bacterium with holin-endolysin-NrtA construct with only Luria-Bertani (LB) broth. According to our previous experiment result, the bacteria don’t grow well in only LB broth medium. Therefore, to make the figure look similar, team TAS Taipei <font class='mark_backbone'>normalizes their data</font> (absorbance at 0 hour is the background). <font class='mark_backbone'>Despite the different of culturing medium, the results both show that our suicide mechanism can work</font>.
| + | |
| − | <a href="https://2017.igem.org/Team:NYMU-Taipei/Collaborations">(see more detail about collabration)</a></p>
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| − | style="font-family:'Acme', sans-serif;font-size:30px;color:#205e1a;height: 30px;">
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| − | Reference
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| − | </a>
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| − | <p></p>
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| − | <div style="font-size:16px;">
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| − | <p style="padding-top:5px;"></p>
| + | |
| − | <ol>
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| − | <p>
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| − | <li>J.W. Allen et al. (2015). Triacylglycerol synthesis during nitrogen stress involves the prokaryotic lipid synthesis pathway and acyl chain remodeling in the microalgae <i>Coccomyxa subellipsoidea</i>. <i>Algal Research, 10,</i> 110–120.
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <li>G. Breuer et al. (2012). The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. <i>Bioresource Technology, 124,</i> 217–226.
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <li>Valledor et al. (2014). System-level network analysis of nitrogen starvation and recovery in <i>Chlamydomonas reinhardtii</i> reveals potential new targets for increased lipid accumulation. <i>Biotechnology for Biofuels, 7:171.</i>
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <li>S. Zhu et al. (2014). Metabolic changes of starch and lipid triggered by nitrogen starvation in the microalga <i>Chlorella zofingiensis</i>. <i>Bioresource Technology, 152,</i> 292–298.
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| − | </p>
| + | |
| − | <p>
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| − | <li>J. Jia et al. (2015). Molecular mechanisms for photosynthetic carbon partitioning into storage neutral lipids in <i>Nannochloropsis oceanica</i> under nitrogen-depletion conditions. <i>Algal Research, 7, </i> 66–77.
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| − | </p>
| + | |
| − | <p>
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| − | <li>S.K. Lenka et al. (2016). Current advances in molecular, biochemical, and computational modeling analysis of microalgal triacylglycerol biosynthesis. <i>Biotechnology Advances, 34,</i> 1046–1063
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| − | </p>
| + | |
| − | <p>
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| − | <li>A.J. Klok et al. (2013). A model for customising biomass composition in continuous microalgae production. <i>Bioresource Technology, 146,</i> 89–100.
| + | |
| − | </p>
| + | |
| − | <p>
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| − | <li>H. Abedini Najafabadi et al. (2015). Effect of various carbon sources on biomass and lipid production of <i>Chlorella vulgaris</i> during nutrient sufficient and nitrogen starvation conditions. <i>Bioresource Technology, 180,</i> 311–317.
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| − | </p>
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| − | <p>
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| − | <li>Yu et al. (2011). Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. <i>Microbial Cell Factories, 10:91.</i>
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| − | </p>
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| − | </ol>
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