Team:OUC-China/Parts

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</ul>
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<div id="da-slider" class="da-slider">
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<div class="da-slide">
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<h2>Overview</h2>
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<div id="slidenav-bar">
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<p>The sensors for next-generation detector of red tide!</p>
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<ul id="social" class="group">
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<a href="" class="da-link">Read more</a>
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<div class="da-img"><img src="" alt="senso" /></div>
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<li class="overview"><a href="https://2012.igem.org/Team:OUC-China"><span>Back</span></a></li>
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<h2>Sensor</h2>
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<p>The sensors for next-generation detector of red tide!</p>
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<a href="https://2012.igem.org/Team:OUC-China/Project/Sensor/AbstractandBackground" class="da-link">Read more</a>
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<div class="da-img"><img src="https://static.igem.org/mediawiki/2012/3/32/Home-sensor.png" alt="sensor" /></div>
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</div>
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<div class="da-slide">
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<h2>Model-Driven Design</h2>
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<p>
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Parameter optimization in ternary system...
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</p>
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<a href="https://2012.igem.org/Team:OUC-China/Modeling/Overview" class="da-link">Read more</a>
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<div class="da-img"><img src="https://static.igem.org/mediawiki/2012/2/26/Home-modeling.png" alt="Home-modeling" /></div>
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</div>
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<div class="da-slide">
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<h2>Making decision through sRNA...</h2>
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<p>The sophisticated  'CPU' of our red tide detector...<br/>
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Engineering CPU for your own "synthia"!</p>
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<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Abstract" class="da-link">Read more</a>
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<div class="da-img"><img src="https://static.igem.org/mediawiki/2012/2/2c/Home-decision-making-device.png" alt="Home-decision-making-device" /></div>
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<h2>Oceanfloat and Oceanfeel<br/><span>----a floating biosenser</span></h2>
</div>
</div>
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</div>
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<section id="project_step">
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<div class="moduletable">
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<h3>Our Project</h3>
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<div>
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<ul id="tab">
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<li class="on" id="tab_1" onclick="switchTab(1)"><img src="https://static.igem.org/mediawiki/2012/8/8f/Ouc-home-step1.png" /><span>step 1</span></li>
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<li id="tab_2" onclick="switchTab(2)"><img src="https://static.igem.org/mediawiki/2012/d/de/Ouc-home-step3.png" /><span>step 2</span></li>
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<li id="tab_3" onclick="switchTab(3)"><img src="https://static.igem.org/mediawiki/2012/a/a4/Ouc-home-steplast.png" /><span>step 3</span></li>
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</ul>
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<div style="clear: both;">&nbsp;</div>
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<div class="tuxing"><img src="https://static.igem.org/mediawiki/2012/e/e2/Ouc-hometab_arrow.png" /></div>
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<ul id="tab_con">
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<li id="tab_con_1">
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<div class="tup"><img src="https://static.igem.org/mediawiki/2012/3/3a/Ouc-home-step1-2.png" /></div>
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<div class="nr">
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<h1>Step 1</h1>
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<p>Our phosphate- and nitrate-sensitive devices are responsible for detection of environmental phosphate and nitrate. Besides that they transform the input signals into "readable information" for decision-making devices.</p>
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<p>much work has been done for characterization and modification of nitrate- and phosphate-sensitive promoters. A whole set of design ideas for TCS modifications are also presented for other igemers. Additionally an actuator that can remove the phosphate is recommended.</p>
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<p><a href="https://2012.igem.org/Team:OUC-China/Project/Sensor/AbstractandBackground"><strong>learn more</strong></a>
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</div>
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<div style="clear: both;">&nbsp;</div>
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</li>
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<li id="tab_con_2">
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<div class="tup"><img src="https://static.igem.org/mediawiki/2012/a/a1/Ouc-home-step3-02.png" /></div>
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<div class="nr">
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<h1>Step2</h1>
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<p>Our decision-making device aims at processing the concentration change of nitrate or phosphate into visible buoyancy increase. Comparator recognizes the difference of two inputs quantitatively ,while ratio sensor could senses a particular ratio between two inputs.</p>
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<p>Both comparator and ratio sensor facilitate a ternary system consisted of two small RNA and GFP mRNA. The concentration change of external two inducers leads to the transcription rate change of small RNA(alpha s)in this ternary system, the interactions between three RNAs will lead to interesting behaviors of our system like sensing the difference between two inputs quantitatively(Comparator) or the ratio of two inputs(Ratio senor).</p>
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<p><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Abstract"><strong>learn more</strong></a>
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</div>
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<div style="clear: both;">&nbsp;</div>
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</li>
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<li id="tab_con_3">
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<div class="tup"><img src="https://static.igem.org/mediawiki/2012/f/fa/Ouc-step3-last.png" /></div>
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<div class="nr">
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<h1>Step3</h1>
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<p>In our project, by transforming the buoyancy device BBa_K737010, the gas vesicle cluster from the genome of Planktothrix rubescens into E.Coli, we succeed in making Escherichia.Coli floating on the surface of the water. It can help the cells produce the gas vesicle inside, which could make densities significant stratification in the culture. Not only the gene cluster but the two parts BBa_K737006 and BBa_K737007, which produce the gas vesicle structure protein, can also make the Escherichia.Coli floating. We use the shorter length of the part achieve a better effect than BBa_K190033 and the gas vesicle application prospect.</p>
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<p><a href="https://2012.igem.org/Team:OUC-China/Project/GVP/GasandBackground"><strong>learn more</strong></a>
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</div>
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<div style="clear: both;">&nbsp;</div>
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</li>
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</ul></div>
</div>
</div>
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<div class="start"></div>
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                <div class="list-box">
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<br/>
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<div class="list-text">
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<br/>
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<h1>Summary</h1>
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<br/>
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<p><strong>Parts submitted:</strong></p>  <p>33 Composite expression modules</p>  
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                    <p>10 Generator</p>
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                    <p>4 Regulatory promoters</p>
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                      <p>  1 Ribosome Binding Sites</p>
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<script type="text/javascript">
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                      <p> 10 Coding proteins</p>
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$(function() {
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                      <p>  10 RNA </p>           
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<p>Total: <strong>68 Parts</strong></p>
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<p>All 68 parts have been submitted to the registry and can be found <a
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href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2012&group=OUC-China">here</a>.</p>
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<br/>
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<h1>Data for favorite parts</h1>
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<p><strong>Gvp cluster(<a href="http://partsregistry.org/Part:BBa_K737010">BBa_K737010</a>)</strong> is a 1 kbp part
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contains 2 open reading frames coding for gas vesicle genes (gvpA gvpC-20) from Bacillus Planktothrix rubescens strain BC-
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Pla 9402. Promotion of the sequence results in expression of gas vesicles, organelles made entirely out of protein. These
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organelles contain gas and therefore provide bouyancy to the cell. This slows the rate of settling of the cells in
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different water layer from Lake Zurich by gas vesicles, see Walsby 2002.</p>
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<div class="image"><img src="https://static.igem.org/mediawiki/2012/d/d5/Ouc-parts1.jpg"/></div>
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<p><span>the expression situation of the Gas vesicle gene cluster in Escherichia coli JM109, from the picture, we can see
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obviously gas vesicle due to the expression of bacteria produce clear separation phenomenon. The picture on the right of
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the experimental group were liquid surface layer and intermediate layer of fluid sampling results, we can see that the
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fluid layer surface bacteria volume density is obviously higher than that of the middle layer of fluid.</span></p>
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<div class="image"><img src="https://static.igem.org/mediawiki/2012/b/bf/Ouc-parts2.png"/></div>
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<p><span>The picture shows the gas vesicle in the cells after expressed in the overall trend. We in the cultivation of the
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cells into logarithmic phase after transferring them to test tube, quiet place and culture 36 hours after using Trace
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stratified take liquid apparatus at the same time to each liquid layer for sampling. Then each layer of fluid samples of
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blood count the number of cells with statistics. Results show that gas vesicle due to the existence of the cell
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concentration in the test tube surface layer of fluid, and control group due to the action of gravity that most cells
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sedimentation in the test tube bottom.</span></p>
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<div class="image"><img src="https://static.igem.org/mediawiki/2012/4/4c/Ouc-parts3.png"/></div>
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<p><span>Graph of OD measurements at 600nm of both experience group and control group. From the experimental results can be
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seen in the liquid surface .There cell number have significant abrupt transformation.</span></p>
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<br/>
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<p><strong>Constitutive GVPA Generator</strong>(<a href="http://partsregistry.org/Part:BBa_K737006">BBa_K737006</a>) It contains a constitutive promoter, GvpA protein , GFP protein and a
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terminator.This part can be used as a control of (J23106+P0412+R0011+B0034+GVPA+E0840). It can express GvpA protein and GFP
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constantly. When it transformed with  (J23106+P0440+R0040+B0034+GVPC+E0840), the bacteria may can float with GFP!</p>
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<p><strong>YtfJ_Comparator</strong>( <a href="http://partsregistry.org/Part:BBa_K737045">BBa_K737045</a>)  The leading
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sequence involved in the YtfJ transcript which can act as the target of the sRNA, spot42. It enables strong competition
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with the galk::GFP to interact with spot42 which shows great potential for constructing sRNA-mediated circuit.</p>
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<p>This device mean to test feasibility of N/P comparator,replacing Nitrate,Phosphate inputs by IPTG,aTc。Small RNA Spot42
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controls the GFP mRNA translation by blocking the RBS in galK.The galK and E0040 is fused together using ClaI site to avoid
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stop condon in standard scar.J23106 is a moderate promotor,and galK’s RBS is a moderate one.</p>
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<div class="image"><img src="https://static.igem.org/mediawiki/2012/9/97/Ouc-parts4.png"/></div>
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<p>Spot42 sRNA is under control of aTc inducible device,the buffer RNA YtfJ is under control of IPTG inducible device.</p>
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<div class="image"><img src="https://static.igem.org/mediawiki/2012/a/a4/Ouc-parts5.jpg"/></div>
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<p><strong>Constitutive GVPC Generator</strong>(<a href="http://partsregistry.org/Part:BBa_K737007">BBa_K737007</a>)It contains a constitutive promoter, GvpC
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protein , GFP protein and a terminator.This part can be used as a control of (J23106+P0440+R0040+B0034+GVPC+E0840). It can
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express GvpC protein and GFP constantly. When it transformed with  (J23106+P0412+R0011+B0034+GVPA+E0840), the bacteria may
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can float with GFP!</p>
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<p>Here is the result of SDS-polyacrylamide gel electrophoresis. We can see GFP and GvpC protein directly. Lane 4 is top10
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control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper
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white boxes are in the position about 26KD are GFP protein (25.8KD). The lower white box in the position of about 20KD is
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GvpC-20 (20KD). Because the MW(molecular weight) of GvpA protein is too small, it’s very difficult to see it on the SDS-
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polyacrylamide gel. But the expression of GFP suggests it may be expressed.
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Some papers say that GvpA is difficult to see is that GvpA is too hydrophobic to run out of sample hole.</p>
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<div class="image"><img src="https://static.igem.org/mediawiki/2012/8/88/Ouc-parts6.jpg"/></div>
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<p><span>Figure1.The result of SDS-polyacrylamide gel electrophoresis. Lane 4 is top10 control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper white boxes are in the position about 26KD are GFP protein (25.8KD). The lower white box in the position of about 20KD is GvpC-20 (20KD).</span></p>
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<p>Because the GvpA protein can’t be seen on the SDS-polyacrylamide gel electrophoresis, we wanted to use GFP to detect the expression of GvpA. We use a polycistron of gvpA(gvpC) and gfp to detect the RFU. We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control. First, the RFU of these parts are rather high. Second, the E.Coli with these two plasmids can float. Third, we can see GvpC on the SDS-polyacrylamide gel. So, we can see the two parts can express Gvp Protein and work well.</p>
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bgincrement : 450
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<div class="image"><img src="https://static.igem.org/mediawiki/2012/8/80/Ouc-parts7.jpg"/></div>
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<p></span>Figure2.We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control.</span></p>
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<p><strong>SrlA_Comparator</strong>(<a href="http://partsregistry.org/Part:BBa_K737046">BBa_K737046</a>)The leading sequence involved in the SrlA transcript which can act as the target of the sRNA, spot42. It enables strong competition with the galk::GFP to interact with spot42 which shows great potential for constructing sRNA-mediated circuit.
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This device mean to test feasibility of N/P comparator,replacing Nitrate,Phosphate inputs by IPTG,aTc.</p>
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<p>Small RNA Spot42 controls the GFP mRNA translation by blocking the RBS in galK.The galK and E0040 is fused together using ClaI site to avoid stop condon in standard scar.J23106 is a moderate promotor,and galK’s RBS is a moderate one.</p>
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<p>Spot42 sRNA is under control of aTc inducible device,the buffer RNA SrlA is under control of IPTG inducible device.</p>
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<p><strong>Spot42</strong> (<a href="http://partsregistry.org/Part:BBa_K737058">BBa_K737058</a>)is a multitarget small RNA that mediates the discoordinate expression of the E.coli galactose operon and other sugar metabolic pathway,such as galK,nanC,ytfJ,srlA,which are the 5’ leader sequence of the corresponding metabolic enzymes.
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It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.Spot42-galK complex degrades in a slow and presently unclear way. The base pairing is initiated by the recognition of seed region,which plays a significant role in the repression efficiency.</p>
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<p><strong>galK:GFP generator</strong>(<a href="http://partsregistry.org/Part:BBa_K737034">BBa_K737034</a>) Compared with the original part, our new part possesses
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new function.Small RNA Spot42 controls the GFP mRNA translation by blocking the RBS in galK. The galK and E0040 is fused together using ClaI site to avoid stop condon in standard scar.J23106 is a moderate promotor,and galK’s RBS is a moderate one.</p>
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<p><strong>GFP generator</strong>(<a href="http://partsregistry.org/Part:BBa_K737068">BBa_K737068</a>) is provided by MIT in 2012 plates for teams. But we couldn’t transfer it successfully. So we constructed it again and submitted it to registry.</p>
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<p><strong>OmpR</strong>(<a href="http://partsregistry.org/Part:BBa_K737067">BBa_K737067</a>) Positively regulated, OmpR-controlled promoter ligated with B0034 can be used to control genetic circuit by light with light-dependent control element. This promoter is taken from the upstream region of OmpC. Phosphorylated OmpR binds to the three operator sites and activates transcription.</p>
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Revision as of 02:22, 27 September 2012

Overview

The sensors for next-generation detector of red tide!

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senso

Sensor

The sensors for next-generation detector of red tide!

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sensor

Model-Driven Design

Parameter optimization in ternary system...

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Home-modeling

Making decision through sRNA...

The sophisticated 'CPU' of our red tide detector...
Engineering CPU for your own "synthia"!

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Home-decision-making-device

Oceanfloat and Oceanfeel
----a floating biosenser

Our Project

  • step 1
  • step 2
  • step 3
 

  • Step 1

    Our phosphate- and nitrate-sensitive devices are responsible for detection of environmental phosphate and nitrate. Besides that they transform the input signals into "readable information" for decision-making devices.

    much work has been done for characterization and modification of nitrate- and phosphate-sensitive promoters. A whole set of design ideas for TCS modifications are also presented for other igemers. Additionally an actuator that can remove the phosphate is recommended.

    learn more

     

  • Step2

    Our decision-making device aims at processing the concentration change of nitrate or phosphate into visible buoyancy increase. Comparator recognizes the difference of two inputs quantitatively ,while ratio sensor could senses a particular ratio between two inputs.

    Both comparator and ratio sensor facilitate a ternary system consisted of two small RNA and GFP mRNA. The concentration change of external two inducers leads to the transcription rate change of small RNA(alpha s)in this ternary system, the interactions between three RNAs will lead to interesting behaviors of our system like sensing the difference between two inputs quantitatively(Comparator) or the ratio of two inputs(Ratio senor).

    learn more

     

  • Step3

    In our project, by transforming the buoyancy device BBa_K737010, the gas vesicle cluster from the genome of Planktothrix rubescens into E.Coli, we succeed in making Escherichia.Coli floating on the surface of the water. It can help the cells produce the gas vesicle inside, which could make densities significant stratification in the culture. Not only the gene cluster but the two parts BBa_K737006 and BBa_K737007, which produce the gas vesicle structure protein, can also make the Escherichia.Coli floating. We use the shorter length of the part achieve a better effect than BBa_K190033 and the gas vesicle application prospect.

    learn more

     







Retrieved from "http://2012.igem.org/Team:OUC-China/Parts"