Difference between revisions of "Team:LN-Shiyan-China/Parts"

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<p>Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <code>&lt;groupparts&gt;</code> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.</p>
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</style>
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<p>Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.</p>
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    <h1><a href="index.html">LN-shiyan-China</a></h1>
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<li><a href="project.html"><i class="glyphicon glyphicon-list-alt"> </i>Project</a></li>
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<li><a href="Parts.html"><i class="glyphicon glyphicon-picture"> </i>Parts</a></li>
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<h3>Parts</h3>
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</div>
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<div class="col-md-8 blog-left">
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    <img src="http://www.dftvip.com/templa/images/o2.jpg" alt="" class="img-responsive">
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  <div class="blog-grid">
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  <h3>Our Submission Parts: </h3>
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<h3>BBa_K1667005</h3>
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    <p>This part includes two individual DNA domains. Constitutive promoter tunes the expression of downstream opdA gene with further help from RNA thermometer. RNA thermometer provides a temperature sensitive post-transcriptional regulation on opdA gene, which initiate the opdA translation around 32°C. OmpA signal peptide guides the secretion of opdA protein to the outside of host strain. Then opdA enzyme specifically degrades organophosphorus pesticide appeared, through hydrolysis. Upon UV light, RecA（SOS）promoter drives the transcription of downstream ccdB suicide gene, whose protein expression interferes DNA sysnthesis and lead to cell dealth. In this way, we can wipe out our genetic engineered bacteria by giving UV lights under manual control to avoid secondary pollution. Without UV light, RecA promoter won’t be activated, so the normal growth and activity of genetic engineering bacteria will be preserved well to release functional opdA protein under temperature control. </p>
 +
<h3>Experimentally Verification Scheme:</h3>
 +
<p>Bacteria strain BL21 (DE3) of colibacillus genetically engineered bacteria which contains our target plasmid goes through extended culture in our LB liquid substrate for sixteen hours under the temperature of 30°C, then centrifuge the culture for five minutes with the speed of 5000rpm under the temperature of 4°C and use sterile MSM (mineral base medium) to wash and sediment the bacteria, then use fresh MSM culture substrate to dilute the bacteria. Inoculate the bacteria to MSM culture substrate which contains 10μg/mL chlorpyrifos or methyl-parathion (the final concentration of bacteria is 106 CFU/mL), and culture it with the speed of 160rpm under the temperature of 37°C while shaking it. Regularly get samples from the solution and measure the concentration of organophosphorus pesticides in the solution. Measure the OD600 of the culture to indicate the growth of the bacteria, use the culture solution which is not inoculated as a blank control while using the colibacillus inoculated with the bacteria strain BL21 (DE3) which does not contain target plasmid as a reference control. Adopt gas chromatography to measure the concentration of organophosphorus pesticides in the culture. </p>
 +
<p>Extraction of organophosphorus pesticides: Get 5mL MSM solution which is inoculated and cultured as above, add 25mL acetonitrile into it and shake it violently for five minutes, then add 2.5g sodium chloride into it and shake it violently, place the solution quietly for 30 minutes then laminarize it, get 1mL organic phase in the upper layer and dry it with pressure blowing concentrator, add 1mL acetone into it to dissolve above dry organic phase again. </p>
 +
<p>Gas chromatography detecting conditions: Use Shimadzu 2014C gas chromatographic analyzer, equip it with fire photometric detector (FPD) and quartz capillary chromatographic column (length of column is 30 meters, inner diameter of column is 0.53mm, thickness of membrane layer is 1μm, RESTEX, USA), flow speed of nitrogen is lmL/min while the flow speeds of air and hydrogen are respectively 81.8mL/min and 3.2mL/min. The temperatures of vaporizing chamber, column and detector are respectively 250°C, 150°C and 250°C. The initial temperature of column is 150°C and should be kept for three minutes. Then the temperature of column should be heated to 250°C using the speed of 8°C/min and the final temperature should be kept for eight minutes. The quantity of input sample is 10μL. </p>
 +
<p>Later, different change influences culture temperatures, PH values and inoculation quantity have on degrading organophosphorus can be set. Among these conditions, the organophosphorus pesticides are not degraded at the temperature below 32°C while they begin to be degraded at the temperature of over 32°C. The degradation effects generally must be good at neutral PH values. Generally, the more the inoculation quantity is, the better the degrading effects are. </p>
  
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<div class="highlight">
 
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<h5>Note</h5>
 
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<p>Note that parts must be documented on the <a href="http://parts.igem.org/Main_Page"> Registry</a>. This page serves to <i>showcase</i> the parts you have made. Future teams and other users and are much more likely to find parts by looking in the Registry than by looking at your team wiki.</p>
 
 
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<h3>Navigation</h3>
 +
<ul>
 +
<li><a href="index.html">Home</a></li>
 +
<li><a href="about.html">About</a></li>
 +
<li><a href="shortcodes.html">Short Codes</a></li>
 +
<li><a href="blog.html">Blog</a></li>
 +
<li><a href="contact.html">Contact</a></li>
 +
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<div class="clearfix"> </div>
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</div>
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</div>
 
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<h5>Adding parts to the registry</h5>
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<p>You can add parts to the Registry at our <a href="http://parts.igem.org/Add_a_Part_to_the_Registry">Add a Part to the Registry</a> link.</p>
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<p>We encourage teams to start completing documentation for their parts on the Registry as soon as you have it available. The sooner you put up your parts, the better you will remember all the details about your parts. Remember, you don't need to send us the DNA sample before you create an entry for a part on the Registry. (However, you <b>do</b> need to send us the DNA sample before the Jamboree. If you don't send us a DNA sample of a part, that part will not be eligible for awards and medal criteria.)</p>
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<h5>What information do I need to start putting my parts on the Registry?</h5>
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<p>The information needed to initially create a part on the Registry is:</p>
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<ul>
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<li>Part Name</li>
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<li>Part type</li>
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<li>Creator</li>
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<li>Sequence</li>
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<li>Short Description (60 characters on what the DNA does)</li>
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<li>Long Description (Longer description of what the DNA does)</li>
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<li>Design considerations</li>
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</ul>
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<p>
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We encourage you to put up <em>much more</em> information as you gather it over the summer. If you have images, plots, characterization data and other information, please also put it up on the part page. </p>
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<h5>Inspiration</h5>
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<p>We have a created  a <a href="http://parts.igem.org/Well_Documented_Parts">collection of well documented parts</a> that can help you get started.</p>
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<p> You can also take a look at how other teams have documented their parts in their wiki:</p>
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<ul>
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<li><a href="https://2014.igem.org/Team:MIT/Parts"> 2014 MIT </a></li>
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<li><a href="https://2014.igem.org/Team:Heidelberg/Parts"> 2014 Heidelberg</a></li>
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<li><a href="https://2014.igem.org/Team:Tokyo_Tech/Parts">2014 Tokyo Tech</a></li>
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<h5>Part Table </h5>
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<groupparts>iGEM2016 Example</groupparts>
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Revision as of 10:17, 18 October 2016

LN-shiyan-China

Parts

Our Submission Parts:

BBa_K1667005

This part includes two individual DNA domains. Constitutive promoter tunes the expression of downstream opdA gene with further help from RNA thermometer. RNA thermometer provides a temperature sensitive post-transcriptional regulation on opdA gene, which initiate the opdA translation around 32°C. OmpA signal peptide guides the secretion of opdA protein to the outside of host strain. Then opdA enzyme specifically degrades organophosphorus pesticide appeared, through hydrolysis. Upon UV light, RecA（SOS）promoter drives the transcription of downstream ccdB suicide gene, whose protein expression interferes DNA sysnthesis and lead to cell dealth. In this way, we can wipe out our genetic engineered bacteria by giving UV lights under manual control to avoid secondary pollution. Without UV light, RecA promoter won’t be activated, so the normal growth and activity of genetic engineering bacteria will be preserved well to release functional opdA protein under temperature control.

Experimentally Verification Scheme:

Bacteria strain BL21 (DE3) of colibacillus genetically engineered bacteria which contains our target plasmid goes through extended culture in our LB liquid substrate for sixteen hours under the temperature of 30°C, then centrifuge the culture for five minutes with the speed of 5000rpm under the temperature of 4°C and use sterile MSM (mineral base medium) to wash and sediment the bacteria, then use fresh MSM culture substrate to dilute the bacteria. Inoculate the bacteria to MSM culture substrate which contains 10μg/mL chlorpyrifos or methyl-parathion (the final concentration of bacteria is 106 CFU/mL), and culture it with the speed of 160rpm under the temperature of 37°C while shaking it. Regularly get samples from the solution and measure the concentration of organophosphorus pesticides in the solution. Measure the OD600 of the culture to indicate the growth of the bacteria, use the culture solution which is not inoculated as a blank control while using the colibacillus inoculated with the bacteria strain BL21 (DE3) which does not contain target plasmid as a reference control. Adopt gas chromatography to measure the concentration of organophosphorus pesticides in the culture.

Extraction of organophosphorus pesticides: Get 5mL MSM solution which is inoculated and cultured as above, add 25mL acetonitrile into it and shake it violently for five minutes, then add 2.5g sodium chloride into it and shake it violently, place the solution quietly for 30 minutes then laminarize it, get 1mL organic phase in the upper layer and dry it with pressure blowing concentrator, add 1mL acetone into it to dissolve above dry organic phase again.

Gas chromatography detecting conditions: Use Shimadzu 2014C gas chromatographic analyzer, equip it with fire photometric detector (FPD) and quartz capillary chromatographic column (length of column is 30 meters, inner diameter of column is 0.53mm, thickness of membrane layer is 1μm, RESTEX, USA), flow speed of nitrogen is lmL/min while the flow speeds of air and hydrogen are respectively 81.8mL/min and 3.2mL/min. The temperatures of vaporizing chamber, column and detector are respectively 250°C, 150°C and 250°C. The initial temperature of column is 150°C and should be kept for three minutes. Then the temperature of column should be heated to 250°C using the speed of 8°C/min and the final temperature should be kept for eight minutes. The quantity of input sample is 10μL.

Later, different change influences culture temperatures, PH values and inoculation quantity have on degrading organophosphorus can be set. Among these conditions, the organophosphorus pesticides are not degraded at the temperature below 32°C while they begin to be degraded at the temperature of over 32°C. The degradation effects generally must be good at neutral PH values. Generally, the more the inoculation quantity is, the better the degrading effects are.

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