Difference between revisions of "Team:Lambert GA/Parts"

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<h2> Part Documentation</h2>
 
<h2> Part Documentation</h2>
  
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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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<h2>PARTS</h2>
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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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<h3>Main purpose of all the constructs:</h3>
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<p> All of the constructs contain these genes: The pelB leader sequence, mature CDA2 gene, and the eGFP gene. The pelB leader sequence is necessary because it lead the CDA gene into the periplasm. The expression of the CDA gene in the periplasm will be non toxic to the E. coli cells. The eGFP will allow us to physically see that the DNA constructs were successfully transcribed and translated.  The CDA protein produced will then be available to use to break down chitin and convert it into chitosan which has many industrial applications.</p>
  
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<div class="highlightBox">
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<h3>How to purify and use:</h3>
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<h4>Note</h4>
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<p>The constructs were ordered online at IDT and they came as gBlocks. (each construct contained 1000 ng). The constructs were resuspended as the instructions that came with the gBlocks stated. Then, they were digested (to eliminate the biobricks) and ligated into pSB1C3 and pSB1T3 plasmids. Once they were ligated into the plasmids, they were transformed into competent E. coli cells so that the genes may be expressed. </p>
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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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</div>
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<h4>Construct 1: Bicistronic</h4>
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<img src="https://static.igem.org/mediawiki/2015/f/f2/Lambert_project_bicistronic_fixed.png" style="width:100%"/>
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<p>The bicistronic construct consists of two promoters: the TetR promoter and the P(Lac) IQ promoter. This construct creates two different mRNA transcripts. One transcript will consist of the pelB leader sequence and the mature CDA2 gene which are both expressed under the TetR promoter while the other transcript will have the eGFP gene which is expressed under the P(Lac)IQ promoter. The eGFP will remain in the cytoplasm while the pelB leader sequence will lead the mature CDA2 gene into the periplasm. This means that the entire E. coli will glow green if the DNA construct is transcribed and translated correctly.</p>
  
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<h4>Construct 2: Bidirectional</h4>
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<img src="https://static.igem.org/mediawiki/2015/a/aa/Lambert_project_bidirectional.png" style="width:100%"/>
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<p>The bidirectional construct only uses one promoter: the TetR promoter. This serves as an advantage because unlike the bicistronic construct, only one antibiotic (tetracycline) is needed to control gene expression. The construct puts two TetR promoters back to back with a multiple cloning site in between them. One TetR promoter controls the pelB + mature CDA2 genes while the other controls the eGFP gene. Once again, this creates two separate mRNA transcripts and the eGFP will be expressed in the cytoplasm while the mature CDA2 gene is led into the periplasm by the pelB leader sequence, just like the bicistronic construct.</p>
  
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<h4>Adding parts to the registry</h4>
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<h4>Construct 3: Fusion</h4>
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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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<img src="https://static.igem.org/mediawiki/2015/e/e2/Lambert_project_fusion.jpg" style="width:100%"/>
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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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<p>The fusion construct also uses one promoter: the TetR promoter. However, unlike all the other constructs, all of the genes are connected together and it only produces one mRNA transcript. Both of the mature CDA2 gene and eGFP gene will be led into the periplasm by the pelB leader sequence. This will mean the eGFP will be hard to see because it will be only produced in the periplasm. This construct was the easiest to design, but the fact that the gene is so long might be problematic.</p>
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<h4>What information do I need to start putting my parts on the Registry?</h4>
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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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<h4>Inspiration</h4>
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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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</ul>
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<h4>Part Table </h4>
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</html>
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<groupparts>iGEM015 Example</groupparts>
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<html>
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</div>
 
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Revision as of 01:40, 17 September 2015

Part Documentation

PARTS

Main purpose of all the constructs:

All of the constructs contain these genes: The pelB leader sequence, mature CDA2 gene, and the eGFP gene. The pelB leader sequence is necessary because it lead the CDA gene into the periplasm. The expression of the CDA gene in the periplasm will be non toxic to the E. coli cells. The eGFP will allow us to physically see that the DNA constructs were successfully transcribed and translated. The CDA protein produced will then be available to use to break down chitin and convert it into chitosan which has many industrial applications.

How to purify and use:

The constructs were ordered online at IDT and they came as gBlocks. (each construct contained 1000 ng). The constructs were resuspended as the instructions that came with the gBlocks stated. Then, they were digested (to eliminate the biobricks) and ligated into pSB1C3 and pSB1T3 plasmids. Once they were ligated into the plasmids, they were transformed into competent E. coli cells so that the genes may be expressed.

Construct 1: Bicistronic

The bicistronic construct consists of two promoters: the TetR promoter and the P(Lac) IQ promoter. This construct creates two different mRNA transcripts. One transcript will consist of the pelB leader sequence and the mature CDA2 gene which are both expressed under the TetR promoter while the other transcript will have the eGFP gene which is expressed under the P(Lac)IQ promoter. The eGFP will remain in the cytoplasm while the pelB leader sequence will lead the mature CDA2 gene into the periplasm. This means that the entire E. coli will glow green if the DNA construct is transcribed and translated correctly.

Construct 2: Bidirectional

The bidirectional construct only uses one promoter: the TetR promoter. This serves as an advantage because unlike the bicistronic construct, only one antibiotic (tetracycline) is needed to control gene expression. The construct puts two TetR promoters back to back with a multiple cloning site in between them. One TetR promoter controls the pelB + mature CDA2 genes while the other controls the eGFP gene. Once again, this creates two separate mRNA transcripts and the eGFP will be expressed in the cytoplasm while the mature CDA2 gene is led into the periplasm by the pelB leader sequence, just like the bicistronic construct.

Construct 3: Fusion

The fusion construct also uses one promoter: the TetR promoter. However, unlike all the other constructs, all of the genes are connected together and it only produces one mRNA transcript. Both of the mature CDA2 gene and eGFP gene will be led into the periplasm by the pelB leader sequence. This will mean the eGFP will be hard to see because it will be only produced in the periplasm. This construct was the easiest to design, but the fact that the gene is so long might be problematic.