Difference between revisions of "Team:SCSU-New Haven/Parts"

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<h1>Parts</h1>
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<p>
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You get the DNA to make the parts. You make the parts to build the system. You build the system to do the thing. You do the thing to make a difference.
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<h2>The Degrader</h2>
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<h3>BBa_K2143003</h3>
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<img src="https://static.igem.org/mediawiki/2016/8/84/SCSU-New_HavenParts_DegraderSimple.jpeg">
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<hr/>
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<p class="header_intro">
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This part contains the Nah Upper Operon from Pseudomonas putida which produces a series of enzymes that degrade Naphthalene into Salicylate. NahA1-NahA4 form the enzyme complex 1,2-Napthalene dioxygenase. The other 5 genes each code for a single enzyme.
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</p>
  
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<div class="sub_section">
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<h3><b>How does it work in your system?</b></h3>
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<p>
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Our project is all about detecting small amounts of an aromatic compound called 1-Mehtylnapthalene (1-MN). While our “Detector” can’t detect that compound, it can detect 3-methylsalicylate (3-MS) which is produced when our “Degrader” part’s enzymes act on 1-MN.
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</p>
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</div>
  
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<div class="sub_section">
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<h3><b>How did you choose this part?</b></h3>
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<p>The operon comes from the NAH7 Pseudomonas putida plasmid. When trying to find a way to detect 1-MN we came across the Naphthalene degradation pathway. We thought, if we can’t detect 1-MN, can we detect anything along the pathway?
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</p>
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</div>
  
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</div><h2>The Detector</h2>
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                                <h3>BBa_K2143001</h3>
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<img src="https://static.igem.org/mediawiki/2016/b/be/SCSU-New_HavenParts_Detector.png">
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<hr/>
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<p class="header_intro">
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Based on the Peking 2013 iGEM team’s Part:BBa_K1031610. NahR is a transcription factor that binds to a specific sequence upstream of the Psal promoter. Psal is then induced by the addition of salicylate like compounds. Our version of NahR has a single amino acid change at position 248 from Arginine to Histidine, this change causes the induction of Psal by NahR to become more sensitive to Salicylate.
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</p>
  
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<div class="column full_size">
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<div class="sub_section">
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<h3><b>How does it work in your system?</b></h3>
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<p>
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Our project is all about detecting small amounts of an aromatic compound called 1-Mehtylnapthalene (1-MN). While our “Detector” isn’t detecting that compound, it is detecting 3-methylsalicylate (3-MS) which is produced when our “Degrader” part’s enzymes work on 1-MN. Once our “Detector” turns on Psal, another part the  “Color Reporter” lets us know.
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</p>
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<img src="https://static.igem.org/mediawiki/2016/a/ac/SCSU-New_HavenLinkParts.png">
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</div>
  
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<div class="sub_section">
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<h3><b>How did you choose this part?</b></h3>
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<p>
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The mutant NahR came from a paper (10.1016/j.jbiotec.2010.09.936) comparing the inducibility of Psal using various NahR mutants. NahR_R248H seemed to show the highest output while still being only inducible by salicylate compounds. 
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</p>
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<img src="https://static.igem.org/mediawiki/2016/8/8b/SCSU-New_HavenParts_WTNahR.png"  height="500px" width="560px">
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<img src="https://static.igem.org/mediawiki/2016/f/f9/SCSU-New_HavenParts_MutNahR.png"  height="500px" width="560px">
  
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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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</div><h2>The Feedback</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>BBa_K2143000</h3> <img src="https://static.igem.org/mediawiki/2016/7/73/SCSU-New_HavenParts_Feedback.png">
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<hr/>
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<p class="header_intro">More NahR mutants! The WT NahR and the mutant version we are using in our “Detector” part requires Salicylate to fully induce Psal. This version can induce Psal without salicylate.  
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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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<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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<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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</div>
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<div class="sub_section">
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<h3><b>How does it work in your system?</b></h3>
 +
<p>
 +
It is used as a positive feedback loop. In our system, we’ve placed this part just downstream of Psal. So, once Psal gets turned on, even if only for a second by a tiny amount of 3-MS, the feedback kicks on, binds upstream of Psal and now Psal is on even if no more 3-MS is present.
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</p>
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<img src="https://static.igem.org/mediawiki/2016/a/a5/SCSU-New_HavenParts_FeedbackWork.png">
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</div>
  
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<div class="sub_section">
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<h3><b>How did you choose this part?</b></h3>
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<p>
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One of our goals with this project was to be able to report very small amounts of 1-MN present on the breath of an individual. We also wanted the indicator to be read without sensitive equipment. Therefore, we needed a strong output using a very low input. With the feedback loop, the input only needs to activate Psal once and NahR_L154F makes more of itself and the reporter downstream. We’ve put two sets of NahR_L154F in this part because our salicylate dependent NahR is still being produced. We thought doubling the concentration of this version would ensure it is the one binding upstream of Psal.
 +
</p>
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</div><h2>The Color Reporter</h2>
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<h3>BBa_K2143002</h3><img style="width:80%" src="https://static.igem.org/mediawiki/2016/1/19/SCSU-New_HavenParts_CR.jpeg">
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<hr/>
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<p class="header_intro"> This is the tryptophanase gene from E. coli!
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</p>
  
 +
<div class="sub_section">
 +
<h3><b>How does it work in your system?</b></h3>
 +
<p>
 +
One of the “Degrader” enzymes 1,2 -Naphthalene dioxygenase catalyzes the conversion of indole to indigo dye. Indole is produced when tryptophanase acts upon L-Tryptophan. We use a strain of E. coli with a nonfunctional tnaA gene, so when our color reporter is activated
 +
</p>
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</div>
  
 +
<div class="sub_section">
 +
<h3><b>How did you choose this part?</b></h3>
 +
<p>
 +
When looking at “Degrader” enzymes we came across the reaction involving indole to indigo dye. We liked using Indigo dye because it is insoluble, if the cells dye or time passes, the results remain.
 +
</p>
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</div><h2>The Whole Device</h2>
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<img style="width:80%" src="https://static.igem.org/mediawiki/2016/b/b1/SCSU-New_HavenParts_WholeSimple.jpeg">
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<hr/>
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<p class="header_intro">
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This is the whole device as it would be placed into PSB1A3. The device must be put into an ampicillin selection plasmid because the tryptophanase deficient chassis of E. coli we will be using already has chloramphenicol resistance markers.
 +
</p>
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<h3>References</h3>
 +
<p> Shin, Hae Ja. "Development of highly-sensitive microbial biosensors by mutation of the nahR regulatory gene." Journal of biotechnology 150.2 (2010): 246-250.</p>
 +
<p>Huang, J. Z., and M. A. Schell. "In vivo interactions of the NahR transcriptional activator with its target sequences. Inducer-mediated changes resulting in transcription activation." Journal of Biological Chemistry 266.17 (1991): 10830-10838. </p>
 +
<p>COLLINS, DESMOND, DEREK BRIGGS, and SIMON CONWAY MORRIS. "Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo." (1983).</p>
 +
<p>Cambray, Guillaume, et al. "Measurement and modeling of intrinsic transcription terminators." Nucleic acids research 41.9 (2013): 5139-5148.
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</p>
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</div>
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{{SCSU-New_Haven/Footer}}

Latest revision as of 22:47, 30 November 2016

SCSU IGEM SCSU IGEM

SCSU iGEM

Parts

You get the DNA to make the parts. You make the parts to build the system. You build the system to do the thing. You do the thing to make a difference.

The Degrader

BBa_K2143003

This part contains the Nah Upper Operon from Pseudomonas putida which produces a series of enzymes that degrade Naphthalene into Salicylate. NahA1-NahA4 form the enzyme complex 1,2-Napthalene dioxygenase. The other 5 genes each code for a single enzyme.

How does it work in your system?

Our project is all about detecting small amounts of an aromatic compound called 1-Mehtylnapthalene (1-MN). While our “Detector” can’t detect that compound, it can detect 3-methylsalicylate (3-MS) which is produced when our “Degrader” part’s enzymes act on 1-MN.

How did you choose this part?

The operon comes from the NAH7 Pseudomonas putida plasmid. When trying to find a way to detect 1-MN we came across the Naphthalene degradation pathway. We thought, if we can’t detect 1-MN, can we detect anything along the pathway?

The Detector

BBa_K2143001

Based on the Peking 2013 iGEM team’s Part:BBa_K1031610. NahR is a transcription factor that binds to a specific sequence upstream of the Psal promoter. Psal is then induced by the addition of salicylate like compounds. Our version of NahR has a single amino acid change at position 248 from Arginine to Histidine, this change causes the induction of Psal by NahR to become more sensitive to Salicylate.

How does it work in your system?

Our project is all about detecting small amounts of an aromatic compound called 1-Mehtylnapthalene (1-MN). While our “Detector” isn’t detecting that compound, it is detecting 3-methylsalicylate (3-MS) which is produced when our “Degrader” part’s enzymes work on 1-MN. Once our “Detector” turns on Psal, another part the “Color Reporter” lets us know.

How did you choose this part?

The mutant NahR came from a paper (10.1016/j.jbiotec.2010.09.936) comparing the inducibility of Psal using various NahR mutants. NahR_R248H seemed to show the highest output while still being only inducible by salicylate compounds.

The Feedback

BBa_K2143000

More NahR mutants! The WT NahR and the mutant version we are using in our “Detector” part requires Salicylate to fully induce Psal. This version can induce Psal without salicylate.

How does it work in your system?

It is used as a positive feedback loop. In our system, we’ve placed this part just downstream of Psal. So, once Psal gets turned on, even if only for a second by a tiny amount of 3-MS, the feedback kicks on, binds upstream of Psal and now Psal is on even if no more 3-MS is present.

How did you choose this part?

One of our goals with this project was to be able to report very small amounts of 1-MN present on the breath of an individual. We also wanted the indicator to be read without sensitive equipment. Therefore, we needed a strong output using a very low input. With the feedback loop, the input only needs to activate Psal once and NahR_L154F makes more of itself and the reporter downstream. We’ve put two sets of NahR_L154F in this part because our salicylate dependent NahR is still being produced. We thought doubling the concentration of this version would ensure it is the one binding upstream of Psal.

The Color Reporter

BBa_K2143002

This is the tryptophanase gene from E. coli!

How does it work in your system?

One of the “Degrader” enzymes 1,2 -Naphthalene dioxygenase catalyzes the conversion of indole to indigo dye. Indole is produced when tryptophanase acts upon L-Tryptophan. We use a strain of E. coli with a nonfunctional tnaA gene, so when our color reporter is activated

How did you choose this part?

When looking at “Degrader” enzymes we came across the reaction involving indole to indigo dye. We liked using Indigo dye because it is insoluble, if the cells dye or time passes, the results remain.

The Whole Device

This is the whole device as it would be placed into PSB1A3. The device must be put into an ampicillin selection plasmid because the tryptophanase deficient chassis of E. coli we will be using already has chloramphenicol resistance markers.

References

Shin, Hae Ja. "Development of highly-sensitive microbial biosensors by mutation of the nahR regulatory gene." Journal of biotechnology 150.2 (2010): 246-250.

Huang, J. Z., and M. A. Schell. "In vivo interactions of the NahR transcriptional activator with its target sequences. Inducer-mediated changes resulting in transcription activation." Journal of Biological Chemistry 266.17 (1991): 10830-10838.

COLLINS, DESMOND, DEREK BRIGGS, and SIMON CONWAY MORRIS. "Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo." (1983).

Cambray, Guillaume, et al. "Measurement and modeling of intrinsic transcription terminators." Nucleic acids research 41.9 (2013): 5139-5148.