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

 
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<h1>Parts</h1>
 
<h1>Parts</h1>
 
<p>
 
<p>
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Herein i have inputted the introductory words to this here section. You see. The project is compromised of parts. Yes PARTS! You would be surprised at how difficult it was to arrive at this conclusion. With the various messons and protons that continously denied our attempts to locate the god part-icle. If you thought, that our attempts were futile, then you stand in err. For why else would I stand here, ready to present the parts that comprise the whole of the matter? 
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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.
 
</p>
 
</p>
 
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<!-- Content -->
 
<!-- Content -->
 
<div id="parts_content">
 
<div id="parts_content">
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<h2>Section Title</h2>
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<h2>The Degrader</h2>
 +
<h3>BBa_K2143003</h3>
 +
<img src="https://static.igem.org/mediawiki/2016/8/84/SCSU-New_HavenParts_DegraderSimple.jpeg">
 
<hr/>
 
<hr/>
 
<p class="header_intro">
 
<p class="header_intro">
−
Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's standard dummy text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting,
+
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.
 
</p>
 
</p>
  
 
<div class="sub_section">
 
<div class="sub_section">
−
<h3>SubSection Title</h3>
+
<h3><b>How does it work in your system?</b></h3>
 
<p>
 
<p>
−
Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's <b>standard dummy</b> text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting,
+
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.  
 
</p>
 
</p>
 
</div>
 
</div>
  
 
<div class="sub_section">
 
<div class="sub_section">
−
<h3>SubSection Title</h3>
+
<h3><b>How did you choose this part?</b></h3>
 +
<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?
 +
</p>
 +
</div>
 +
 
 +
</div><h2>The Detector</h2>
 +
                                <h3>BBa_K2143001</h3>
 +
<img src="https://static.igem.org/mediawiki/2016/b/be/SCSU-New_HavenParts_Detector.png">
 +
<hr/>
 +
<p class="header_intro">
 +
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.
 +
</p>
 +
 
 +
<div class="sub_section">
 +
<h3><b>How does it work in your system?</b></h3>
 
<p>
 
<p>
−
Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's <b>standard dummy</b> text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting,
+
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.
 
</p>
 
</p>
 +
<img src="https://static.igem.org/mediawiki/2016/a/ac/SCSU-New_HavenLinkParts.png">
 
</div>
 
</div>
  
 
<div class="sub_section">
 
<div class="sub_section">
−
<h3>SubSection Title</h3>
+
<h3><b>How did you choose this part?</b></h3>
 
<p>
 
<p>
−
Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's <b>standard dummy</b> text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting,
+
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. 
 
</p>
 
</p>
−
</div><h2>Section Title</h2>
+
<img src="https://static.igem.org/mediawiki/2016/8/8b/SCSU-New_HavenParts_WTNahR.png"  height="500px" width="560px">
 +
<img src="https://static.igem.org/mediawiki/2016/f/f9/SCSU-New_HavenParts_MutNahR.png"  height="500px" width="560px">
 +
 
 +
</div><h2>The Feedback</h2>
 +
<h3>BBa_K2143000</h3> <img src="https://static.igem.org/mediawiki/2016/7/73/SCSU-New_HavenParts_Feedback.png">
 
<hr/>
 
<hr/>
−
<p class="header_intro">
+
<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.
−
Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's standard dummy text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting,
+
 
</p>
 
</p>
  
 
<div class="sub_section">
 
<div class="sub_section">
−
<h3>SubSection Title</h3>
+
<h3><b>How does it work in your system?</b></h3>
 
<p>
 
<p>
−
Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's <b>standard dummy</b> text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting,
+
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.
 
</p>
 
</p>
 +
<img src="https://static.igem.org/mediawiki/2016/a/a5/SCSU-New_HavenParts_FeedbackWork.png">
 
</div>
 
</div>
  
 
<div class="sub_section">
 
<div class="sub_section">
−
<h3>SubSection Title</h3>
+
<h3><b>How did you choose this part?</b></h3>
 
<p>
 
<p>
−
Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's <b>standard dummy</b> text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting,
+
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>
 +
</div><h2>The Color Reporter</h2>
 +
<h3>BBa_K2143002</h3><img style="width:80%" src="https://static.igem.org/mediawiki/2016/1/19/SCSU-New_HavenParts_CR.jpeg">
 +
<hr/>
 +
<p class="header_intro"> This is the tryptophanase gene from E. coli!
 +
 +
</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>
 
</p>
 
</div>
 
</div>
  
 
<div class="sub_section">
 
<div class="sub_section">
−
<h3>SubSection Title</h3>
+
<h3><b>How did you choose this part?</b></h3>
 
<p>
 
<p>
−
Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's <b>standard dummy</b> text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting,
+
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>
 
</p>
−
</div>
+
</div><h2>The Whole Device</h2>
 +
                               
 +
<img style="width:80%" src="https://static.igem.org/mediawiki/2016/b/b1/SCSU-New_HavenParts_WholeSimple.jpeg">
 +
<hr/>
 +
<p class="header_intro">
 +
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>
 +
<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.
 +
</p>
 
</div>
 
</div>
 
</div>
 
</div>

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.