Difference between revisions of "Team:NAWI-Graz/Design"

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{{NAWI-Graz}}
 
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By talking about your design work on this page, there is one medal criterion that you can attempt to meet, and one award that you can apply for. If your team is going for a gold medal by building a functional prototype, you should tell us what you did on this page.
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<p>This is a prize for the team that has developed a synthetic biology product to solve a real world problem in the most elegant way. The students will have considered how well the product addresses the problem versus other potential solutions, how the product integrates or disrupts other products and processes, and how its lifecycle can more broadly impact our lives and environments in positive and negative ways.</p>
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If you are working on art and design as your main project, please join the art and design track. If you are integrating art and design into the core of your main project, please apply for the award by completing this page.
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<h2 class="mt-xl mb-none"><span class="alternative-font font-size-md">Vector</span></h2>
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To run our project in the best manner, we have chosen a high-copy plasmid, pUC19 plasmid, which is compatible with RFC10 standard.
 +
</p>
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<h2 class="mt-xl mb-none"><span class="alternative-font font-size-md">Inserts</span></h2>
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The next thing was creating our inserts. First of all we had to keep in mind that we work with the RFC10 standard. To ensure the correct codon usage we got rid of various restriction sides.  We created two inserts for each of our two strains. One insert is coding for the toxin and the second insert is coding for the antitoxin.  
 
</p>
 
</p>
 +
<p>
 +
Our antitoxin has a very low half-life and for this reason we used a constitutive promoter. The same was used for our chromo protein, which should give us the possibility to detect the growth of the right bacteria without other measuring methods.
 +
 +
</p>
 +
<p>
 +
The second insert, carrying the toxin, has to be inducible to make sure our E.coli cells have enough time to produce the antitoxin before being killed by the toxin. Furthermore, the Dam-Methylase, which is responsible for a higher mutation rate should not be expressed from the beginning. So we decided to use the tac promoter which is inducible by IPTG. Unfortunately, we were not aware of the leakiness of tac at that time.
 +
</p>
 +
 +
<h2 class="mt-xl mb-none"><span class="alternative-font font-size-md">Assembly</span></h2>
 +
<p>
 +
Concerning assembly of the parts, we decided that the two inserts should work in the opposite direction. Hence we could use one bidirectional double-terminator in the middle of the two inserts. This design ensured in case the terminator does not work only the antitoxin is expressed by the constitutive promoter.
 +
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Revision as of 09:15, 12 October 2016

MENU ▤

NAWI-Graz @ iGem - Description

Vector

To run our project in the best manner, we have chosen a high-copy plasmid, pUC19 plasmid, which is compatible with RFC10 standard.

Inserts

The next thing was creating our inserts. First of all we had to keep in mind that we work with the RFC10 standard. To ensure the correct codon usage we got rid of various restriction sides. We created two inserts for each of our two strains. One insert is coding for the toxin and the second insert is coding for the antitoxin.

Our antitoxin has a very low half-life and for this reason we used a constitutive promoter. The same was used for our chromo protein, which should give us the possibility to detect the growth of the right bacteria without other measuring methods.

The second insert, carrying the toxin, has to be inducible to make sure our E.coli cells have enough time to produce the antitoxin before being killed by the toxin. Furthermore, the Dam-Methylase, which is responsible for a higher mutation rate should not be expressed from the beginning. So we decided to use the tac promoter which is inducible by IPTG. Unfortunately, we were not aware of the leakiness of tac at that time.

Assembly

Concerning assembly of the parts, we decided that the two inserts should work in the opposite direction. Hence we could use one bidirectional double-terminator in the middle of the two inserts. This design ensured in case the terminator does not work only the antitoxin is expressed by the constitutive promoter.

-->

Sponsoring

Location

NAWI Graz
Petersgasse 12
8010 Graz, AUT

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