Difference between revisions of "Team:UNOTT/Design1"

 
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   <figure><img src="https://www.iconexperience.com/_img/g_collection_png/standard/256x256/brain.png"><figcaption style="color: #ffffff;">Brainstorm</figcaption></figure>
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   <figure><a href="https://2017.igem.org/Team:UNOTT/Design1"><img src="https://www.iconexperience.com/_img/g_collection_png/standard/256x256/dna.png"><figcaption style="color: #ffffff;">Plasmid Design</figcaption></figure>
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   <figure><a href="https://2017.igem.org/Team:UNOTT/Design2"><img src="https://www.iconexperience.com/_img/g_collection_png/standard/128x128/key.png"><figcaption style="color: #ffffff;">Key. coli Design</figcaption></figure>
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   <figure><a href="https://2017.igem.org/Team:UNOTT/Brainstorms"><img src="https://www.iconexperience.com/_img/g_collection_png/standard/256x256/brain.png"><figcaption style="color: #ffffff;">Brainstorm</figcaption></figure>
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   <figure><a href="https://2017.igem.org/Team:UNOTT/Design1"><img src="https://www.iconexperience.com/_img/g_collection_png/standard/256x256/dna.png"><figcaption style="color: #ffffff;">Design Process</figcaption></figure>
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   <figure><a href="https://2017.igem.org/Team:UNOTT/Design2"><img src="https://www.iconexperience.com/_img/g_collection_png/standard/128x128/key.png"><figcaption style="color: #ffffff;">Key. coli Plasmid Design</figcaption></figure>
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   <figure><a href="https://2017.igem.org/Team:UNOTT/Design3"><img src="https://www.iconexperience.com/_img/g_collection_png/standard/256x256/window_key.png"><figcaption style="color: #ffffff;">Key Transport Design</figcaption></figure></a><figure><a href="https://2017.igem.org/Team:UNOTT/Design4"><img src="https://static.igem.org/mediawiki/2017/3/35/T--UNOTT--ROCKET.png"><figcaption style="color: #ffffff;">Future Improvements</figcaption></a></figure>
  
 
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<h1>Design</h1>
 
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Although our project is applied in the idea of Key. coli, we are actually looking at creating a general system for creating combinations of metabolites and reporters. We knew that we wanted to design a method where an expandable range of products can be expressed at various levels in order to create a large amount of combinations with randomness coming from the assortment of these promoters to different reporters.
 
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<h1>Design Process</h1>
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<h2> The Prerequisite</h2>
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<p><br>The idea of how to make bacteria which could be used as a security system evolved over several weeks. The key requirement of the system is the ability to generate <b>many random</B> combinations of expression outputs to ensure security.
 +
</p><br><br>
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<h2>Transposons</h2>
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<br><p>One of the first ideas regarding how this could be achieved was with the use of transposons. <br><br>
 +
Transposable elements are DNA fragments that can change their position within a genome. This creates mutations resulting in different levels/suppression of expression of certain genes. The use of transposons would yield bacteria with various phenotypes, which could be used in the <i>Key. coli</i> security system. A target site-specific Tn7 transposon could be used for this purpose. This bacterial mobile DNA segment inserts at high-frequency into a single specific site, called attTn7 in <i>E. coli</i><sup>1</sup>. <br><br><br>
 +
<img src="https://static.igem.org/mediawiki/2017/8/87/Deseign_pic_1.png"; style="float:left;"><br><br>Credit: Phillip Dumesic, UCSF (Adapted from Transposon by Lauren Solomon, Broad Institute
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<br><br><br><br><br><br><br><br><br><br>
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<p>The Tn7 system has been commonly used to generate random mutation libraries within a broad range of organisms whereby the machinery of the transposon system is expressed by the host organism. Unfortunately, this is not a good option in our case; expression of the transposition machinery in the host organism will create an inevitable bias, with transposition events most likely resulting in configurations that are associated with a smaller metabolic cost.
 +
<br><br>
 +
A solution to this problem would be to do the transposition in vitro. However, further research revealed several disadvantages with this idea. The mechanisms of Tn7 recombination are complicated, requiring many proteins for successful transposition. Although we thought this would be interesting to test, we came across difficulties with sourcing the TsnD subunit of the Tn7 transposon. As expression and purification of this subunit was not realistic within the timeframe we had, we decided to pursue a different method.<br><br>
 +
<h2>CRISPRi (dcas9 mediated gene repression)</h2><p>
 +
<br>The next idea on how to obtain different phenotypes of bacteria was the use of RNA interference. In this process RNA molecules inhibit gene expression on the transcriptional level.
 +
<br><br>
 +
An extensive literature search revealed that the CRISPR interference system seems to be a reliable and predictable RNA interference mechanism. CRISPRi influences gene expression primarily at the transcriptional level and allows sequence-specific control of gene expression. This method utilises the CRISPR pathway and a catalytically inactive Cas9 (dCas9) protein. In the traditional type II CRISPR system, Cas9 introduces double-stranded breaks in specific genomic sequences, guided by a short guide RNA (sgRNA). dCas9 is engineered to lack nuclease activity enabling repurposing of the system for genomic DNA targeting without cleavage and therefore allowing precise transcription regulation<sup>2</sup>.
 +
<br><br>
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<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2017/3/3e/T--UNOTT--cas9sgRNAcomplex.png"; style="float:center;"></p>
 
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Initially, we thought of the idea of using transposons to shuffle different strength promoters to assort them randomly to various reporters to give various levels of products. EXPLAIN IDEA HERE. Although we thought this would be really interesting to test, we came accross difficulties with sourcing the TsnD subunit of the Tn7 transposon. As expression and purification of this subunit was not realistic within the timeframe we had so we decided a new method would be needed.
+
<br><br><br>As a result, we came up with an idea that a variety of reporters could be under the control of promoters which can be targeted by sgRNAs and dCas9. The sgRNA-dCas9 complex can be targeted to bind to the sequence of the promoter. By doing so, it physically interferes with translation, inhibiting the transcription machinery from recognising and binding to the promoter.
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</p>
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<br><br>
 +
<sup>1</sup>Peters, J. E. (2014). Tn7. Microbiology Spectrum, 2(5). doi:10.1128/microbiolspec.mdna3-0010-2014<br>
 +
<sup>2</sup>Dominguez, A. A., Lim, W. A., & Qi, L. S. (2016). Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nature Reviews. Molecular Cell Biology, 17(1), 5–15. http://doi.org/10.1038/nrm.2015.2
  
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<p> Next we looked at using RNAi or CRISPRi. After a literature search, CRISPRi seemed to be more reliable and predictable than RNAi so we decided to use it. We came up with an idea that a variety of reporters within a plasmid could be under the control of promoters which can be targeted by guide RNAs. CRISPR interference (CRISPRi) uses dCas9 which is a nuclease-deficient enzyme that uses the RNA-guided DNA binding of Cas9 but represses expression by interfering with RNA polymerase binding instead of cutting the DNA. By linking gRNA targeted promoters up to the genes for various reporters we can control the level of expression of these reporters by providing a targeting or non-targeting gRNA to give an ON/OFF switch.
 
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<h5>Proof of concept</h5>
 
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<p>Initially we will construct a promoter-gRNA library, then 3 of these promoters to a reporter and test the effect of the corresponding gRNA on repression levels. In further work, we have designed an assembly method where a pool of promoters could be used so that the promoter used would be random.
 
 
</p>
 
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<p> Each individual Promoter-Reporter-Terminator brick contains interchangable parts. The three parts are linked together with Bsa1 sites so that there is no preference for any part when ligating together. This allows randomness to be added later. This method is used also for the construction of Promoter-sgRNA-Terminator bricks so that this could be randomised later on. The bricks are then flanked by a prefix and suffix, and these are flanked by restriction sites ABCD on either end. Digestion of bricks with A+B, B+C, and C+D allows any brick to be placed in any position within the plasmid but it would be pre-determined. This means that the no one promoter-reporter-terminator brick would be limited to one specific place in the plasmid, which allows another level of randomness in assembly as we would not know which reporter was being placed where, which could also affect expression levels.</p>
 
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<p> We chose to express the reporters and dCas9 on a low copy plasmid due to possible toxicity.</p>
 
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<h5>sgRNA expression plasmid</h5>
 
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<p>This plasmid is high copy so that the sgRNAs can be in excess.</p>
 
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<h3><u>Future work</u></h3>
 
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We can think of a few ways by which we could expand the possible combinations for our system:
 
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<ul>
 
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<li>Introducing more possible reporters/products into the bricks</li>
 
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<li>Characterising more and more promoter-gRNA combinations</li>
 
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<li>Using gRNAs with single point mutations in the seed region which could give different levels of repression</li>
 
  
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<p style="text-align: center;"><img src="https://static.igem.org/mediawiki/2017/6/67/UNOTT2017-jkeydesign.png" alt="" width="700" height="500" />&nbsp;</p>
 
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Latest revision as of 03:24, 2 November 2017






Design Process

The Prerequisite


The idea of how to make bacteria which could be used as a security system evolved over several weeks. The key requirement of the system is the ability to generate many random combinations of expression outputs to ensure security.



Transposons


One of the first ideas regarding how this could be achieved was with the use of transposons.

Transposable elements are DNA fragments that can change their position within a genome. This creates mutations resulting in different levels/suppression of expression of certain genes. The use of transposons would yield bacteria with various phenotypes, which could be used in the Key. coli security system. A target site-specific Tn7 transposon could be used for this purpose. This bacterial mobile DNA segment inserts at high-frequency into a single specific site, called attTn7 in E. coli1.




Credit: Phillip Dumesic, UCSF (Adapted from Transposon by Lauren Solomon, Broad Institute









The Tn7 system has been commonly used to generate random mutation libraries within a broad range of organisms whereby the machinery of the transposon system is expressed by the host organism. Unfortunately, this is not a good option in our case; expression of the transposition machinery in the host organism will create an inevitable bias, with transposition events most likely resulting in configurations that are associated with a smaller metabolic cost.

A solution to this problem would be to do the transposition in vitro. However, further research revealed several disadvantages with this idea. The mechanisms of Tn7 recombination are complicated, requiring many proteins for successful transposition. Although we thought this would be interesting to test, we came across difficulties with sourcing the TsnD subunit of the Tn7 transposon. As expression and purification of this subunit was not realistic within the timeframe we had, we decided to pursue a different method.

CRISPRi (dcas9 mediated gene repression)


The next idea on how to obtain different phenotypes of bacteria was the use of RNA interference. In this process RNA molecules inhibit gene expression on the transcriptional level.

An extensive literature search revealed that the CRISPR interference system seems to be a reliable and predictable RNA interference mechanism. CRISPRi influences gene expression primarily at the transcriptional level and allows sequence-specific control of gene expression. This method utilises the CRISPR pathway and a catalytically inactive Cas9 (dCas9) protein. In the traditional type II CRISPR system, Cas9 introduces double-stranded breaks in specific genomic sequences, guided by a short guide RNA (sgRNA). dCas9 is engineered to lack nuclease activity enabling repurposing of the system for genomic DNA targeting without cleavage and therefore allowing precise transcription regulation2.




As a result, we came up with an idea that a variety of reporters could be under the control of promoters which can be targeted by sgRNAs and dCas9. The sgRNA-dCas9 complex can be targeted to bind to the sequence of the promoter. By doing so, it physically interferes with translation, inhibiting the transcription machinery from recognising and binding to the promoter.

1Peters, J. E. (2014). Tn7. Microbiology Spectrum, 2(5). doi:10.1128/microbiolspec.mdna3-0010-2014
2Dominguez, A. A., Lim, W. A., & Qi, L. S. (2016). Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nature Reviews. Molecular Cell Biology, 17(1), 5–15. http://doi.org/10.1038/nrm.2015.2