Difference between revisions of "Team:EPFL/Description"

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                        <h2 class="lead animate-box text-center">intelligene</h2>
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                            Far far away, behind the word mountains, far from the countries Vokalia and Consonantia.
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<img style="max-width: 100%;" src="https://static.igem.org/mediawiki/2016/4/4a/Nihms645765f1.jpg">
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<p><strong>Description summary:</strong></p>
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<p>CRISPR-Cas9 has already revolutionized synthetic biology. To build upon this development we aim to implement digital-like circuits in yeast using a CRISPR-associated RNA scaffold system (Zalatan et al, 2015). Recently, a study published the use of the modular software CELLO which automates the design of DNA circuits using transcription factors in E. coli. As a proof of concept we will modify CELLO to use our dCas9 transistors in yeast for a so-called half-adder system, using AND and XOR gates, that we can then experimentally assess. With this approach we hope to pave the way for even more complex biological circuits in yeasts.</p>
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                            In the 2016’s edition of iGEM, we, the EPFL team, decided to work on biological circuits.  
−
<p><strong>What have we done?</strong></p>
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                            Our main goal was, on one hand, to improve the already-existing tools used for the design of biological circuits.  
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<p>We started brainstorming in December and quickly decided to work on the creation of a biological circuit.<br />We were inspired by the EPFL&rsquo;s 2015 iGEM team, who worked on bioLOGIC. This system uses a catalytically dead version of Cas9 fused with an RNA Polymerase recruiting element (VP64) to create transistors, and depending on the identity of the promoter that dCas9-VP64 binds, it will either be repressed or activated.</p>
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                            On the other hand, we aspired to automate the design of such circuits by improving a new software called Cello, which would save valuable time and reduce the effort of scientists and researchers working on this topic all over the world.
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<p>At first, we created brainstorming groups to find applications of the project. The idea of creating a half-adder stood out from the rest for its possible applications as well as its suitability as a proof of concept. Later, we discovered a program called CELLO that automates the design of DNA based logic circuits.</p>
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                            In order to achieve this, the system required two important things: a modular way to create circuits and a software that allows the automation of circuit design.
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<p>At this point, we split into two groups. The first group worked on the design of the system, the second on the understanding of Cello&rsquo;s software in order to implement it with our system.</p>
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<img src="https://upload.wikimedia.org/wikipedia/commons/thumb/d/d9/Half_Adder.svg/200px-Half_Adder.svg.png" align="right" />
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            </section>
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The project was defined as to create simple gates using biological parts. We wanted to use d-Cas9 to target specific sequences of promoters and therefore be able to activate or repress the expression of the genes controlled by them. In order to build biosensors, we imagined a system that allows our gates to respond differently to various environments, such as presence of galactose. <br />We also want to implement our system in yeast as they are well representation of mammalian cells and easy to handle. With this system we aim to create an half-adder which correspond to a XOR and an AND gate linked together.</p>
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<p>As mentioned before, we also plan to modify CELLO to be able to design genetic circuits in yeast using it. Fortunately, CELLO has a modular nature, allowing us to do this easily. CELLO has a User Constraint File that enables users to pass the program information about this system it is designing the circuit for. This file includes information pertaining to the species, the reactivity of gates to inputs, and the plasmids used. In order to obtain this new information, we plan on characterizing our system and gates using photometric experiments.</p>
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<p>During the process of designing our system, we stumbled upon a paper outlining a more intuitive way to activate and inhibit genes with dCas9, and we decided to improve our project using its results.</p>
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                    <div class="col-md-10 col-md-offset-1">
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<p>This paper describes synthetic dcas9-based transcriptional programs in yeast. Instead of having the dcas9 unit fused to an activator or repressor protein, the guide RNA is extended to include an effector protein recruitment site, so that scaffold RNAs that encode both target locus and regulatory action.</p>
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                        <h2>General idea of the project</h2>
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<p>Using a dCas9 based system with scaffold guide RNAs offers numerous advantages with regards to previous biological circuit designing systems. Firstly, using gRNAs as parts of gates, instead of transcription factors reduces toxicity related to transcription factor density in the nucleus. In addition, our system can be even more complex than systems based on transcription factors since the amount of connections between gates are not limited by the amount of transcription factors available. Finally, the use of scaffolding RNAs simplifies design, since we can have just one dCas9, and it will also hopefully lead to more predictable repression and activation in the system. </p>
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<p>
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<img src="https://static.igem.org/mediawiki/2016/3/34/Repression_schema_expectation.png" style="max-width: 100%;" />
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</p>
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                    <div class="col-md-10 col-md-offset-1">
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</div>
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                        <p class="sub-lead">
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</div>  
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                            We based our project on CRISPR/(d)Cas9, a system largely used for genome engineering. Taking the catalytically dead dCas9 from this system allows the targeted binding of almost any DNA sequence. dCas9 is a mutant of the original Cas9 protein that lost its ability to cut DNA, but can still bind to DNA sequences using a specific guide RNA (gRNA). The only limitation for the target is that it has to lie next to a 3-base-pairs “PAM” sequence, NGG, N being any nucleotide. At the molecular level, the gRNA encodes the target sequence which is then ‘loaded’ into the dCas9, which, in turn then, ‘opens up’ the two DNA strands at the target sequence. Changing the sequence of the gRNA means changing which DNA sequence is targeted. In this way, it is possible to target different regions of a promoter. Moreover, dCas9 can be fused with activating transcriptional effectors to increase the expression of the promoter it binds to. This allows for the activation of the downstream gene that is associated to the promoter region.
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                        <p class="sub-lead">
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                            The main disadvantage of this system was that it only relied on allosteric repression to turn genes off by not allowing the polymerase to bind to the targeted promoter. This type of repression is not entirely reliable and is not performed by a “real” repressor, making it less efficient.  
 +
                        </p>
 +
                        <p class="sub-lead">
 +
                            With these limitations in mind, we set out to develop a system that was more intuitive, precise, and dynamic.
 +
                        </p>
 +
                    </div>
 +
                    <div class="spacer h20"></div>
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                    <div class="col-md-5 col-md-offset-1">
 +
                        <div class="spacer"></div>
 +
                        <figure>
 +
                            <img src="img/figures/dCas9_CRISPRi.png" />
 +
                            <figcaption class="text-center">FIGURE 1: Two ways to act on the expression of a promoter through CRISPR/dCas9. Figure adapted from Zalatan et al. 2015.</figcaption>
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                        </figure>
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                    <div class="col-md-5">
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                        <div class="spacer"></div>
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                        <!--TODO-->
 +
                        <figure>
 +
                            <img src="img/figures/dCas9_scRNA.png" />
 +
                            <figcaption class="text-center">FIGURE 1: Two ways to act on the expression of a promoter through CRISPR/dCas9. Figure adapted from Zalatan et al. 2015.</figcaption>
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                        </figure>
 +
                    </div>
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                </div>
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            </section>
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        </div>
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        <div class="simple-page animate-box">
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            <section>
 +
                <div class="container">
 +
                    <div class="col-md-10 col-md-offset-1">
 +
                        <h2>How to obtain a <b>modular</b> system?</h2>
 +
                        <div class="spacer h20"></div>
 +
                        <hr/>
 +
                        <div class="spacer h20"></div>
 +
                        <p class="sub-lead">
 +
                            Modularity is the ability of a system to be composed of simple pieces that can then be recombined to form another one. It is also the key to obtaining an easy-to-use and understandable tool. Something that was unavailable until recently, was a truly modular way to achieve complex biological circuits composed of many different gates. The choice of the promoter was pivotal as the ability to use only one promoter in entire circuits would significantly simplify the system many times over. We had to find a promoter with distinct regions that could be targeted by different gRNAs: some for gene activation and other for repression. The sequences of those regions would also have to be modifiable to avoid cross-talk between all the gRNAs composing a given biological circuit.  
 +
                        </p>
 +
                        <!-- TODO: immagine modularità -->
 +
                    </div>
 +
            </section>
 +
        </div>
 +
        <div class="simple-page animate-box">
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            <section>
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                <div class="container">
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                    <div class="col-md-10 col-md-offset-1">
 +
                        <h2 class="">
 +
                            Making it work when you want to,
 +
                            means making it <b>inducible</b>
 +
                        </h2>
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                        <div class="spacer h20"></div>
 +
                        <hr/>
 +
                        <div class="spacer h20"></div>
 +
                        <p class="sub-lead">
 +
                            As we now solved the issue of modularity of our system, it would be a lost opportunity to only end up with a tool that is not able to adapt to its surrounding. Adapting to its surrounding means requiring inducible promoters that do not interfere with the vital functions of the cell but still detect the molecule of interest. These promoters would allow researchers to achieve functional biosensors for many applications by triggering distinct responses in the cell according to the signals they detect.
 +
                            <br/>
 +
                            <br/>
 +
                            <img src="img/figures/inducibility_concept.png" />
 +
                        </p>
 +
                        <!-- TODO: immagine modularità -->
 +
                    </div>
 +
                </div>
 +
            </section>
 +
        </div>
 +
        <div class="simple-page animate-box">
 +
            <section>
 +
                <div class="container">
 +
                    <div class="col-md-10 col-md-offset-1">
 +
                        <h2 class="">Finding the right organism to work with</h2>
 +
                        <div class="spacer h20"></div>
 +
                        <hr/>
 +
                        <div class="spacer h20"></div>
 +
                        <p class="sub-lead">
 +
                            We choose to work in yeasts for two main reasons: firstly, the promising results regarding transcription activation of genes using the new CRISPR/dCas9 system from Zalatan et al. 2015 were obtained in yeast, so we knew that our activation experiments on our modular promoter should function accordingly.
 +
                        </p>
 +
                        <p class="sub-lead">
 +
                            Second, yeast is a eukaryotic system which means that the results can be predictably extrapolated to mammalian cells, without their high cost and long replication time. This makes it appealing for many other researchers as they may take advantage of our system to develop new ways to detect and cure diseases in humans for example.  
 +
                        </p>
 +
                    </div>
 +
                </div>
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            </section>
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        </div>
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        <div class="simple-page animate-box">
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            <section>
 +
                <div class="container">
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                    <div class="col-md-10 col-md-offset-1">
 +
                        <h2 class="">How to <b>automate</b> the process? </h2>
 +
                        <div class="spacer h20"></div>
 +
                        <hr/>
 +
                        <div class="spacer h20"></div>
 +
                        <p class="sub-lead">
 +
                            Designing biological logic gates is one of the biggest challenges of synthetic biology. Using the recently developed software Cello (Nielsen et al., Science 2016 )[REF], we aimed to automate this process and thus allow people to construct logic gates in biological circuits in an easier way. To implement Cello with our system, we needed to characterize the gates and to adapt Cello to the CRISPR-associated RNA scaffold system.  
 +
                        </p>
 +
                        <p class="sub-lead">
 +
                            The characterization of gates was done by photospectroscopy experiments. To adapt the software to the scaffold system, we used the modular nature of Cello. Here, new data regarding new organisms, gates, and expression plasmids can be easily implemented through a user constraint file. This however is not particularly trivial and does not help in user experience. We therefore also decided to integrate a graphic interface to the software. It allows the program to be more accessible to people who cannot code in Verilog, the language initially used by the software. Finally, it also allows the user to predict the results of his/her circuit before going to the lab and test it under real conditions.
 +
                        </p>
 +
                        <!-- TODO: immagine cello -->
 +
                    </div>
 +
                </div>
 +
            </section>
 +
        </div>
 +
        <div class="simple-page animate-box">
 +
            <section>
 +
                <div class="container">
 +
                    <div class="col-md-10 col-md-offset-1">
 +
                        <h2 class="">Going further with activation and repression: <br/><b>proof of concept</b></h2>
 +
                        <div class="spacer h20"></div>
 +
                        <hr/>
 +
                        <div class="spacer h20"></div>
 +
                        <p class="sub-lead">
 +
                            As a proof of concept, we chose to bring together all the preceding steps to build a simple logic gate: the NOT gate. [Once built, we will characterize this gate and compare our biological output with the theoretical one given by Cello .] This NOT gate will naturally be inducible by galactose. The final step of our project should prove that all the foregoing work done functions accordingly and can simply be integrated in the automatically biological circuits design. The NOT gate is also interesting because in electrical circuits, one can build any possible gate with a NOT and a XOR gate.  From all of this, reaching the final goal of creating even more complex biological circuits in living cells is only a few steps away.
 +
                        </p>
 +
                        <!-- TODO: immagine gate logico + biologico -->
 +
                    </div>
 +
                </div>
 +
            </section>
 +
        </div>
 +
        <div class="simple-page animate-box">
 +
            <section>
 +
                <div class="container">
 +
                    <div class="col-md-10 col-md-offset-1">
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                        <h2 class="">Cool! But is it useful?</h2>
 +
                        <div class="spacer h20"></div>
 +
                        <hr/>
 +
                        <div class="spacer h20"></div>
 +
                        <p class="sub-lead">
 +
                            Sure it is! Designing and constructing gates is one of the trickiest steps in the synthetic biology. And with this project, it just became easier! Once your gates are designed and ready, you can make a lot of things such as strong biosensors, or even detection methods for diseases or substances. And these are only few examples of the large applications of biological circuits.
 +
                        </p>
 +
                    </div>
 +
                </div>
 +
            </section>
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{{RISE_foot}}

Revision as of 12:09, 19 October 2016

iGEM EPFL 2016

intelligene

Far far away, behind the word mountains, far from the countries Vokalia and Consonantia.

In the 2016’s edition of iGEM, we, the EPFL team, decided to work on biological circuits. Our main goal was, on one hand, to improve the already-existing tools used for the design of biological circuits. On the other hand, we aspired to automate the design of such circuits by improving a new software called Cello, which would save valuable time and reduce the effort of scientists and researchers working on this topic all over the world. In order to achieve this, the system required two important things: a modular way to create circuits and a software that allows the automation of circuit design.

General idea of the project

We based our project on CRISPR/(d)Cas9, a system largely used for genome engineering. Taking the catalytically dead dCas9 from this system allows the targeted binding of almost any DNA sequence. dCas9 is a mutant of the original Cas9 protein that lost its ability to cut DNA, but can still bind to DNA sequences using a specific guide RNA (gRNA). The only limitation for the target is that it has to lie next to a 3-base-pairs “PAM” sequence, NGG, N being any nucleotide. At the molecular level, the gRNA encodes the target sequence which is then ‘loaded’ into the dCas9, which, in turn then, ‘opens up’ the two DNA strands at the target sequence. Changing the sequence of the gRNA means changing which DNA sequence is targeted. In this way, it is possible to target different regions of a promoter. Moreover, dCas9 can be fused with activating transcriptional effectors to increase the expression of the promoter it binds to. This allows for the activation of the downstream gene that is associated to the promoter region.

The main disadvantage of this system was that it only relied on allosteric repression to turn genes off by not allowing the polymerase to bind to the targeted promoter. This type of repression is not entirely reliable and is not performed by a “real” repressor, making it less efficient.

With these limitations in mind, we set out to develop a system that was more intuitive, precise, and dynamic.

FIGURE 1: Two ways to act on the expression of a promoter through CRISPR/dCas9. Figure adapted from Zalatan et al. 2015.
FIGURE 1: Two ways to act on the expression of a promoter through CRISPR/dCas9. Figure adapted from Zalatan et al. 2015.

How to obtain a modular system?

Modularity is the ability of a system to be composed of simple pieces that can then be recombined to form another one. It is also the key to obtaining an easy-to-use and understandable tool. Something that was unavailable until recently, was a truly modular way to achieve complex biological circuits composed of many different gates. The choice of the promoter was pivotal as the ability to use only one promoter in entire circuits would significantly simplify the system many times over. We had to find a promoter with distinct regions that could be targeted by different gRNAs: some for gene activation and other for repression. The sequences of those regions would also have to be modifiable to avoid cross-talk between all the gRNAs composing a given biological circuit.

Making it work when you want to, means making it inducible

As we now solved the issue of modularity of our system, it would be a lost opportunity to only end up with a tool that is not able to adapt to its surrounding. Adapting to its surrounding means requiring inducible promoters that do not interfere with the vital functions of the cell but still detect the molecule of interest. These promoters would allow researchers to achieve functional biosensors for many applications by triggering distinct responses in the cell according to the signals they detect.

Finding the right organism to work with

We choose to work in yeasts for two main reasons: firstly, the promising results regarding transcription activation of genes using the new CRISPR/dCas9 system from Zalatan et al. 2015 were obtained in yeast, so we knew that our activation experiments on our modular promoter should function accordingly.

Second, yeast is a eukaryotic system which means that the results can be predictably extrapolated to mammalian cells, without their high cost and long replication time. This makes it appealing for many other researchers as they may take advantage of our system to develop new ways to detect and cure diseases in humans for example.

How to automate the process?

Designing biological logic gates is one of the biggest challenges of synthetic biology. Using the recently developed software Cello (Nielsen et al., Science 2016 )[REF], we aimed to automate this process and thus allow people to construct logic gates in biological circuits in an easier way. To implement Cello with our system, we needed to characterize the gates and to adapt Cello to the CRISPR-associated RNA scaffold system.

The characterization of gates was done by photospectroscopy experiments. To adapt the software to the scaffold system, we used the modular nature of Cello. Here, new data regarding new organisms, gates, and expression plasmids can be easily implemented through a user constraint file. This however is not particularly trivial and does not help in user experience. We therefore also decided to integrate a graphic interface to the software. It allows the program to be more accessible to people who cannot code in Verilog, the language initially used by the software. Finally, it also allows the user to predict the results of his/her circuit before going to the lab and test it under real conditions.

Going further with activation and repression:
proof of concept

As a proof of concept, we chose to bring together all the preceding steps to build a simple logic gate: the NOT gate. [Once built, we will characterize this gate and compare our biological output with the theoretical one given by Cello .] This NOT gate will naturally be inducible by galactose. The final step of our project should prove that all the foregoing work done functions accordingly and can simply be integrated in the automatically biological circuits design. The NOT gate is also interesting because in electrical circuits, one can build any possible gate with a NOT and a XOR gate. From all of this, reaching the final goal of creating even more complex biological circuits in living cells is only a few steps away.

Cool! But is it useful?

Sure it is! Designing and constructing gates is one of the trickiest steps in the synthetic biology. And with this project, it just became easier! Once your gates are designed and ready, you can make a lot of things such as strong biosensors, or even detection methods for diseases or substances. And these are only few examples of the large applications of biological circuits.