Difference between revisions of "Team:Toronto/Description"

 
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<h1>Description</h1>
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<div class="content" id="content-main"><div class="row"><div class="col col-lg-8 col-md-12"><div class="content-main"><h1>Wetlab</h1>
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<p>The wet lab study a LacILOV/cI repressor circuit for the purpose of controlling CRISPR gene editing. The LacILOV repressor is a light-activated fusion protein which represses downstream transcription of sgRNA necessary for CRISPR-Cas9 targeting and the cI repressor in the dark, but allows transcription in blue light. The cI repressor prevents transcription of the anti-CRISPR protein AcrIIA4spy, thus forming a complementary &quot;OFF&quot; command for the LacILOV-controlled sgRNA &quot;ON&quot; switch. The switch will be validated in E. coli cells in a circuit, using the response YFP and mCherry to measure its kinetics via fluorescence and qPCR assays. Finally, CRISPR activity will be tested by targeting the araC gene with CRISPRi for silencing, and measuring the resulting population levels in an arabinose environment. Hello World!!</p>
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<h3 class="text-yellow">Light-activated gene expression</h3>
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<p>Light is an attractive mode of gene regulation that provides high spatio-temporal resolution with relatively low levels of toxicity. In order to add to the genetic toolbox, we characterized a novel light-activated gene regulation system that combines the DNA-binding region of LacI with the light inducible LOV (Light Oxygen Voltage) domain. The characterization assay was performed by measuring the fluorescence output of a LacILOV-regulated reporter under blue light illumination. We then computationally modelled the structure of our protein and identified key mutations to optimize its activity.</p>
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<h3 class="text-cyan">Identifying and informing stakeholders</h3>
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<p>In order to inform future applications of our tool, we identified key stakeholders that would be impacted by potential uses of LacILOV including experts, businesses, the public and future scientists. To this end, we developed resources to promote interest and meaningful interdisciplinary dialogue between researchers and the public. This was achieved through a podcast series exploring the interaction of synthetic biology with different disciplines, a synthetic biology workshop for burgeoning scientists and an iconathon to promote collaborations between scientists and artists.</p>
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<h3 class="text-red">Applying LacILOV to human gene editing</h3>
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<p>However, in order to demonstrate the utility of our tool, we decided to focus on human gene editing, an area that would benefit from stringent gene regulation. We designed and modelled a light activated switch to control CRISPR-Cas9 activity by putting guide RNAs and anti-CRISPR proteins under LacILOV-regulated promoters. We then investigated the ethical and technical concerns of our stakeholders through an interview series involving scientists, engineers, physicians, advocacy groups and religious leaders. Based on technical feedback, we identified an accurate light delivery system as a key technical barrier to validating our tool in mammalian cultures, the next step in its translation to the clinic. We therefore designed and prototyped hardware to pave the way for our system to be used in stem cell cultures. Secondly, based on the different opinions on the ethical applications of human gene editing, we identified the need for clear ethical guidelines. Using the recommendations of the 2017 report by the National Academy of Science, we developed an ethical guide for future iGEM teams.
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<p>Our dry lab team is responsible for developing a model of the gene circuit designed by the wet lab. Previous studies have illustrated the stochastic nature of gene regulatory networks, indicating the complexity of interactions. Thus, stochastic model algorithms will be used to simplify the complexity of gene regulation. Specifically, the dry lab team will recreate the gene pathway and determine the rate kinetics of the many outputs of our system, using the data provided by the wet lab. Next, the dynamics of our CRISPR-Cas 9 system will be analyzed with MATLAB Symbio Library. Finally, our wet lab team will implement the newly designed genetic circuit using the optimized model created by the dry lab.</p>
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<h1>Policy &amp; Practices</h1>
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<p>While the light activated CRISPR-Cas 9 system developed in this project should provide scientists with greater control and accuracy when gene editing, its effects on a larger scale (at the level of multicellular organisms such as humans or plants) are more contested. The indefinite nature of this technology has resulted in barriers to integrating it into mainstream healthcare and policy discourse.</p>
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<p>Although mathematical modeling will attempt to increase the accuracy of our gene editing system it is important to understand and address societal apprehensions to integrating it into mainstream discourse. To investigate these barriers our team will create a dialogue with individuals who represent diverse cultural, economic, and religious backgrounds. These will include local politicians, healthcare professionals, members of religious communities, and end users who will be affected by the inclusion of this technology into mainstream healthcare. Through this dialogue we hope to provide a diverse understanding of the manner in which this technology will shape, and is shaped by, the political and social landscape of Canada. Our team also seeks to engage the broader public in dialogue on synthetic biology by making it more accessible in three ways. The first is by educating and engaging high school students through a summer camp which incorporates activities such as protein modeling, coding, and case studies. Through this camp we seek to provide practical skills to students and foster an interest in synthetic biology and introduce the social impacts of it. The second will pair scientists and artists for a daylong Iconathon event. Not only will the icons created through this event be used to enrich the currently meager synthetic biology icon repository, it will also act as a way to educate artists about synthetic biology in an interdisciplinary fashion. Finally, a five episode podcast related to synthetic biology will engage industry professionals, specialists, and our own team members to educate a wider audience.</p>
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Latest revision as of 20:44, 14 December 2017

Description

Light-activated gene expression

Light is an attractive mode of gene regulation that provides high spatio-temporal resolution with relatively low levels of toxicity. In order to add to the genetic toolbox, we characterized a novel light-activated gene regulation system that combines the DNA-binding region of LacI with the light inducible LOV (Light Oxygen Voltage) domain. The characterization assay was performed by measuring the fluorescence output of a LacILOV-regulated reporter under blue light illumination. We then computationally modelled the structure of our protein and identified key mutations to optimize its activity.

Identifying and informing stakeholders

In order to inform future applications of our tool, we identified key stakeholders that would be impacted by potential uses of LacILOV including experts, businesses, the public and future scientists. To this end, we developed resources to promote interest and meaningful interdisciplinary dialogue between researchers and the public. This was achieved through a podcast series exploring the interaction of synthetic biology with different disciplines, a synthetic biology workshop for burgeoning scientists and an iconathon to promote collaborations between scientists and artists.

Applying LacILOV to human gene editing

However, in order to demonstrate the utility of our tool, we decided to focus on human gene editing, an area that would benefit from stringent gene regulation. We designed and modelled a light activated switch to control CRISPR-Cas9 activity by putting guide RNAs and anti-CRISPR proteins under LacILOV-regulated promoters. We then investigated the ethical and technical concerns of our stakeholders through an interview series involving scientists, engineers, physicians, advocacy groups and religious leaders. Based on technical feedback, we identified an accurate light delivery system as a key technical barrier to validating our tool in mammalian cultures, the next step in its translation to the clinic. We therefore designed and prototyped hardware to pave the way for our system to be used in stem cell cultures. Secondly, based on the different opinions on the ethical applications of human gene editing, we identified the need for clear ethical guidelines. Using the recommendations of the 2017 report by the National Academy of Science, we developed an ethical guide for future iGEM teams.

General Pages

Wet Lab

Dry Lab

Human Practices

iGEM Toronto