Difference between revisions of "Team:EPF Lausanne"
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To overcome these limitations, an ideal in vivo logic element should be modular, reusable, and orthogonal - i.e avoiding unwanted cross-talk with its host organism as well as other elements of the engineered circuit.</p> | To overcome these limitations, an ideal in vivo logic element should be modular, reusable, and orthogonal - i.e avoiding unwanted cross-talk with its host organism as well as other elements of the engineered circuit.</p> | ||
<h2>Cas9 Logic Gates</h2> | <h2>Cas9 Logic Gates</h2> | ||
| − | <p>Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease that targets and | + | <p>Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease that targets and cleaves any DNA sequence complementary to its guide RNA (gRNA). Our project will be based upon a derivative of this technology : catalytically “dead” Cas9 (dCas9) that lack the ability to cleave DNA. When fused to a RNA polymerase (RNAP) recruiting element (e.g. the omega subunit of RNAP in E. Coli or VP64 in eukaryotes), chimeric dCas9 can act as a programmable transcription activator. In addition, activating dCas9 may also act as a DNA transcription inhibitor: depending on its gRNA-determined binding site, it has been shown in yeasts to sterically hinder RNAP recruitment to promoter sequences.<br> |
Exploiting dCas9-omega/VP64’s ambivalence, we propose the creation of gRNA-controlled switch-like elements analogous to transistors. The state of the switch would be solely dependent on the position of dCas9 relative to the promoter. The content of the gRNA-targeted sequences might therefore be designed such that each transistor is orthogonal to other logic elements. Using gRNA to make what could be seen as “biological wires”, we also hope to achieve chainability of the transistors and thus complexification of bio-circuits.</p> | Exploiting dCas9-omega/VP64’s ambivalence, we propose the creation of gRNA-controlled switch-like elements analogous to transistors. The state of the switch would be solely dependent on the position of dCas9 relative to the promoter. The content of the gRNA-targeted sequences might therefore be designed such that each transistor is orthogonal to other logic elements. Using gRNA to make what could be seen as “biological wires”, we also hope to achieve chainability of the transistors and thus complexification of bio-circuits.</p> | ||
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Revision as of 07:34, 16 July 2015
Biologic Orthogonal GRNA-Implemented Circuit
This summer, the EPFL iGEM team strives to pave the way for simpler implementation of digital circuits in vivo. Using the newly discovered dCas9 as a synthetic transcription factor, we aim to design biocompatible transistor-like elements. Our ultimate goal is to make cells smarters by assembling these transistors into logic gates that are both chainable and parallelizable in a homogenous logic framework.
Thinking Binary
Boolean Logic is the bedrock of the digital revolution. Developed by George Boole in the mid-19th century, it is based on a simple set of values: 0 (“FALSE”) or 1 (“TRUE”). Modern computers represent all forms of information using strings of the same 0s and 1s (also named “Bits”). The processing of bits is handled by logical transistors - which can be seen as electronically controllable switches. Elementary logic operation are performed using cleverly assembled transistors. Such assemblies are named “logic gates”. Gates are the bricks with which complex behaviour is produced.
Biological computers
Since the early 2000’s, multiple synthetic biological gates have been built, revolutionizing our ability to dictate the way organisms react to stimuli. Their applications range from intelligent biosensors to cellular therapeutics with improved in vivo targeting and curing.
Unfortunately, the development of programmable cells has been hampered by difficulties in the multiplication and chaining of logic elements. This has hindered the complexification of bio-circuits as well as the automation and flexibility of their design.
To overcome these limitations, an ideal in vivo logic element should be modular, reusable, and orthogonal - i.e avoiding unwanted cross-talk with its host organism as well as other elements of the engineered circuit.
Cas9 Logic Gates
Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease that targets and cleaves any DNA sequence complementary to its guide RNA (gRNA). Our project will be based upon a derivative of this technology : catalytically “dead” Cas9 (dCas9) that lack the ability to cleave DNA. When fused to a RNA polymerase (RNAP) recruiting element (e.g. the omega subunit of RNAP in E. Coli or VP64 in eukaryotes), chimeric dCas9 can act as a programmable transcription activator. In addition, activating dCas9 may also act as a DNA transcription inhibitor: depending on its gRNA-determined binding site, it has been shown in yeasts to sterically hinder RNAP recruitment to promoter sequences.
Exploiting dCas9-omega/VP64’s ambivalence, we propose the creation of gRNA-controlled switch-like elements analogous to transistors. The state of the switch would be solely dependent on the position of dCas9 relative to the promoter. The content of the gRNA-targeted sequences might therefore be designed such that each transistor is orthogonal to other logic elements. Using gRNA to make what could be seen as “biological wires”, we also hope to achieve chainability of the transistors and thus complexification of bio-circuits.