Difference between revisions of "Team:EPF Lausanne/Interlab"

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           <p>In science, and in the field of synthetic biology in particular, characterizing new devices turns out to be as important as conceiving them. This not only provides a “how to use” guide for future users of your part but also allows the discovery of biologically relevant information about how it functions. When it comes to iGEM, the importance of characterization reaches huge proportion since thousands of new parts are registered each year. As a matter of fact, last year the competition launched its first InterLab Study, inviting every participating team to collaborate to measure previously existing devices. In addition to providing robust and statistically useful data, the InterLab Study aims at assessing how those measurements vary between labs. How similar are data from two teams using the same protocol? How well are the ratios conserved using two different measurement equipment? This year, these questions will be answered for the three constructs each team was given. They each contained a promoter from the widely used Anderson promoter collection that controlled the expression of a GFP. Each construct is described below. We contributed this year by measuring the three constructs in biological triplicates with a flow cytometer, which allowed us to assess the cell-to-cell variability of our samples. As part of the extra-credit assignment, we also provided technical triplicates of our data, thus determining the precision of the measurements. </p>
 
           <p>In science, and in the field of synthetic biology in particular, characterizing new devices turns out to be as important as conceiving them. This not only provides a “how to use” guide for future users of your part but also allows the discovery of biologically relevant information about how it functions. When it comes to iGEM, the importance of characterization reaches huge proportion since thousands of new parts are registered each year. As a matter of fact, last year the competition launched its first InterLab Study, inviting every participating team to collaborate to measure previously existing devices. In addition to providing robust and statistically useful data, the InterLab Study aims at assessing how those measurements vary between labs. How similar are data from two teams using the same protocol? How well are the ratios conserved using two different measurement equipment? This year, these questions will be answered for the three constructs each team was given. They each contained a promoter from the widely used Anderson promoter collection that controlled the expression of a GFP. Each construct is described below. We contributed this year by measuring the three constructs in biological triplicates with a flow cytometer, which allowed us to assess the cell-to-cell variability of our samples. As part of the extra-credit assignment, we also provided technical triplicates of our data, thus determining the precision of the measurements. </p>
 
           <h2>Thinking Binary</h2>
 
           <h2>Thinking Binary</h2>
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          <p>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.</p>
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          <h2>Biological computers</h2>
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          <p>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.<br>
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          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.<br>
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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>
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          <h2>Cas9 Logic Gates</h2>
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          <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>
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          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 12:32, 11 September 2015

EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

Interlab study

Biologic Orthogonal GRNA-Implemented Circuit

In science, and in the field of synthetic biology in particular, characterizing new devices turns out to be as important as conceiving them. This not only provides a “how to use” guide for future users of your part but also allows the discovery of biologically relevant information about how it functions. When it comes to iGEM, the importance of characterization reaches huge proportion since thousands of new parts are registered each year. As a matter of fact, last year the competition launched its first InterLab Study, inviting every participating team to collaborate to measure previously existing devices. In addition to providing robust and statistically useful data, the InterLab Study aims at assessing how those measurements vary between labs. How similar are data from two teams using the same protocol? How well are the ratios conserved using two different measurement equipment? This year, these questions will be answered for the three constructs each team was given. They each contained a promoter from the widely used Anderson promoter collection that controlled the expression of a GFP. Each construct is described below. We contributed this year by measuring the three constructs in biological triplicates with a flow cytometer, which allowed us to assess the cell-to-cell variability of our samples. As part of the extra-credit assignment, we also provided technical triplicates of our data, thus determining the precision of the measurements.

Thinking Binary

J23101
J23106
J23117
EPFL 2015 iGEM bioLogic Logic Orthogonal gRNA Implemented Circuits

NOT PROOFREAD