Team:ETH Zurich/project/overview

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(Implementation in E. coli)
(Implementation in E. coli)
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=== Implementation in ''E. coli'' ===
=== Implementation in ''E. coli'' ===
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Mosai''coli'' involves three main constructs per cell, one for quorum sensing, one for production of integrases, and the last with the integrase based XOR logic gate to perform computation. Each cell can receive two orthogonal AHLs - the rhlI and luxI products.  The rhl-AHL or the lux-AHL received by the cell bind to their corresponding receptor proteins  rhlR and luxR, thus activating them. The luxR-lux-AHL or rhlR-rhl-AHL complex bind to the corresponding promoters Prhl and Plux on the second plasmid and positively regulate the expression of two integrases phiC31 and Bxb1 respectively. Additionally, we use riboswitches with the integrases to to reduce the leakiness of promoters Plux or Prhl<sup>[[#refWilliams|[10]]]</sup>.
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Mosai''coli'' involves three main constructs per cell, one for quorum sensing, one for production of integrases, and the last with the integrase based XOR logic gate to perform computation. Each cell can receive two orthogonal AHLs - the RhlI and LuxI products.  The Rhl-AHL or the Lux-AHL received by the cell bind to their corresponding receptor proteins  RhlR and LuxR, thus activating them. The LuxR-Lux-AHL or RhlR-Rhl-AHL complex bind to the corresponding promoters Prhl and Plux on the second plasmid and positively regulate the expression of two integrases phiC31 and Bxb1 respectively. Additionally, we use riboswitches with the integrases to to reduce the leakiness of promoters Plux or Prhl<sup>[[#refWilliams|[10]]]</sup>.
[[File:ETHZurich molbio simplified.png |500px|center|]]
[[File:ETHZurich molbio simplified.png |500px|center|]]
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The XOR gate present on the third plasmid comprises an asymmetric transcription terminator flanked by two pairs of opposing recombination sites recognised by phiC31 and Bxb1 respectively. ''gfp'' and ''luxI'' (or ''rhlI'') genes are adjacent to the XOR gate and under control of the same promoter. In the absence of both integrases, the terminator blocks transcription. Expression of either integrase alone inverts the DNA encoding the terminator and allows transcription of ''luxI'' (or ''rhlI'') and ''gfp''. Presence of both integrases inverts the terminator twice bringing it back to its original orientation. Thus, transcription is blocked again<sup>[[#refBonnet|[9]]]</sup>.
The XOR gate present on the third plasmid comprises an asymmetric transcription terminator flanked by two pairs of opposing recombination sites recognised by phiC31 and Bxb1 respectively. ''gfp'' and ''luxI'' (or ''rhlI'') genes are adjacent to the XOR gate and under control of the same promoter. In the absence of both integrases, the terminator blocks transcription. Expression of either integrase alone inverts the DNA encoding the terminator and allows transcription of ''luxI'' (or ''rhlI'') and ''gfp''. Presence of both integrases inverts the terminator twice bringing it back to its original orientation. Thus, transcription is blocked again<sup>[[#refBonnet|[9]]]</sup>.
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Colonies of such cells are placed in a grid in a 3D-printed millifluidic chip. Each colony can exist in one of two states - ON and OFF. The cells are OFF if they do not produce any GFP and luxI (or rhlI) and ON when they produce GFP and luxI (or rhlI). The luxI or rhlI expressed catalyse the production of the corresponding AHL molecules which diffuse out and are propagated to the colonies in the next row. Each colony updates its state by integrating signals from its neighbours (colonies in the previous rows). We expect to see complex fluorescent patterns, such as the Sierpinski triangles after several rows of colonies on the grid have updated their states.
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Colonies of such cells are placed in a grid in a 3D-printed millifluidic chip. Each colony can exist in one of two states - ON and OFF. The cells are OFF if they do not produce any GFP and LuxI (or RhlI) and ON when they produce GFP and LuxI (or RhlI). The LuxI or RhlI expressed catalyse the production of the corresponding AHL molecules which diffuse out and are propagated to the colonies in the next row. Each colony updates its state by integrating signals from its neighbours (colonies in the previous rows). We expect to see complex fluorescent patterns, such as the Sierpinski triangles after several rows of colonies on the grid have updated their states.
=== Biological tools: quorum sensing and integrases ===
=== Biological tools: quorum sensing and integrases ===

Revision as of 06:18, 12 August 2014

iGEM ETH Zurich 2014

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