Team:ETH Zurich/project/overview/implementation

From 2014.igem.org

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Mosaicoli involves three main plasmid 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 N-acyl homoserine latones (AHLs) - the LuxI product N-(3-oxohexanoyl)-L-homoserine (3OC₆-HSL) and the LasI product N-(3-oxododecanoyl)-L-homoserine (3OC₁₂-HSL). The 3OC₆-HSL or the 3OC₁₂-HSL received by the cell bind to their corresponding receptor proteins LuxR and LasR, thus activating them. The LuxR-3OC₆-HSL or LasR-3OC₁₂-HSL complexes bind to the corresponding promoters Plux and Plas on the second plasmid and induce the expression of two integrases Bxb1 or ΦC31, respectively. Additionally, we use riboregulators to reduce leakiness in the expression of the integrase genes from the promoters P_lux or P_las^[10].

The integrases act on an asymmetric transcription terminator present present on the third plasmid, that contains the XOR gate. The terminator is located between a constitutive promoter and the start of two consecutive downstream genes in one operon, luxI and gfp. If the terminator is in its "functional” orientation, expression from these genes is not possible, because it is prematurely terminated. Further, the terminator is flanked by two pairs of opposing recombination sites recognised by ΦC31 and Bxb1, respectively. In the absence of both integrases, the terminator is in its functional orientation and thus blocks transcription. Expression of either integrase alone inverts the DNA encoding the terminator. It is no longer in its functional orientation and transcription of luxI (or lasI) and gfp is enabled. Presence of both integrases inverts the terminator twice bringing it back to its original functional orientation. Thus, transcription is blocked again^[9]. After the integrases acted on their corresponding recombination sites, the sites recombine in a way leaving the integrases unable to invert the region a second time.

The XOR gate present on the third plasmid comprises an asymmetric transcription terminator flanked by two pairs of opposing recombination sites recognised by ΦC31 and Bxb1 respectively. gfp and luxI (or lasI) 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 lasI) and gfp. Presence of both integrases inverts the terminator twice bringing it back to its original orientation. Thus, transcription is blocked again^[9].

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 LasI) and ON when they produce GFP and LuxI (or LasI). The LuxI or LasI 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.