Team:Fatih-Medical/DJcoli/Design

• Module must be modular that is compatible to different systems.

To make enrichment systems, several inducible promoters and quorum sensing devices can be found in the Registry. Commonly, Lux promoter system has been used for years by iGEMers in different projects. But, to establish an efficient and effective intercommunication system, many more alternative devices should be submitted to the Registry. This year we assembled a new receiver-sender device cassette by using the existing parts. Las promoter (pLas) is an inducible promoter that becomes active in the presence of LasR protein and 3OHSL6 which is the product of an enzyme coded by LasI gene. In previous years a receiver device was assembled by using Lux promoter system. (BBa_F1610, BBa_F2620) We propose alternative sender and receiver devices by using Las promoter system.

• Module must become active when only the first module begins to work.

In Sherlocoli segment, we mentioned about our TEV protease inducible tumor cell recognition system. After our bacterium finds the cell, the enrichment process should begin to work in order to avoid false negative responses. Therefore, we designed an anchor-inducer protein complex fixed together with a TEV protease cleavage site. When TEV protease become active in the course of binding to EpCAM protein, this anchor-inducer complex will be cleaved and inducer will be released free.

The sender system will, then, become active by the inducer after recognition occurs. Our sender consists of two devices: a regulator protein generator and sender PoPS generator. In our design, sender device uses Las promoter components. Constitutive promoter produces 3OHSL6 by LasI gene which is built up in the cell. (Figure 1) After freeing the LasR, the inducer part of anchor-inducer complex, HSL forms a complex with LasR (Figure 2) which activates the pLas in order to generate PoPS response. (In our system, the PoPS response is to produce 3OHSL12, the activator for Lux promoter in the receiver cell.) (Figure 3) The production of 3OHSL12 will lead to the diffusion into the media that results as the induction of receiver cells which ends with a marker protein (fluorescent protein for example) in order to amplify the recognition signal. (Figure 4, Figure 5, Figure 6 and Figure 7)

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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• The enrichment process must be visualized by alternative methods.

To ensure that our enrichment and amplification system is working, we need to characterize our system by using alternative markers. We intend to proof the mechanism via performing fluorescent dependent assays to obtain quantitative data, making visible results by carrying out X-Gal assays etc.

• Other bacteria must proceed to the CTC to amplify the signal.

Another goal we want to accomplish is to make bacteria to move towards the sender cell and tumor cell. Instead of producing marker protein in the receiver cells, we aim to produce CheZ in order to induce chemotaxis. The more inducer (HSL) is produced by the sender, the more the receiver moves towards it. By conducting this, we hope to amplify the recognition signal dramatically.