<aside> <img src="/icons/light-bulb_gray.svg" alt="/icons/light-bulb_gray.svg" width="40px" /> Overview

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The Responder Cell produces and releases a chemical signal molecule into the environment, in response to a small molecule input. This provides an example of integrated detection (as in the Detector Cells) and emission (as in the Emitter Cells), a first step toward synthetic cells that can provide closed-loop control within an environment. As in the IV-HSL Emitter Cell, the Responder Cell is based on a paper by Jefferson M Smith, Denis Hartmann, and Michael J. Booth: Engineering cellular communication between light-activated synthetic cells and bacteria.

In response to anhydrotetracycline (aTc), the Responder Cell produces and releases N-isovaleryl-L-homoserine lactone (IV-HSL). aTc transits the synthetic cell membrane and activates expression of the enzyme BjaI by binding with the tet repressor (TetR), which otherwise represses expression at one or more tet operator (tetO) sites within the bjaI transcription unit. Expressed BjaI produces IV-HSL from two substrate molecules, S-adenosylmethionine (SAM) and isovaleryl coenzyme A (IV-CoA). The IV-HSL transits the membrane where it can be detected by co-cultured E. coli receiver cells which produce green fluorescent protein, enabling readout. Further detail is available in the documentation for the TetR Inducible Module and IV-HSL Emitter Module.

<aside> <img src="/icons/subtask_gray.svg" alt="/icons/subtask_gray.svg" width="40px" /> Design

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The Responder Cell implements the aTc→IV-HSL Responder Module within a synthetic cell. BjaI expression is controlled by a dual-tetO operator site within the module construct, which maintains tight repression to prevent production of IV-HSL in the off state. This is required due to the low concentration of IV-HSL required to induce the E. coli receiver cells, and because of the amplifying effect of the BjaI enzyme (one enzyme can produce many molecules of IV-HSL from the substrates).

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Usage

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Protocol

Protocol: aTc IV-HSL Responder Cell

Modules

• aTc→IV-HSL Responder Module—implements IV-HSL production in response to an aTc input within a PURE cytosol.

DNA Components

• pT7-tetO-tetO-bjaI — Nucleus v0.3.0 Distribution Plate upcoming. Expresses the BjaI enzyme to produce IV-HSL.
• bjaR-GFP-native —Nucleus v0.3.0 Distribution Plate upcoming. E. coli native receiver module; responds to IV-HSL by producing GFP.

Key Materials

<aside> <img src="/icons/chart-line_gray.svg" alt="/icons/chart-line_gray.svg" width="40px" /> Performance Data

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