Responder Cell_ aTc to IV-HSL.png

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The IV-HSL Responder module integrates the TetR Inducible Module and the IV-HSL Emitter Module to create this responder module, which generates the small molecule isovaleryl-l-homoserine lactone (IV-HSL) in response to anhydrotetracycline (aTc).

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The module is implemented as a single genetic construct, encoding the BjaI enzyme under control of the tet operator. A second construct allows E. coli to act as a IV-HSL receiver and report the activity of the responder module.

There are three variants of the responder module. pT7-tetO-bjaI is the simplest construct, however it has poor dynamic range—leaky expression in the off state produces sufficient IV-HSL to induce receiving E. coli cells. The other two variants resolve this issue: tetO-pT7-tetO-bjaI (the “sandwich” operator) and pT7-tetO-tetO-bjaI (the “train” operator) provide tight repression in the off state and improve dynamic range (Lutz and Bujard, 1997). We use pT7-tetO-tetO-bjaI to implement the Responder Cell.

As in the IV-HSL Emitter Module, we use bjaR-GFP-native as a receiver module, expressing GFP in response to IV-HSL in E. coli cells.

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The IV-HSL emitter module may be implemented by assembling the pT7-tetO-tetO-bjaI responder construct within a standard PURE reaction, following Assemble PURE Reactions. Add equimolar amounts of the substrates SAM and IV-CoA at 0.3 uM and 0.08 uM final concentration, respectively.

The responder module may also be generated as linear DNA from the IV-HSL Emitter Module pT7-bjaI construct, using the primers listed below.

DNA Parts

Protein Components

Cell Components