aTc IV-HSL Responder Cell

Overview

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.

Design

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).

Usage

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

Name	Product	Manufacturer	Part #	Price	Link
TetR	Tet repressor purified protein	MedChemExpress	HY-P71520	$280	[link]
SAM	S-adenosylmethionine (SAM)	NEB	B9003S	$45	[link]
IV-CoA	Isovaleryl coenzyme A lithium salt hydrate	Millipore Sigma	I9381-10MG	$348	[link]
IV-HSL	3-Methyl-N-[(3S)-tetrahydro-2-oxo-3-furanyl]butanamide	LGC	TRC-M282980-50MG	$171	[link]

Performance Data

Responder Cells were constructed following Protocol: aTc IV-HSL Responder Cell and co-cultured with E. coli containing the bjaR-GFP-native IV-HSL receiver plasmid. We performed time-series confocal microscopy (Revvity Operetta CLS) over 6 hours, collecting red (Rhodamine-B) and green (GFP) fluorescence, and brightfield images at 40x magnification across multiple fields per well, such that the entirety of each well was imaged. Timepoints were approximately 15 minutes apart.

Responder Cell, 8 hour endpoint images of EggPC liposomes containing PURE in M9 media. Left liposome (orange) and E.coli-expressed GFP (green) channels. Right E.coli-expressed GFP (green) channel. + positive control; - negative control (no DNA); induced responder cell module, induced with aTc in the outer solution; uninduced responder cell module, without inducer.

Responder Cell positive control timeseries. EggPC liposomes containing PURE + IV-HSL in M9 media; aTc induced. Liposome membrane (orange) and E. coli-expressed GFP (green).

Responder Cell negative control timeseries. EggPC liposomes containing PURE in M9 media; aTc induced. Liposome membrane (orange) and E. coli-expressed GFP (green).

Responder Cell sample timeseries (induced). EggPC liposomes containing PURE, responder module DNA, IV-CoA and SAM, in M9 media; aTc induced. Liposome membrane (orange) and E. coli-expressed GFP (green).

Responder Cell sample timeseries (uninduced). EggPC liposomes containing PURE, responder module DNA, IV-CoA and SAM, in M9 media. Liposome membrane (orange) and E. coli-expressed GFP (green).

Resources and References

Smith, J. M., Hartmann, D. & Booth, M. J. Engineering cellular communication between light-activated synthetic cells and bacteria. Nature Chemical Biology vol. 19 1138–1146 (2023) [link]
Lindemann, A. et al. Isovaleryl-homoserine lactone, an unusual branched-chain quorum-sensing signal from the soybean symbiont Bradyrhizobium japonicum. Proceedings of the National Academy of Sciences vol. 108 16765–16770 (2011) [link]

Credits

Jefferson Smith & Michael Booth (Oxford / UCL)
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