The IV-HSL Emitter module is a set of two genetic constructs. The first produces a type of homoserine lactone called IV-HSL through an expressed enzyme pathway. The second activates expression in response to IV-HSL, enabling amplification of the signal.
The IV-HSL production module is a genetic construct pT7-bjaI that encodes the enzyme BjaI. This enzyme converts S-adenosylmethionine (SAM) and isovaleryl coenzyme A (IV-CoA) into N-isovaleryl-l-homoserine lactone (IV-HSL). IV-HSL is a membrane-permeable acyl homoserine lactone.
The IV-HSL receiver module is an E. coli genetic construct bjaR-GFP-native that encodes the transcription factor bjaR that promotes downstream gene expression in response to picomolar concentrations of IV-HSL [Lindemann, 2011].
pT7-bjaI is MoClo compatible as a Level 0 CDS part, and may be assembled into a Level 1 transcription unit using BsaI.
The IV-HSL emitter module may be implemented by assembling the pOpen-pT7-bjaI DNA construct into 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.
DNA Parts
pT7-bjaI—Nucleus v0.2.0 Distribution Plate upcoming.bjaR-GFP-native—Nucleus v0.2.0 Distribution Plate upcoming.
Protein Components
- N/A
Cell Components
- XL-10 Gold
Reaction Construction
Sample | Negative control | Positive control | ||
Component | Volume (uL) | Volume (uL) | Volume (uL) | Notes |
PURE Solution A | 12 | 12 | 0 | PURE energy solution: small molecules |
PURE Solution B | 9 | 9 | 0 | PURE proteins and ribosomes |
RNAse Inhibitor | 1.5 | 1.5 | 0 | Prevents RNAse activity |
(~200 ng/uL) | 1.5 | 0 | 0 | DNA encoding green fluorescent protein |
SAM (5mM) | 1.8 | 1.8 | 0 | |
IV-CoA (5mM) | 0.48 | 0.48 | 0 | |
OptiPrep | 1.5 | 1.5 | 1.5 | Adds density for phase-transfer |
IV-HSL (10 uM) | 0 | 0 | 0.3 | |
3M Glucose | 0 | 0 | 8.46 | |
ddH2O | 2.22 | 3.72 | 19.74 | |
Total | 30 | 30 | 30 |
The IV-HSL emitter module was validated in NEB PURExpress reactions as assembled using the Assemble PURE Reactions protocol. The performance of the emitter module containing 200 ng/uL pT7-bjaI plasmid DNA along with reaction substrates SAM and IV-CoA was compared against a positive control containing 10 uM IV-HSL (LGC Standards, TRC-M28298. The negative control contained the substrates only without pT7-bjaI plasmid DNA.
PURE reactions were incubated for 4 hours, and added to E. coli receiver cell cultures (containing the bjaR-GFP-native plasmid) in log-phase. E. coli GFP fluorescence was measured over six hours using a BioTek Cytation 5 plate reader at 5 minute timepoints.
bjaR-GFP-native
bjaR-GFP-native at steady state. Expression of the fluorescent reporter in XL-10 Gold cells containing bjaR-GFP-native is equivalent for bulk PURE reactions containing pT7-bjaI plasmid DNA and substrates and emitter cells containing IV-HSL and no plasmid DNA. Both show a significant response over the negative control.
- Papers
- 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]
- Jefferson Smith & Michael Booth (Oxford / UCL)
- b.next