Overview¶

Quorum sensing allows bacteria to regulate gene expression in response to population density by secreting diffusible acyl-homoserine lactones (AHSLs). Responding to these signals offers a route to developer cells that detect and act on the presence of bacteria. Here we reconstitute AHSL sensing in a nucleus–cytosol cell-free system using the EsaR repressor. EsaR binds the esa box operator placed downstream of a T7 promoter, where it sterically blocks T7 RNA polymerase and silences transcription of a downstream fluorescent reporter (mNeonGreen, mNG). Addition of the cognate ligand 3-oxo-C6-HSL relieves EsaR–DNA binding and derepresses reporter production. Previous reconstitutions of EsaR/esa box in PURExpress co-expressed the repressor at the same time as the operator-controlled reporter DNA and achieved only a weak on/off response. We decouple these two steps: EsaR is first expressed cell-free, then pre-incubated with the reporter construct so that operator sites are occupied before the template enters the reaction, deepening the ‘off’ state. We further expanded the dynamic range by systematically varying the molar ratio of pre-expressed EsaR to reporter DNA, the reaction temperature, and the molar competition between the two DNA constructs. Together these results establish a tunable, ligand-inducible transcriptional switch that equips developer cells to respond to bacterial quorum signalling.

DNA Constructs¶

Three linear DNA templates were used throughout this work: the EsaR(D91G) template used to pre-express EsaR, and two EsaO-mNG reporter constructs carrying either a single or a dual EsaO operator. Linear templates were amplified using PCR and cleaned-up prior to their addition into cytosol reactions.

Results¶

At 12nM, T7-EsaO-mNG DNA is not repressed by cell-free expressed EsaR¶

To determine the ratio of pre-expressed EsaR repressor to sensor construct that gives a well-defined ‘on’/’off’ state, we titrated increasing amounts of pre-expressed EsaR protein into nucleus–cytosol reactions containing one of two esa-mNeonGreen reporter constructs, carrying either one or two esa box operators. The reporter construct was held at 12.2 nM (10 ng/µL) in all reactions.

No difference between the ‘on’ and ‘off’ states was observed under those conditions. This suggests either that the pre-expressed EsaR was non-functional, or that the reporter construct remained in excess even at the highest EsaR-to-DNA ratio tested.

Source:EsaR Repressor Titration

Lower concentrations of T7-EsaO-mNG DNA activates repression¶

To test whether the EsaO-mNG DNA templates had previously been in excess, we titrated the reporter construct concentration while holding pre-expressed EsaR at the highest achievable concentration in each reaction. Repression was lost above 1 nM DNA, whereas 0.5 nM and 0.1 nM gave fold-changes in reporter fluorescence of approximately 2 and 1.5 respectively.

Even at roughly ten-fold lower DNA concentrations, these reactions achieved fluorescence yields comparable to the constitutively expressed control. We hypothesise this is because the pre-expressed EsaR preparation carries over unspent energy components as well as transcription and translation factors, so each addition of repressor also supplies additional capacity to the reaction.

0.1 nM DNA

0.5 nM DNA

1 nM DNA

3 nM DNA

Fold induction

Source:Sensor DNA Titration

Source:Sensor DNA Titration

Source:Sensor DNA Titration

Source:Sensor DNA Titration

Source:Sensor DNA Titration

Overnight pre-expression of EsaR reduces overall yield whilst maintaining sensor dynamic range¶

To test whether the yield increase depends on unspent energy components carried over from the EsaR pre-expression reaction, we pre-expressed EsaR at 30 °C for 17 h rather than 37 °C for 3 h before adding it to fresh nucleus–cytosol reactions containing sensor DNA. EsaR volumes added to each reaction were kept consistent with previous experiments, and sensor DNA was limited to 0.1 nM, 0.5 nM and 1 nM. Relative to the previous experiment, the fold-change between ‘on’ and ‘off’ states remained consistent at each DNA concentration, while overall fluorescent yield across all conditions decreased. We hypothesise that byproducts accumulating during the extended pre-expression such as inorganic phosphate affect the subsequent reactions and lowered overall yield.

Timecourse

Fold change (- AHSL / + AHSL)

End-point

Source:DNA Titration, Spent EsaR

Source:DNA Titration, Spent EsaR

Source:DNA Titration, Spent EsaR

Magnesium addition doesn’t affect dynamic range but increased pre-incubation does¶

The incubation of sensor DNA and EsaR was supplemented with 0 mM, 5 mM, 10 mM or 20 mM magnesium before addition to the cell-free reaction, on the expectation that magnesium would improve EsaR–DNA binding and strengthen repression. Sensor DNA was incubated with EsaR for 1 h at 37 °C before addition to each reaction, rather than the 15 min used previously. Magnesium did not alter the dynamic range at any concentration tested, and 20 mM markedly reduced overall yield. The extended pre-incubation did improve the switch: the fluorescent fold-change rose to approximately five across all DNA concentrations, compared with around two previously.

Timecourse

Fold change

Source:Plot Raw Curves

Source:Plot Raw Curves

EsaR DNA template competition improves sensor dynamic range but reduces yield¶

To raise the final EsaR concentration, additional EsaR template was added to the sensor reaction, so that EsaR continued to be expressed alongside the reporter. Previously, EsaR was present only as carryover from the pre-expression step; here, pre-expressed repressor provides immediate operator occupancy while the added template sustains EsaR production during the reaction.

Adding pre-expressed EsaR also carries over EsaR template, giving a nominal final concentration of 3.4 nM, a 34-fold excess over the 0.1 nM reporter template. To deepen the ‘off’ state further, 5.3 nM EsaR template was added by diluting the reporter DNA stock in EsaR PCR template rather than water, bringing the total EsaR template in the reaction to 8.7 nM, an 87-fold excess over reporter.

Expressing additional EsaR limited overall fluorescence yield, as resources were diverted away from reporter production. This came with an improved dynamic range: the fold-change between ‘on’ and ‘off’ states almost doubled, reflecting a deeper ‘off’ state.

Normalized curves

Fold change

Source:EsaR Template Competition

Source:EsaR Template Competition

25C reactions were incubated in a thermocycler giving a 5.8 fold change as the strongest sensor after over night incubation (Figure 15: 19 800 / 3 400 ≈ 5.8, checked against the figure’s own bars, below).

Lower reaction temperatures increase sensor dynamic range¶

Cell-free reactions were run at the ratios established above, with sensor DNA at 0.1 nM and EsaR template at 3.4 nM. Reactions were incubated at 25 °C rather than 37 °C, on the reasoning that a lower temperature would both strengthen EsaR–DNA binding and slow T7 kinetics, limiting reporter expression from templates that were insufficiently repressed. Reactions were incubated overnight in a thermocycler and read on a plate reader the following day, giving an increased fold-change of ~ 5.8.

Source:EsaR Template Competition

GUVs encapsulating [EsaO]2-mNG sense AHSL in solution¶

Images show the final timepoint of a six-hour incubation at 37 ˚C, processed in Python with matplotlib using a consistent vmin/vmax across all wells. GUVs in the +AHSL conditions are markedly brighter than the -AHSL conditions, demonstrating that GUV-encapsulated [EsaO]2-mNG senses AHSL in solution and derepresses mNG production.

Source:Microscopy Analysis

What’s next¶

• Engineer the T7-EsaR(D91G)-Term construct for increased yield. Modify the 5’ UTR sequence and codon optimise to maximise the amount of protein produced.

• Assess whether it is possible to increase repression further by adding a third EsaO box. Furthermore, search the literature / perform random mutagenesis to determine whether EsaO mutations can promote increased EsaR binding.

• Purchase / recombinantly purify EsaR protein. Add purified EsaR to cytosol reactions containing [EsaO]2-mNG in >100x excess in an attempt to achieve maximal gene repression. Test in bulk reactions first and then in GUVs.

• Once optimised, test [EsaO]2-PLA1 in the colour change module to confirm a visible colour change upon GUVs sensing AHSL.

Methods¶

Reactions across the EsaR titration, sensor DNA titration, and overnight EsaR pre-expression experiments below shared a common assembly: a mastermix of Nucleus cytosol was prepared without DNA or water, per that experiment’s own reaction composition table. Pre-expressed EsaR(D91G) was mixed with the DNA construct(s) and incubated at 37 ˚C for 15 min — this incubation step was lengthened to 1 h starting with the magnesium sweep experiment and in every experiment after it — then topped up with water and added to the cytosol mastermix. Reactions were split in half, AHSL added to one half to a final concentration of 5 µM, then distributed in triplicate into a 384-well plate and read on a platereader set to detect GFP at 37 ˚C. Each section below states only what changed from this base procedure.

Reagents¶

Titrating pre-expressed EsaR into cytosol reactions containing mNG with single or dual EsaO operators¶

EsaR(D91G) was pre-expressed for 3 hrs at 37 ˚C and held at 4 ˚C. Varying amounts of pre-expressed EsaR were mixed with 320.5 ng of either T7-[EsaO]-mNG-T7term or T7-[EsaO]2-mNG-t7term, to a total reaction volume of 65 µL. Full reaction composition is in Table 3.

Titrating single and dual operator EsaO-mNG DNA against fixed pre-expressed EsaR¶

EsaR(D91G) was pre-expressed for 3 hrs at 37 ˚C and held at 4 ˚C, as above. This time, 14 µL of pre-expressed EsaR was held fixed while sensor DNA concentration was varied across T7-[EsaO]-mNG-T7term and T7-[EsaO]2-mNG-t7term, to a total reaction volume of 65 µL. Full reaction composition is in Table 4.

Comparing unspent and spent pre-expressed EsaR added to [EsaO]2-mNG DNA reactions¶

EsaR(D91G) was instead pre-expressed for 17 hrs at 30 ˚C and held at 4 ˚C. 14 µL of pre-expressed EsaR was mixed with varying concentrations of T7-[EsaO]2-mNG-t7term only, to a total reaction volume of 70 µL — AHSL was added as 0.35 µL of 0.5 mM stock, for the same 5 µM final concentration as before. Full reaction composition is in Table 5.

Effect of additional Mg on EsaR-dependent repression of [EsaO]2-mNG DNA¶

Optimising EsaR repression by preeincubating EsaR with the DNA with a higher Mg concentration

Preexpressing EsaR for 3-4h

And then add the DNA with the PURE reaction for 1h at 37 to ensure full repression

The log continues:

Mix of DNA and EsaR pure reaction: Final is 0.332 DNA stock at 22ng/µL 2.6 overall* 3.5 Mg concentrations: 5, 10, 20 locally: Stocks of Mg: X20 = 346.7mM X10 = 173.3mM X5 = 86.6mM

Effect of increasing EsaR DNA concentration on [EsaO]2-mNG DNA repression¶

We ran our EsaR repression reactions at the same conditions as before with 0.1nM DNA template and preincubating the DNA for 1h at 37C with a cell free pre expressed EsaR (15nM DNA, 3h at37C).

EsaR DNA was 15nM. Final EsaR concentration is 3.4nM in the cell free reaction where 2.27µL of EsaR were added and around 0.33 of the DNA stock -> 2.6µL of EsaR-DNA mixture added on 7.4µL mix of Nucleus cytosol+RNase inhibitor and AHSL(5µM)/ DI water (0.15µL)

To improve the amounts of EsaR in the final reaction and introduce a higher off switch:

we added more EsaR DNA template:

We diluted the mNG DNA template not in DI water but in the DNA template of EsaR:

Of mixing a 2x DNA stock with the raw PCR template that was at 320nM in a 50:50 manner so we have 5.3nM extra added enhancing the 34x excess of DNA template up to 77x

Effect of lower reaction temperature on EsaR-dependent repression of [EsaO]2-mNG DNA¶

Testing EsaR-dependent repression of [EsaO]2-mNG DNA in GUVs¶

2 mg of POPC were dried under nitrogen and further dried under vacuum for at least two hours. Lipids were resuspended in 500 µL mineral oil and the mixture was sonicated at room temperature for around 20 minutes.

EsaR was pre-expressed in a cell-free reaction at a template concentration of 24 nM, double the 12 nM used previously, resulting in a final concentration of 5 nM. The reporter construct, (esa)₂-mNG, was held at 0.1 nM. Following completion of EsaR pre-expression, the reporter DNA was incubated with the pre-expressed EsaR for approximately 1 h at 37 °C prior to encapsulation. The EsaR reaction itself was incubated for 3 h at 37 °C before being supplemented with the DNA and incubated for a further 1 h. This mixture was used as a supplement to the inner solution of the GUVs, as an EsaR-DNA mixture.

The inner solution was prepared as 20 µL. The water-in-oil emulsion was prepared by adding 200 µL of lipid-in-oil mixture onto the inner solution and pipetting up and down 9 times. The emulsion was added onto 250 µL of outer solution. GUVs were formed by spinning the two phases in a centrifuge at 15 °C for 20 min at 9,000 g. The resulting pellet was resuspended in 100 mL outer solution. 7.5 µL of GUV solution were added into 22.5 µL of outer solution in a 384-well plate and imaged overnight at 37 °C.

Resources¶

Reaction composition, one sidecar per day. No sidecar exists for 2026-10-01 or 2026-10-07, because neither folder has a build file.

• experiments/build-composition-20260924.csv

• experiments/build-composition-20260925.csv

• experiments/build-composition-20260926.csv

Analysis notebooks.

• experiments/20260924-repressor-test/platereader.ipynb

• experiments/20260925-dna-titration/platereader.ipynb

• experiments/20260926-dna-titration-esar-spent/platereader.ipynb

• experiments/20261001-mg-osmo-sweep/20261001-mg-osmo-sweep.ipynb

• experiments/20261007-esar-template-competition/platereader.ipynb

Platemaps.

• experiments/20260924-repressor-test/build - 20260924_Platemap.csv

• experiments/20260925-dna-titration/20260924-dna-titration-esar-fixed.csv

• experiments/20260926-dna-titration-esar-spent/20260924-esao2-dna-titration.csv

• experiments/20261001-mg-osmo-sweep/20261001-mg-sweep-platemap.csv

• experiments/20261007-esar-template-competition/20261008-esao-mng-esar-ahsl.csv

Raw instrument data.

• experiments/20260924-repressor-test/20260924-151252-cytation5-pure-timecourse-gfp-repressor_test.txt

• experiments/20260925-dna-titration/20260925-153022-synergy2-pure-timecourse-gfp-DNA_titration.txt

• experiments/20260926-dna-titration-esar-spent/20260926-115326-synergy2-pure-timecourse-gfp-DNA_titration_EsaR_spent.txt

• experiments/20261001-mg-osmo-sweep/20261001-181311-cytation5-pure-timecourse-gfp-JM-MB-Mg-Osmo-Sweep.txt

• experiments/20261007-esar-template-competition/20261007-170653-YH Cytosol comparison_CH.txt