Overview¶

We tested TetR repression of the pT7-tetO-plamGFP reporter in bulk cell-free reactions, and the relief of that repression across a seven-point anhydrotetracycline (aTc) titration.

Four bulk reactions were assembled: a no-DNA control, an unrepressed reaction with reporter plasmid but no TetR, and two repressed reactions containing 500 nM or 1000 nM purified TetR. Each bulk reaction was aliquoted into a 384-well plate and supplemented with aTc to a final concentration of 0, 0.25, 0.35, 0.5, 1, 5 or 10 µM, in triplicate, giving 28 conditions and 84 reaction wells.

Protocol¶

Genetic circuit¶

The reporter plasmid pT7-tetO-plamGFP (b.next, Nucleus tetR-aTc Detector module) places a tetO operator between the T7 promoter and the plamGFP coding sequence. Purified His-tagged TetR (MedChemExpress, HY-P71520A) binds tetO and blocks transcription; aTc binds TetR and releases it from the operator, restoring plamGFP expression. The output is therefore a graded GFP signal set by the aTc concentration. The full plasmid map is in Plasmid map.

Plasmid map¶

Reagents and stocks¶

Reactions were run in b.next Nucleus Base Cytosol, a PURE-type reconstituted system supplied as a premade reagent kit, and assembled following the Nucleus Assemble Base Cytosol process. Per 10 µL of reaction the component ratios match that reference composition: 3.0 µL SMix, 1.0 µL tRNA, 1.2 µL PMix, 1.8 µL ribosomes and 0.5 µL RNase inhibitor. The reporter and repressor are the Nucleus tetR-aTc Detector module. Stocks and sources are listed in Table 1.

Master mix¶

Components common to all four conditions (SMix, tRNA, PMix, Ribosomes, RIM, Sucrose) were combined into a single master mix in a 1.5 mL Eppendorf tube, sufficient for four 380 µL reactions. Amounts were obtained by summing the per-reaction requirements in Table 2. Sucrose was weighed directly into the tube as 203 mg of solid, the remaining components were added on top, and the sucrose was dissolved by pipette mixing.

The master mix was mixed gently by pipette and set aside while the DNA, TetR and water tubes were prepared, then split between them as described below. Apart from the 10 min pre-incubation, everything was kept on ice from assembly until the plate went into the reader.

Reaction conditions¶

Each bulk reaction was brought to a final volume of 380 µL. Volumes in Table 3 are per tube; the first six rows (SMix, tRNA, PMix, Ribosomes, RIM, Sucrose) make up the master mix volume added to each tube, as 318.44 µL to the pre-incubated plasmid, TetR, and water mixes.

Specifically, plasmid, TetR, and water were combined in their own tube per condition (61.56 µL in every case) and incubated at 37 °C for 10 min to allow TetR to bind the operator. The same volume of master mix, 318.44 µL, was then added to every tube and mixed gently.

aTc titration and plate layout¶

Each bulk reaction was dispensed at 10 µL per well into a 384-well plate, followed by 1 µL of aTc prepared at 11×, for an 11 µL final volume at the stated final aTc concentration. Each of the four conditions was plated across all seven aTc levels in triplicate.

Rows B–E carry the four conditions in order (B = no DNA, C = unrepressed, D = 500 nM TetR, E = 1000 nM TetR), plated as shown in Figure 1. Columns are grouped in threes by aTc level, as in Table 4:

Four fluorescein standards (S1–S4, at 0, 0.5, 1 and 2 µM) were plated in duplicate in row G, columns 2–9, for conversion of arbitrary fluorescence units to molar equivalents of fluorescein (MEF).

Row A and columns 1 and 23 carried no samples and were filled with water, to buffer the outermost wells against evaporation and edge effects over the 6 h read.

Data acquisition and analysis¶

Reactions were plated in a Greiner 384-well small-volume NoBind plate and read on a BioTek Cytation 3 plate reader (serial 130723A) held at 37 °C (gradient 0 °C). GFP fluorescence was recorded as a kinetic run over 6 h at 5 min intervals, with excitation at 485 nm and emission at 528 nm, top optics, gain 75. The light source was xenon flash at high lamp energy, normal read speed, 100 ms delay, 10 measurements per data point, read height 10.75 mm. The incubator was switched off at the end of the kinetic run.

Endpoint values are the final timepoint of the 6 h kinetic run, reported as raw fluorescence and plotted as the mean ± standard deviation of three wells per condition. No-DNA reactions are plotted alongside the samples at each aTc concentration rather than subtracted from them, so that any aTc-dependent change in background is visible directly; across the whole titration this background stayed between 40 and 90 RFU. Fluorescein standards at 0, 0.5, 1 and 2 µM were read on the same plate and give 31,805 RFU per µM for converting these values to molar equivalents of fluorescein. Where fold repression is quoted it is the unrepressed endpoint mean divided by the repressed endpoint mean at the same aTc concentration.

Results¶

TetR represses the reporter tightly at both concentrations, and aTc relieves that repression fully, but the inducer concentration needed scales with the amount of repressor. Endpoint values are in Table 5, plotted in Figure 2.

Without inducer, endpoint fluorescence fell from 21,662 RFU in the unrepressed reaction to 541 RFU at 500 nM TetR and 399 RFU at 1000 nM. Adding aTc restored expression in both cases, and by 5 µM neither repressed condition was distinguishable from the unrepressed one.

The two repressor concentrations separate across the middle of the titration. At 0.25 µM aTc the 500 nM reaction had already recovered to 6,587 RFU, about 30% of full expression, while the 1000 nM reaction was still almost fully repressed at 876 RFU. The 1000 nM curve reaches the same point near 0.5 µM aTc, so doubling the repressor shifts the induction curve roughly two-fold to the right in aTc.

The no-DNA control stayed between 43 and 87 RFU across the titration, so aTc contributes no meaningful fluorescence of its own at these concentrations and does not inhibit expression: the unrepressed reaction held at 21,662–22,871 RFU from 0 to 10 µM aTc.

dataset link:

Platemap link:

Conclusion¶

The tetR-aTc module works as specified in Nucleus Base Cytosol, repressing expression to near background at both 500 and 1000 nM TetR and recovering cleanly to the unrepressed level on induction. Both repressor concentrations are usable, so the choice between them is a choice about where the switch should sit: 500 nM gives a more sensitive detector that responds at 0.25 µM aTc, while 1000 nM gives a tighter OFF state and a threshold closer to 0.5–1 µM.

For circuits that need a sharp, late-triggering switch, 1000 nM is the better setting. For a detector meant to report low aTc, 500 nM is. The useful inducer range for either is 0.25–1 µM, which sits below the 2.5–5 µM the module specification recommends and well below the concentrations where aTc’s own color interferes with the GFP channel.

Two things are worth noting about scope. The titration is coarse between 0.5 and 5 µM, so the thresholds quoted here are read off the measured points rather than interpolated; a finer spacing across 0.1–1 µM would pin down where each curve turns over. And this write-up looks only at the 6 h endpoint, though the run recorded fluorescence every 5 min throughout. Those kinetic traces are available from the same plate and would show whether the repressed conditions differ in onset time as well as in final level.

Sources¶

• Assemble Base Cytosol — Nucleus process, component names and reference composition

• Detector: tetR-aTc — Nucleus module specification for pT7-tetO-plamGFP and TetR

• Make Ribosomes, Make Small Molecule Mix, Make tRNAs, Protein Mix — Nucleus component processes

• Yadav, S. (2025). First Nucleus Cytosol Testing: Evaluation of Nucleus Cytosol v0.5 Through deGFP Expression. Yadav (2025)

• RNase Inhibitor, Murine — New England Biolabs #M0314L

• TetR repressor protein — MedChemExpress HY-P71520A