Overview¶

Why two stages. Expressing PLA1 inside the same compartment it is meant to lyse confounds the two events: a vesicle functions improperly may have, failed to lyse, or lysed so early that nothing accumulated. Running expression to completion in bulk first, then delivering a fixed 4 µL dose to intact vesicles, separates the question “was the enzyme made?” from the question “does the enzyme lyse the membrane?”

The reporter. GUVs are loaded with β-galactosidase (LacZ) in the inner solution. Intact vesicles keep the enzyme sequestered; a breached membrane releases it into the external volume where it meets its substrate. The readout is therefore a gain of signal on lysis, with the vesicle acting as its own negative control until the moment it fails.

The genetic circuit. Both PLA constructs place the coding sequence downstream of a tetO operator, so purified TetR blocks transcription and aTc relieves that block. Two backbones are compared: PJL-PLA and pOpen-PLA. The three states per plasmid give the full regulatory picture in one pass — the expressed arm sets the maximum lytic activity available, the repressed arm asks how tight the off state is, and the induced arm asks how much of the expressed activity aTc can recover.

Solution versus hydrogel. The same GUV preparation is assayed free in outer solution and embedded in PEG-NB hydrogel. The hydrogel is cast in 1 M HEPES, and the vesicle inner solution is built to match it osmotically (≈397 mM sucrose plus ≈892 mM HEPES). Comparing the two environments asks whether the matrix slows the arrival of enzyme at the membrane, mechanically supports vesicles that would otherwise burst, or has no effect at all.

Controls. pOpen-deGFP runs the circuit without a lytic product, so any signal it produces in the lysis readout is background rather than enzymatic membrane damage. The no-DNA arm reports what the cell-free mix alone does to vesicles — a 4 µL addition of a concentrated reaction brings salts, PEG, detergent-like components and surfactant-active proteins to the membrane, and some vesicle loss from that alone is expected. Externally supplied TetR tests the purified protein prep separately.

What a successful run looks like. β-galactosidase release from both PLA constructs in their expressed state, clearly above the deGFP and no-DNA arms; little or no release from the repressed arms; and release from the induced arms that recovers a substantial share of the expressed signal. The deGFP arm should show the expected repression and induction in its own fluorescence channel, confirming the circuit worked in the reactions that fed the assay.

Materials¶

Stocks to prepare in advance¶

Prepare these before the day of the experiment; everything after the lipid film is time-sensitive.

Lipid films can be dried the day before and left under vacuum overnight.

Part 1 — Express or repress the plasmids, 3 h at 37 °C¶

Twelve 25 µL reactions: four plasmids × three regulatory states. All twelve take the same master mix, so it is batched once and split.

Conditions¶

All reactions carry 1 nM plasmid except the no-DNA arms.

Assembly, volumes in µL per reaction¶

aTc is added to the induced reactions only, to 1 µM final, within the water or TetR allowance.

Steps¶

1. Thaw cell-free components on ice and keep them there throughout.

2. Weigh out the solid sucrose for the batch and vortex it into the Smix until fully dissolved, then add tRNA, Pmix, ribosomes and RIM in that order.

3. Combine plasmid DNA, TetR and water for each condition in its own tube and incubate 10 min at 37 °C, so TetR binds tetO before transcription can start. The expressed and no-DNA arms carry water only but go through the same incubation.

4. Add aTc to the induced tubes to 1 µM final.

5. Add 21 µL of master mix to each pre-incubated tube. Mix by gentle pipetting; do not vortex the assembled reaction.

6. Incubate 3 h at 37 °C. Hold on ice afterwards and use the same day.

7. Reserve an aliquot of each deGFP reaction to read GFP fluorescence directly, confirming expression and repression in this batch.

Part 2 — Prepare β-galactosidase-loaded GUVs¶

The inner solution is built on 1 M HEPES so that it matches the hydrogel the vesicles will sit in. Eight lipid films supply the 720 µL of resuspended GUVs this plate consumes.

Inner solution, 200 µL¶

This pairs with the outer solution from the deGFP GUV protocol (400 mM glucose, 800 mM HEPES) and with PEG-NB cast in 1 M HEPES, so all three phases sit at comparable osmolarity.

Steps¶

1. Prepare 8 lipid films and dissolve each in 200 µL mineral oil as in the deGFP GUV protocol.

2. Add 20 µL of the β-galactosidase inner solution to each cold lipid/oil mixture. Vortex 30 s, then rest on ice 5 min.

3. Layer each emulsion over 100 µL outer solution and rest on ice 5 min.

4. Centrifuge 15 min at 18,000 rcf, 4 °C.

5. Remove the mineral oil, collect each pellet with a fresh tip, and pool the eight preparations.

6. Resuspend to 720 µL total in outer solution. Keep on ice.

7. Before plating, confirm by microscopy that vesicles are intact and that the β-galactosidase is inside rather than free in the suspension — a leaky preparation will read as lysis in every well.

Part 3 — Plate the vesicles in solution and in hydrogel¶

Each well receives 10 µL of GUV suspension plus 10 µL of either outer solution or PEG-NB precursor. Columns 3–5 are the hydrogel arm; columns 6–8 are the solution arm.

Plate layout, 384-well¶

12 conditions × 3 replicates × 2 environments = 72 wells, consuming 720 µL of GUV suspension.

Steps¶

1. Block the wells with 2% w/v BSA for at least 20 min, then remove and rinse with outer solution.

2. Dispense 10 µL of GUV suspension per well, mixing the stock gently between rows so vesicles do not settle out of the pipette.

3. Add 10 µL of PEG-NB precursor to columns 3–5 and 10 µL of outer solution to columns 6–8.

4. Crosslink the hydrogel columns under blue light. Hold under blue light for 60 seconds to crosslink.

5. Add 2 µL of 5 mM CPRG per well for a final concentration of ~0.5 mM.

6. Let the plate equilibrate to the read temperature before adding enzyme.

Part 4 — Deliver the cell-free reactions and read lysis¶

1. Add 4 µL of the matching 3 h cell-free reaction to the outside of each well, following the layout above.

2. Read kinetically at 37C and at absorbance 575 nm.

3. Read for long enough to separate the no-DNA background from genuine lysis — the background rises slowly as the cell-free addition perturbs membranes, while enzymatic lysis should appear as a faster, earlier rise.

Plasmid reference — pOpen-TetO-PLA¶

The expression cassette places the tetO operator immediately downstream of the T7 promoter, so bound TetR occludes the polymerase at initiation rather than acting as a roadblock during elongation.

The terminator and backbone sequences are not transcribed in the cell-free reaction, but the full sequence is kept here so the construct is traceable from the protocol.

Results¶

Evaporation, most likely from inadequate plate sealing over the 10 h read, cost us the in-solution arm entirely. The results below are from the crosslinked PEG-NB arm only.

First, adding TetR-His raised CPRG conversion in conditions that contain no phospholipase at all. Both the no-DNA and deGFP arms show this. The most likely cause is Tween 80 in the MedChemExpress TetR storage buffer, which would permeabilize the membrane directly. Every TetR-containing well therefore receives a detergent dose alongside the repressor, and the induced wells receive it too.

Second, constitutive pOpen-PLA was indistinguishable from constitutive deGFP. If PLA1 were being expressed and were active, the constitutive arm should be the most lytic condition on the plate; instead it sits at the non-lytic baseline. The induced pOpen-PLA condition did give clearly higher conversion than either its constitutive or repressed counterpart, which is the one result consistent with functional PLA1 — but that same condition is the one carrying both TetR buffer and aTc, so the detergent confound cannot be separated from genuine lytic activity with the controls run here.

Third, we predict that the reason the no DNA lysis was so high was because without DNA, no protein was synthesized and that with protein synthesis comes a decrease in osmolarity. So the No DNA GUVs are undergoing high osomotic shock than the other GUVs that got spent cell free reaction.

PJL-PLA underperformed pOpen-PLA across the board.

Conclusion¶

This run does not yet establish that cell-free expressed PLA1 lyses GUVs. The assay reported a strong, reproducible signal, but the signal tracks the presence of TetR-His rather than the presence of a phospholipase gene, and the constitutive PLA1 condition — which should have been the most lytic well on the plate — was no more lytic than the non-lytic deGFP control. The elevated conversion in induced pOpen-PLA is the only observation pointing toward working PLA1, and it is confounded by the detergent carried in with the repressor.

For the next attempt, remove Tween 80 from the TetR preparation with a detergent-removal spin column. pOpen-PLA will be carried forward over PJL-PLA.