Detector: 3OC6-HSL (EsaR)

Overview¶

The EsaR Detector Module senses the bacterial quorum signal 3-oxo-C6-HSL and starts transcription in response. EsaR is a repressor. It binds the EsaO operator, which sits between a T7 promoter and the gene behind it, and blocks T7 RNA polymerase from transcribing that gene. Adding 3-oxo-C6-HSL releases EsaR from the DNA, and expression recovers. The Module therefore detects its analyte by derepression: the regulator is bound by default, and the analyte removes it.

The analyte has three names. AHL, AHSL and 3-oxo-C6-HSL all name N-(3-oxohexanoyl)-L-homoserine lactone, the same ligand.

EsaR is a repressor, despite what the database entry is called. UniProt P54293 titles the entry “Transcriptional activator protein EsaR” for the Pantoea stewartii subsp. stewartii protein, 249 residues. The protein represses, and every result below is a repression result. Read the entry for the sequence, not for the mechanism.

Reference Composition¶

The repressor reaches the reaction as protein, not as a template to transcribe. It is expressed first in its own cell-free reaction, 3 h at 37 °C, then held at 4 °C until use. It is then mixed with the reporter construct and held again before that mixture enters the working reaction. Occupying the operator before transcription can start is what makes the “off” state deep; co-expressing repressor and reporter together gives a much weaker switch.

Expected Behavior¶

Cytosols¶

3-oxo-C6-HSL binds EsaR and relieves repression of the gene behind the operator. The Module was characterized on a fluorescent reporter, mNeonGreen, read against a fluorescein ladder.

Repression fails when the reporter template is in excess, not because the repressor is dead. The first titration moved the repressor against a reporter held at 10 ng/µL and found no difference between the induced and uninduced states, at either operator count. Reversing the titration — moving the reporter against a fixed repressor dose — recovers repression. Above 1 nM reporter there is no repression. At 0.1 nM the two-operator reporter gives about 2-fold and the one-operator reporter about 1.5-fold. Each gives less at 0.5 nM than at 0.1 nM.

Yield does not fall when the reporter is cut. Reactions at roughly a tenth of the usual template reach fluorescence yields comparable to the constitutively expressed control. The likely reason is that each dose of pre-expressed repressor also carries over unspent energy and transcription and translation factors, so adding repressor adds capacity as well.

The Module is expected to switch more deeply the longer the repressor and the reporter are held together before the reaction starts. A 15 min hold gives about 2-fold. Extending it to 1 h gives about 3-fold, with individual replicates between 2.2 and 4.2.

Magnesium is not expected to improve the dynamic range, and too much reduces it. Supplementing the repressor–reporter hold with 0, 5 or 10 mM magnesium changes the dynamic range at none of those levels. At 20 mM the dynamic range roughly halves, and the loss falls almost entirely on the induced state.

More repressor template deepens the “off” state and costs yield. Carrying over pre-expressed repressor also carries over its template, to about 3.4 nM. Diluting the reporter stock in repressor template rather than water adds a further 5.3 nM, bringing the total to 8.7 nM. The fold change rises from 2.8 to about 3.8. Overall fluorescence falls roughly four-fold in the induced state, because expressing more repressor diverts resources away from the reporter.

A lower reaction temperature gives the strongest switch recorded. Running the reaction at 25 °C overnight in a thermocycler instead of at 37 °C gives about 5.8-fold, from 19,800 against 3,400 RFU. The reasoning is that a lower temperature both strengthens EsaR–DNA binding and slows T7, which limits expression from any template that is not fully repressed.

How the repressor is prepared changes the result, so the two preparations are not interchangeable. Expressing the repressor for 3 h at 37 °C and for 17 h at 30 °C give the same fold change at each reporter dose, but the longer preparation lowers overall yield. The likely cause is byproducts accumulating over the longer incubation, inorganic phosphate among them. The 3 h preparation is the reference.

Cells¶

The Module works inside a synthetic cell. The two-operator reporter was encapsulated in GUVs at 0.1 nM with pre-expressed repressor, and 3-oxo-C6-HSL was supplied in the outer solution at 5 µM. GUVs in the induced wells are markedly brighter than the uninduced ones, so the encapsulated Module senses an analyte that starts outside the membrane.

GUVs were made by emulsion transfer. POPC was dried under nitrogen, dried further under vacuum for at least two hours, resuspended in 500 µL mineral oil and sonicated at room temperature for about 20 min. The inner solution was 20 µL. 200 µL of the lipid-in-oil mixture was added onto it and pipetted up and down nine times, and the emulsion was layered onto 250 µL of outer solution. GUVs were spun at 9000 g for 20 min at 15 °C.

For this run the repressor was pre-expressed from template at 24 nM, double the usual, giving a final repressor-reaction concentration of 5 nM. The reporter was held with the pre-expressed repressor for about 1 h at 37 °C before encapsulation.

Requirements¶

Requires pT7 transcription and translation (e.g. Base Cytosol).

Requires the repressor to be supplied as protein, expressed in a separate reaction beforehand. Expressing the repressor and the reporter together in one reaction gives a much weaker switch, because the operator is not occupied before transcription begins.

Requires the repressor and the reporter template to be held together at 37 °C for 1 h before the reaction starts.

Requires the reporter template to be held at or below 0.5 nM. Above 1 nM the reporter is in excess of the repressor and no repression is seen.

Requires the analyte to reach the repressor. 3-oxo-C6-HSL crosses a POPC membrane well enough to induce an encapsulated reaction from the outer solution, so no membrane pore is needed.

Materials¶

Credits¶

Developed by Charlie Newell, Manuel Bibrowski, Jonah McDonald, Niall McIntyre and Julia Purrinos De Oliveira.

Drawn from the DevNote Quorum Sensing in Nucleus Cytosol using EsaO/EsaR Transcriptional Regulation.

Supported by¶

DevNotes that support this specification include:

Quorum Sensing in Nucleus Cytosol using EsaO/EsaR Transcriptional Regulation