Detector: tetR-aTc

Overview¶

The TetR inducible expression module is a set of two genetic constructs that encode tetracycline-inducible gene expression: pT7-tetR, encoding the TetR repressor protein, and pT7-tetO-plamGFP, encoding a reporter gene under an inducible T7 promoter.

pT7-tetO-plamGFP constitutively expresses the open reporter plamGFP in the absence of repressor protein. The inducible promoter is also a MoClo Level 0 ‘P’ part and may be assembled into a Level 1 transcription unit with other MoClo-compatible genes. Addition of TetR protein — either as a purified protein or via constitutive expression of pT7-tetR — inhibits expression through steric occlusion of the tetO operator site. Addition of anhydrotetracycline (aTc) causes allosteric release of TetR from tetO, recovering expression. aTc is membrane-permeable, so the alpha-hemolysin membrane pore is not required for induction.

Schematic of the TetR inducible expression module. TetR represses expression from pT7-tetO-plamGFP; aTc relieves repression by binding TetR and causing its release from the tetO operator. — Schematic of the TetR inducible expression module. TetR represses expression from `pT7-tetO-plamGFP`; aTc relieves repression by binding TetR and causing its release from the tetO operator.

Reference Composition¶

DNA

Cytosol

Name	Length (bp)	File
`pT7-tetR`	2877	pOpen-tetR.gb
`pT7-tetO-plamGFP`	2954	pOpen-pT7-tetO.gb

Assemble pT7-tetO-plamGFP into a standard PURE reaction. Add purified TetR protein to a final concentration of 500 nM, or include the pT7-tetR DNA construct. Add aTc inducer at 2.5 µM to 5 µM for effective induction. Volumes in µL.

Component	Master Mix (µL)
PURExpress Solution A	4
PURExpress Solution B	3
RNase Inhibitor	0.5
`pT7-tetO-plamGFP` (10 nM)	0.5
TetR (10 µM)	0.5
Master Mix Total	9

Component	Per Reaction (µL)
Master Mix	9
Inducer	1
Total	10

Expected Behavior¶

Cytosols¶

The TetR module was validated in NEB PURExpress reactions. Purified repressor protein (MedChemExpress, HY-P71520A) and anhydrotetracycline inducer (Cayman Chemical, 10009542) were added at the final concentrations indicated. pT7-tetO-plamGFP plasmid DNA was added at 0.5 nM.

Repression follows a roughly linear trend between 125 and 750 nM TetR and saturates around 500 nM, though it can be further improved up to 2000 nM. An inducer concentration of 2.5 µM to 5 µM provides effective induction well below saturating or toxic aTc levels. Note that aTc’s yellow color overwhelms GFP fluorescence at concentrations greater than 50 µM to 100 µM, and high concentrations may negatively affect expression generally.

In vitro repression with TetR

Kinetics

Endpoint

In vitro induction with aTc

Kinetics

Endpoint

Cells¶

The TetR-aTc Detector module in the Base Cell.

TetR detector synthetic cells were induced at multiple anhydrotetracycline concentrations and imaged over 12 h with approximately 22 min per timepoint.

Microscopy Images

Fluorescence Intensity

TetR detector synthetic cells induced at multiple anhydrotetracycline concentrations. 8 timepoints displayed per condition, approximately 22 min apart, over 12 h total. First row: induction using 625 nM, 312.5 nM, and 0 nM (fully repressed) aTc introduced into the outer buffer. Second row: induction with 2500 nM aTc in the inner solution and positive control without TetR repression. — TetR detector synthetic cells induced at multiple anhydrotetracycline concentrations. 8 timepoints displayed per condition, approximately 22 min apart, over 12 h total. **First row:** induction using 625 nM, 312.5 nM, and 0 nM (fully repressed) aTc introduced into the outer buffer. **Second row:** induction with 2500 nM aTc in the inner solution and positive control without TetR repression.

The TetR detector cell functions when induced with low-nanomolar aTc concentrations. Higher concentrations begin to inhibit expression or confound analysis due to background aTc fluorescence and membrane localization.

Requirements¶

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

Implementations¶

Responder: aTc → IV-HSL: aTc relieves TetR repression to drive BjaI expression.

Credits¶

Developed by Yen-Yu Hsu (b.next).