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Overview

The deGFP Reporter Module produces deGFP, a green fluorescent protein.

Schematic
Designs
A schematic representation of PURE converting template DNA into a fluorescent reporter.

A schematic representation of PURE converting template DNA into a fluorescent reporter.

Cytosols

Reference Composition

This composition was evaluated in this DevNote

ComponentStock ConcentrationFinal Concentration in Reaction
PMix15 mg/mL1.8 mg/mL
SMix4X1X
Ribosomes10 μ\muM1.8 μ\muM
tRNAvaries3.5 mg/mL
pOpen-deGFPvaries3 nM
Magnesium acetate200 mMvaries

Expected Behavior

Kinetics
Endpoint
Translation kinetics of Cytosol and PURExpress reactions using two different pOpen-deGFP DNA preps. Cytosol w/o DNA refers to the Cytosol reaction lacking the pOpen-deGFP template. Data in DevNote.

Translation kinetics of Cytosol and PURExpress reactions using two different pOpen-deGFP DNA preps. Cytosol w/o DNA refers to the Cytosol reaction lacking the pOpen-deGFP template. Data in DevNote.

Cytosol Protocols

Step-by-Step Cytosol Protocol

Implemented using Nucleus Cytosol v0.5

Cytosol Materials

Implemented using Nucleus Cytosol v0.5

Cells

Reference Composition

Cytosol
Membrane
Outer Solution
ComponentInput concentrationUnitFinal concentrationUnitVolume for one reaction (µL)
SMix3.33×1×12
PMix15mg/mL1.80mg/mL4.8
Ribosomes10µM1.8µM7.2
pOpen-deGFP DNA124nM3nM0.95
tRNA35mg/ml3.5mg/ml4
Magnesium acetate200mM8mM1.6
Optiprep1.32mg/µL0.043mg/µL1.33
RNase Inhibitor40000U/mL2000U/mL2
Water6.12
Total volume (µL)40

Expected Behavior

Image 1
Image 2
Combined green (488 nm) and red (561 nm) fluroescence channels. Timepoint 0 (t = 0) corresponds to 30 min after preparation of the inner solution, reflecting the time required to prepare liposomes.

Combined green (488 nm) and red (561 nm) fluroescence channels. Timepoint 0 (t = 0) corresponds to 30 min after preparation of the inner solution, reflecting the time required to prepare liposomes.

Protocols

Step-by-Step Cell Protocol

Implemented using Nucleus Cytosol v0.5

Cell Materials

Implemented using Nucleus Cytosol v0.5

References
  1. Yadav, S. (2025). First Nucleus Cytosol Testing: Evaluation of Nucleus Cytosol v0.5 Through deGFP Expression. 10.63765/fppr8928