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Figure 1. The OpenCFPS™ E. coli Core Kit runs with low DNA concentrations using either plasmid or linear DNA. Different DNA concentrations and formats were tested; endpoint GFP after 12 h at 29 °C in 10 µL reactions is shown as a function of DNA concentration (left). Transcription (Tx) and translation (Tl) from 4 nM plasmid DNA or 8 nM linear DNA are also shown (right).
Plasmid DNA (lot D000126041501) used was pS-T7-sfGFP r1. This was generated by Biozilla (Ho Chi Minh City, Vietnam) as a gigaprep, eluted in 10mM Tris pH 8.5, quantified by absorbance at 260 nm, and normalized to 200 nM. Plasmid DNA contains an Malachite green aptamer sequence. Full sequences are provided.
Linear DNA (lot 260318) used was PCR amplified (1.6 mL) against pS-T7-sfGFP r1 with Vazyme 2x RapidTaq Mix (Vazyme, Nanjing, China) and purified by salt and buffer exchange into water using a 50kDa Amicon Ultra-4 (Merck, Darmstadt, Germany). This was quantified by the AccuGreen High Sensitivity dsDNA Quantitation Kit (Biotium, California, USA) as 633 nM and normalized to 200 nM. Linear DNA contains an Malachite green aptamer sequence. Sequences are provided.
Cell-free reactions were set up in duplicate at 10 uL on a 384-well flat-bottom plate 0720111 (Corning, New York, USA) using Extract E0002 lot E000225121803 and Buffer B0001 lot B000126022403, where each reaction is Extract (30%), Buffer (40%), 1 mM Malachite Green, DNA, and water. Reactions were set up using the Formulatrix Mantis v3.3 ACC RFID (Formulatrix, Massachusetts, USA). DNA concentrations were independently dispensed and were varied from 0 nM to 32 nM. On the same plate, duplicates of dilutions of fluorescein in water using 100 µM stock (in 10mM Tris-Cl, pH 9, lot 260126) were dispensed as a control. After dispensing, the plate was spun down, covered with a clear non permeable MicroAmp Clear Adhesive Film (ThermoFisher, California, USA) with a cut out for the wells to be read, then covered with a BreatheEasier film (Diversified Biotech, Massachusetts, USA). This plate was further covered with a wet paper towel, and then read in a Biotek Synergy Neo2 (Agilent, California, USA) from 425 ex/528 em gain 35 (monochromator) and 610 ex / 650 em gain 140 (monochromator) in a kinetic read at 29°C, with continuous orbital shaking (fast, 1 mm).
After ~ 12 hours, the kinetic data was collected and analyzed by ChatGPT 5.6 (OpenAI, California, USA). To obtain Relative Fluorescein Equivalent (RFE) units in µM, signal collected in plate reader was calibrated to a curve-fit to the fluorescein standards collected. Transcription (Tx) signal is represented by afu from 610/650 read, and Translation (Tl) signal is represented as RFE in µM.
Duplicate fluorescein dilution wells were dispensed on the same plate from a 100 µM stock in water (10mM Tris-Cl, pH 9, lot 260126) and read in the 425 ex/528 em gain 35 channel.
Those standards were used to fit the calibration curve that converts endpoint fluorescence into Relative Fluorescein Equivalent (RFE) units in µM for the Tl readout. Tx is reported separately as afu from the raw 610/650 read.
Colored wells show either maximum raw 610/650 signal across the kinetic read for Tx or endpoint RFE for Tl. Switch readouts or click a filled well to inspect the sample.
Fluorescein controls (F100-F1) are shown in gray and excluded from the color scale so calibration wells do not bias the Tx/Tl sample readouts; click a gray control well to inspect its values.