Apr 30, 2026
Version 1
Synergy H1 microplate reader: florescence assay V.1
- Alexander Ge1,
- Donglin Liu1
- 1Boston University
Protocol Citation: Alexander Ge, Donglin Liu 2026. Synergy H1 microplate reader: florescence assay. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoezd9l4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol. Has not been tested in wet-lab.
Created: April 30, 2026
Last Modified: April 30, 2026
Protocol Integer ID: 316040
Keywords: biotek synergy h1 plate reader, synergy h1, scoring genetic circuit, genetic circuit, ph1, pb1, florescence, fluorescence, psb1c3 backbone, promoters pa1, h1, pp1
Disclaimer
This protocol describes Path A, one of three enumerated designs submitted for EC552 HW2 (Spring 2026). It is provided for academic and instructional purposes within the EC552 course and the DAMP Lab teaching workflow; it is not a validated production protocol and has not been benchmarked against published assembly references.
Step 15 (Synergy H1 Fluorescence Assay) is not currently a canvas operation; we are publishing it on protocols.io as our HW2 protocol contribution.
Abstract
This is a high-scoring genetic circuit assembled from 11 Cello-format parts (promoters PA1, PA2, PB1, PB2, PH1, PP2 and repressor CDSs A1, B1, B2, H1, H2) into the pSB1C3 backbone. The design is enumerated from a 3,996-design GOLDBAR space analyzed in HW1 and corresponds to three_design_(3930), the highest-scoring design in the library (true score 3.462). Notably, this design excludes P2, S1, and PP1 — the three parts identified as most strongly negatively correlated with circuit performance under our re-analyzed Knox rule evaluation. Assembly proceeds through a 14-step DAMP Lab canvas workflow (primer rehydration → PCR amplification → cleanup → Modular Gibson Assembly → transformation into electrocompetent E. coli DH5α → colony screening → miniprep → TapeStation sizing → Illumina NextSeq 2000 sequence verification → glycerol stock → 96-well induction culture), followed by a fluorescence readout on a BioTek Synergy H1 plate reader (Ex 485 / Em 528 nm) — the latter constituting our HW2 protocols.io contribution since no equivalent operation currently exists in the canvas library.
Materials
Promoters: PA1, PA2, PB1, PB2, PH1, PP2
CDS: A1, B1, B2, H1, H2
Backbone: pSB1C3 (chloramphenicol)
Procedure
Rehydrate Primers with IDTE Buffer; resuspend to 100 µM stock
PCR using Q5 polymerase, Q5 buffer, 50 µL reaction, 12 samples (including NTC), 30 cycles [98°C 10s / 60°C 30s / 72°C 30s/kb]
Perform Gel Electrophoresis with 1% agarose, 100 V, for 30 minutes
Clean Up & Concentrate using QIAquick kit, elute 30 µL in kit-provided buffer (EB)
Modular Gibson Assembly with NEBuilder HiFi, 12 fragments (including pSB1C3 backbone), 50°C, 60 minutes, 20 µL
Transformation & Plating using DH5α electrocompetent, 1.8 kV, 1 hour SOC recovery, plate on LB + cm 25 µg/mL
Colony PCR with 8 colonies + NTC, VR/VF2 primers, Taq, 25 cycles
Gel Electrophoresis with 1% agarose, 100 V, for 45 minutes
Overnight Inoculum: Pick 3 positives → 5 mL LB + cm, 37°C / 250 rpm / 16 hours
Miniprep using QIAprep Spin, elute 50 µL EB
TapeStation with Genomic DNA ScreenTape, dilute to 50 ng/µL, 1 µL load
Illumina NextSeq 2000 using Nextera XT, 2×150 paired-end, 100× coverage
Glycerol Stock with 25% glycerol, 1 mL aliquots, −80°C
Induction in 96-well black/clear, 200 µL LB + cm + inducer, induce at OD 0.3–0.5, 5 hours at 37°C; inducer: Arabinose 0.2% (w/v) or constitutive
Synergy H1 Fluorescence: Ex 485 nm / Em 528 nm, gain 75, kinetic 15 min × 5 hr, normalize F/OD600