Experiments

This page describes the research, experiments, and protocols we used in our project. It is designed to provide sufficient information for other teams to replicate our work.

Experiments

Experiment Overview

1 - Plasmid Preparation

The optogenetic activator plasmid was constructed by assembling the fragments into the pLVX-Hygro vector digested by XhoI/XmaI. The dual-hormone expression plasmid was synthesized and constructed by Tsingke Biotechnology using pGL3basic vector. Plasmids were extracted from E. coli cultures using the alkaline lysis method, followed by purification via silica-column chromatography. DNA concentrations were quantified via spectrophotometry, yielding high-purity plasmid preparations ranging from 1246.1 to 1311 ng/µL.

2 - Cell Culture & Transfection

HEK-293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (V/V) fetal bovine serum (FBS) and 1% (V/V) penicillin/streptomycin solution. Cells are digested and passaged using 0.05% trypsin digestion solution when reaching 80% confluence. It was ensured that no more than 10 passages of HEK-293T cells were used in the experiments.

3 - Plasmid Transfection

HEK-293T cells were transfected with plasmids using Lipofectamine 3000 according to the manufacturer's protocol.

4 - Blue light LEDs activation

Transfected cells were incubated in dark or illuminated with 480 nm pulsatile blue light (1 s ON, 5 s OFF; light intensity: ~ 500 μW as measured with a Thorlabs PM160T Wireless Power Meter) by using a custom designed light-emitting diode (LED) array.

5 - Confocal Imaging

Cells for confocal imaging were plated into 35 mm glass bottom dish. All confocal images were acquired on an Olympus FV3000 confocal laser scanning microscope equipped with a ×60 NA 1.42 oil immersion objective and 405/488/561/640 nm lasers. For live-cell imaging, cells were maintained at 37℃ and 5% CO2 in a humidified chamber.

6-Total RNA extraction and RT-qPCR

Total RNA was isolated with Trizol reagent and quantified. Reverse transcription was conducted as follows with the Fast All-in-One RT Kit, according to the manufacturer’s instructions. Quantitative PCR of cDNA was performed using Realtime PCR Super mix SYBR green on the Light Cycler 480 real-time PCR instrument (Roche Life Science). The program for qPCR amplifications was as follows: 95℃ for 5 min, followed by 40 cycles at 95℃ for 10s, 60℃ for 30s. All samples were normalized to the housekeeping gene RPLP0 as the endogenous control and determined by the standard 2-ΔΔCt method to obtain relative mRNA expression levels.

Protocol

Purpose: to amplify the target DNA fragment from an existing plasmid.
Steps:
  1. Reaction mixture preparation:
    10×PCR buffer: 5 μL
    2.5mM MgSO₄: 3 μL
    2mM dNTP: 5 μL
    Forward primer (10μM): 1.5 μL
    Reverse primer (10μM): 1.5 μL
    Template plasmid: 1 μg
    KOD plus: 1 μL
    ddH₂O: 32 μL
  2. Initial denaturation: 94°C for 2 minutes
  3. Amplification Cycles (repeat 35 times):
    • Denaturation: 98°C for 10 seconds
    • Annealing: Tm for 30 seconds
    • Extension: 68°C for 30 seconds per kb
  4. Final Extension: 68°C for 7 minutes
  5. Hold: 4°C indefinitely

Purpose: to linearize the vector plasmid by restriction enzyme digestion at specific cleavage sites.
Steps:
  1. Reaction mixture preparation:
    rCutSmart buffer: 5 μL
    Template plasmid: 2 μg
    Restriction Enzyme 1 (XhoI): 1 μL
    Restriction Enzyme 2 (XmaI): 1 μL
    ddH₂O: 41 μL
  2. Digest at 37°C for 1 hour

Purpose: to confirm the size of DNA fragments such as PCR products and separate DNA molecules by their molecular weight.
Steps:
  1. Prepare a 1% agarose gel by dissolving 0.3 g of agarose in 30 mL of 1× TAE buffer, then add 3 μL of Gel Red. Heat the mixture for 1 minute using microwave heating, pour it into a casting mold, and allow it to solidify at room temperature
  2. Add 6× Loading Buffer to the DNA samples, place the agarose gel in the electrophoresis tank, and load the samples into the wells
  3. Run the gel at 120 V for 30 minutes

Purpose: to extract high-purity DNA fragments from the gel following electrophoresis.
Steps:
  1. Excise the DNA band from the gel and transfer it to a 1.5 mL microcentrifuge tube
  2. Add 700 μL of Buffer GM and incubate at 50°C until the gel slice is completely dissolved
  3. Transfer the solution to a spin column and centrifuge at 12,000 rpm for 1 minute
  4. Discard the flow-through, add 700 μL of Buffer WB, and centrifuge at 12,000 rpm for 1 minute
  5. Repeat step 4
  6. Discard the flow-through and centrifuge at 12,000 rpm for 1 minute
  7. Place the spin column into a new microcentrifuge tube and air-dry for 1 minute
  8. Add 20 μL of ddH₂O preheated to 50°C, incubate at 50°C for 5 minutes, then centrifuge at 12,000 rpm for 1 minute
  9. Quantify the DNA concentration using a microvolume spectrophotometer

Purpose: to assemble one or multiple DNA fragments with a vector into a new plasmid.
Steps:
  1. Reaction mixture preparation: combine 5 μL of 2× Basic Assembly Mix with insert fragments and linearized backbone at a 1:5 molar ratio, then adjust the total volume to 10 μL with ddH₂O
  2. Incubate at 50°C for 15 minutes, then store at 4°C

Purpose: to introduce the homologous recombination product into competent cells and culture them on bacterial plates.
Steps:
  1. Thaw 50 μL of DH5α competent cells on ice, add 2 μL of the homologous recombination product, and incubate on ice for 30 minutes
  2. Heat shock at 42°C for 30 seconds, then immediately transfer to ice for 2 minutes
  3. Plate the competent cell mixture
  4. Incubate at 37°C for 16 hours, then store at 4°C

Purpose: to isolate high-purity plasmid DNA from E. coli.
Steps:
  1. Centrifuge the bacterial culture at 4°C and 4500 rpm for 30 minutes, discard the supernatant, and invert the tube to drain
  2. Add 8 mL of RES buffer (+RNase) to resuspend the pellet, then add 8 mL of LYS buffer and invert the tube 5 times. Allow the mixture to stand at room temperature for 5 minutes
  3. Add 12 mL of EQU buffer along the wall of the spin column
  4. Add 8 mL of NEU buffer to the mixture from step 2, then shake and mix until the solution becomes colorless. Transfer the mixture to spin column and allow it to drain completely by gravity flow
  5. Add 5 mL of EQU buffer along the inner wall of the spin column, and allow it to drain completely by gravity flow
  6. Remove the spin column, add 8 mL of WASH buffer to the adsorption column, and allow it to drain completely by gravity flow
  7. Add 5 mL of ELU buffer preheated to 50°C to the adsorption column, and collect the eluate in a 15 mL centrifuge tube
  8. Add 3.5 mL of isopropanol to the centrifuge tube, shake vigorously to mix thoroughly, and incubate at -20°C for 20–30 minutes
  9. Centrifuge the tube at 4°C and 4500 rpm for 30 minutes
  10. Carefully remove the supernatant, wash the precipitated plasmid DNA with 70% ethanol, and transfer it to a 1.5 mL microcentrifuge tube
  11. Centrifuge the plasmid pellet at 4°C and 12,000 rpm for 1 minute, discard the supernatant, and air-dry the pellet
  12. Add 200 μL of ddH₂O and resuspend the plasmid by pipetting
  13. Quantify the concentration of the plasmid solution using a microvolume spectrophotometer, adjusted to 1000 ng/μL with ddH₂O, and stored at 4°C

Purpose: to deliver the target plasmid into the cells
Steps:
  1. For 12-well plates and 35 mm glass-bottom dishes, dilute 2.25 μL of Lipofectamine 3000 in 75 μL of Opti-MEM
  2. Dilute 1.5 μg of plasmid and 3 μL of P3000 in 75 μL of Opti-MEM
  3. Combine the solutions from steps 1 and 2, mix thoroughly by pipetting, and incubate at room temperature for 10-15 minutes
  4. Add the mixture to the cell culture medium and gently swirl to ensure even distribution

Purpose: to observe the fluorescence-labeled protein dynamics in living cells
Steps:
  1. Clean lenses with ethanol, apply immersion oil
  2. Place the 29 mm glass-bottom dish at the center of the stage
  3. Focus cells using coarse and fine knobs, select target cells
  4. Capture single images or time-series images

Purpose: to extract high-purity total RNA from HEK-293T cells
Steps:
  1. For 12-well plates, aspirate the culture medium, rinse the cells with ice-cold PBS, add 400 μL of TRIzol lysis reagent to each well, swirl the plate to ensure uniform coverage
  2. Incubate at room temperature for 5 minutes to complete lysis, mix the lysate by pipetting and transfer it to 1.5 mL microcentrifuge tubes
  3. Add 80 μL of chloroform, invert the tube vigorously for 15 seconds, and incubate at room temperature for 3 minutes
  4. Centrifuge at 12,000 rpm for 15 minutes at 4°C
  5. Pipette 180 μL of the upper aqueous phase into a new 1.5 mL microcentrifuge tube, add 200 μL of isopropanol, mix gently, and incubate at -20°C overnight
  6. Centrifuge at 12,000 rpm for 10 minutes at 4°C
  7. Discard the supernatant, add 400 μL of 75% ethanol solution prepared with DEPC-treated water, and centrifuge at 7500 rpm for 5 minutes at 4°C
  8. Remove as much supernatant as possible, then air-dry the pellet at room temperature for 5–10 minutes
  9. Add 20 μL of DEPC-treated water and mix thoroughly by pipetting
  10. Quantify the RNA concentration using a microvolume spectrophotometer and store the solution at -80°C

Purpose: to reverse transcribe RNA into cDNA
Steps:
  1. Take 500-1000 ng of total RNA, add 4 μL of DNase, and adjust the volume to 16 μL with DEPC-treated water. Mix thoroughly by pipetting
  2. Incubate at room temperature for 5 minutes
  3. Add 4 μL of 5× RT Mix and mix thoroughly by pipetting
  4. Incubate at 42°C for 15 minutes, then dilute the product 10-fold with ddH₂O, and stored at 4°C

Purpose: to quantify the expression level of target genes in samples
Steps:
  1. Reaction mixture preparation:
    2×SYBR Green qPCR Mix: 10 μL
    cDNA: 1.5 μL
    Forward primer (10μM): 0.4 μL
    Reverse primer (10μM): 0.4 μL
    ddH₂O: 7.7 μL
  2. Initial denaturation: 95°C for 5 minutes
  3. 40 cycles:
    • Denaturation: 95°C for 10 seconds
    • Annealing/Extension: 60°C for 30 seconds
    • Melting curve
For full protocols, see our Lab Notebook.