Experiments

Transformation


After receiving the gene synthesized by Synbio Technologies, 2 μg of the powder in the vial was dissolved with 20 μL of MilliQ water, forming a solution with a concentration of 100 μg/μL. It is kept at -20°C. Bacterial transformation was performed in order to produce multiple copies of DNA for experiments.

  1. Mix 1 μL of the plasmid DNA with 200 μL of E. coli BL21 cells.
  2. Put the bacterial mixture in ice for 30 minutes.
  3. Incubate the bacterial mixture at 42°C for 90 seconds.
  4. Put in the ice again for 1-2 minutes.
  5. Add 1mL of LB medium to the bacterial mixture.
  6. Incubate the mixture at 37°C at 200 rpm for 1 hour.
  7. Plate 200 μL and all of the bacterial mixture onto a LB agar plate containing 34 mg/mL chloramphenicol.
  8. Incubate the plates at 37°C overnight.

Colony PCR

  1. Set up the following reaction mixture:
    Mastermix Components (for 11 reactions) μL
    2× EasyTaq 5.5
    Forward primer 2.2
    Reverse primer 2.2
    Distilled water 39.6 (variable)
    DNA template variable
    Total volume 110

  2. Aliquot 9 μL into each tube. Pick bacterial colonies with a sterile tip and add them to each reaction mix.

  3. PCR programme was set as follow:
    1. DNA samples were first heated at 95°C for 5 minutes, and then the cycle began.
    2. Reaction mixture was heated to a temperature of 95°C for 30 seconds to denature.
    3. The samples were annealed at a temperature of 55°C for 30 seconds.
    4. The temperature was then increased to 72°C for 1 minute for DNA extension and complete one cycle.
    5. The cycle was repeated 30 times.
    6. After the cycle, the DNA was incubated at 72°C for 5 minutes.
    7. The DNA was stored at 4°C.

AHL preparation

We have ordered 3 synthetic AHL molecules from commercial companies, named N-butyryl L- homoserine lactone (C4HSL), N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo- dodecanoyl L-homoserine lactone (3OC12-HSL). The AHL is prepared by dissolving it in DMSO to result in a 10mM stock solution and the Eppendorf is labelled as tube 1.

Molecular Mass (g/mol) Volume of DMSO used to make a 10 mM stock solution (μL)
30C4-HSL 171.2 584.1
3OC10-HSL 269.3 371.3
3OC12-HSL 297.3 336.3

The preparation of AHLs in seven concentrations is done by serial dilution. 9 eppendorfs are prepared with 450 μl milliQ water each, each is diluted with 50 μl of the previous eppendorf.


Eppendorf AHL Concentration
2 10-5 10 mM
3 10-6 1.0 mM
4 10-7 0.1 mM
5 10-8 0.01 mM
6 10-9 0.001 mM
7 10-10 0.0001 mM
8 10-11 0.00001 mM
9 10-12 0.000001 mM

Bacterial Subculture

  1. Inoculate a single colony of the biosensor into 5 mL of LB broth with 34 mg/mL chloramphenicol and incubate overnight in a shaking incubator at 37°C and 210 rpm.
  2. Transfer 4 mL overnight culture into 100 mL LB broth with 34 mg/mL chloramphenicol and incubate in a shaking incubator at 37°C and 220 rpm until OD600 = 0.3.
  3. 2 mL of cell culture was then transferred into a 15 mL culture tubes for AHL induction with three synthetic AHLs (3OC12-HSL, C10-HSL and C4-HSL respectively) at seven concentrations (1.0 × 10-5, 1.0 × 10-6, 1.0 × 10-7, 1.0 × 10-8, 1.0 × 10-9, 1.0 × 10-10, 1.0 × 10-11M). The culture tube with MilliQ water added was used as a negative control.

AHL Induction

  1. Add 2 mL of cell cultures with 20 μL of three synthetic AHLs (3OC12-HSL, C10-HSL and C4-HSL respectively) at seven concentrations (1.0 × 10-5, 1.0 × 10-6, 1.0 × 10-7, 1.0 × 10-8, 1.0 × 10-9, 1.0 × 10-10, 1.0 × 10-11M). The culture tube with 20 μL of MilliQ water added was used as a negative control.
  2. To test the effect of tap water and sea water on the performance of the engineered biosensors, 0.2 mL of tap water and sea water spiked with 20 μL 3OC12-HSL, C10-HSL and C4-HSL were added to culture tubes containing 1.8 mL of cell cultures.
  3. Incubate culture tubes in a shaking incubator at 37°C and 220 rpm for 2 h.

Washing Cells with Phosphate Buffered Saline (PBS)

  1. Add three aliquots of the 300 μL culture into the Eppendorf.
  2. Centrifuge the cells at 13,500 rpm for 2 minutes to collect the cell pellets.
  3. Discard the supernatant and wash the cells with 500 μL 1× PBS.
  4. Vortex and centrifuge for 2 minutes.
  5. Repeat steps 2-4 once.
  6. Discard all the supernatant to collect the cell pellets.

Hydrogel Preparation and Resuspension

  1. Add 98 mL milliQ water to 2 g of sodium alginate powder.
  2. Heat the mixture until the powder is completely dissolved.
  3. Let the hydrogel cool down at room temperature.
  4. Add 200 μL hydrogel to each pellet.
  5. Pipette up and down to mix well.
  6. Transfer the hydrogel with biosensor cells to a transparent 96-well plate.

X-gal Induction

  1. Add 10 μL of 10 mg/mL X-gal solution into each well and mix well.
  2. Incubate the plate at room temperature to allow blue color development.
  3. Take photos of the 96-well plate in five minute intervals.

Obtaining Data Using ImageJ

Download ImageJ from https://imagej.net/ij/download.html and Readplate 3.0 from https://forum.image.sc/t/readplate-3-0-release/42008.

  1. Set the contrast and saturation of the image to 100 to articulate subtle differences in color intensity.
  2. Open the ImageJ software. Go to "Analyze" → "Set Measurements". Select "Area", "Mean gray value", "standard deviation", "modal gray value" and "Min & max gray value". Select the results to be 5 decimal places. These are selected to improve the accuracy of quantifying the color intensity even if the color is unevenly distributed in a well.
  3. "File" → "Open" and choose the image from your computer.
  4. Drag the rectangle such that the vertices of the rectangle are on the center of the wells A1, A12, H1 and H12.
  5. "Plugins" → "Macros" → "Run"
  6. From "Readplate_wr", select "source code" → "Readplate 3.0". Select 96 for the number of wells.
  7. Select "Red" for "Channel"
  8. For the "parameters", use the default settings.
  9. Check whether the grid generated by the software fits each well. If the grid is at the center of each well, and the color intensity is evenly distributed in each well, click "OK". Else, click "cancel" and increase the "Diameter of main circle (in pixels)" in Step 8.
  10. Click "OK" in "Checking Results", then, select "Yes" in save results.
  11. Name your file with the type of document as .csv or .xlsx to compile a spreadsheet.
Experiments| HKGTC - iGEM 2025