Team:NYMU-Taipei/Experiments/Protocols

Promoter Testing

These are the protocols we used for promoter testing

General Gene Reporter Assay protocol

This is our general setup for gene reporting assays.

Previous-night Preparation (~10m-60min)

Previous night:

• Culture overnight (~16hrs) at 37C and shaking at 180rpm 2-3ml of single colonies of things to be measured.

Preparation (can be done the previous night too):

• Determine the number of measurement points = number of items x number of replicates
• Prepare that amount of 15ml round-bottom tubes and 1.5uL microcentrifuge tubes.
• Add 2ml of LB + the relevant antibiotics in each round-bottom tube.

Pre-start (~30mins)

Preparation:

• Prewarm the LB+antibioics to the temperature to test at (e.g. 20mins prewarming in the incubator takes it up to 37C).
• Get some ice buckets and pre-cool down the 1xPBS

Culturing (2~3mins)

1. Add any relevant chemicals to the prewarmed round-bottom tubes containing 2ml LB.
2. Dilute the overnight culture to an OD600 of about 0.0325
3. Incubate at the required conditions (usually 37C and 180rpm).
4. Repeat for every replicate.

Measurement point retrieval

For each culture tube:

1. Take out the relevant culture and put it on ice
2. Extract 1.3ml from the culture tube and put it in an 1.5uL microcentrifuge tube
3. Centrifuge for 1min at 16.2g (13.2rpm on tabletop centrifuge)
4. WASH: Tip out the supernatant and add 650uL of 1xPBS, resuspend, then centrifuge again.
5. WASH again.
6. Carefully remove and discard all the supernatant, resuspend in 1.3ml of 1xPBS.
7. Store at 4C until measuring

Measuring

Requires black plates for measuring fluorescence and transparent plates for measuring OD600. For each measurement point, load 200uL each into 3 black wells and 3 transparent wells (i.e. 3 technical replicates each for fluorescence and OD600). Measure the fluorescence on the black plate with the settings depending on your machine. We use excitation/emission wavelength of 485/525 (100ms per measurement). Measure the OD600 on the transparent plate.

H₂0₂ Promoter Gene Reporter Assay

For measuring promoters under different H202 stress concentrations: (e.g.: 0.01mM, 0.05mM, 0.1mM, 0.5mM, 1mM, 2mM, 2.5mM, 5mM).

• Add the H202 just before adding the bacteria.

For reporting assays using 2ml round-bottom tubes:

• First serially dilute the relevant concentrations from the stock (35%) everytime the experiments start.
• Add 13.4uL of the relevant concentration of H202 into the tube.