Protocol

XIII. Test the function of convertor with the liquid handling robot

Day 0

1. Preparation of autoinducer (Rpa signal molecules) solution at different concentration.

Dilute the autoinducers in an 8-well PCR strip with DMSO to make the final concentration in each tube (mol•L-1):

10^-7 10^-8 10^-9 10^-10 0 (DMSO & LB only)

Since that every well is set to contain 50ml samples, working solution concentration in 1ml LB medium is:

10^-4 10^-5 10^-6 10^-7 0 (DMSO & LB only),

 

Note:

• For every Homoserine Lactate autoinducer, it’s recommended to prepare all working solution at the same time to ensure the reproducibility of the experiments.

• The concentration gradient working solution should be stored at 4°C.

• Most Homoserine Lactate autoinducers have a non-polar tail, we choose DMSO as the solvent.

• DO NOT use solvent with high volatility such as Chloroform. Using such solvents will led to great variation in concentration.

Day 1

2. The night before testing, grow bacteria culture overnight for the signal receiver in M9 Media.

3. The night before testing, grow bacteria culture overnight for the signal convertor in LB media.

Day 2

4. Dilute all the signal convertor cultures to OD₆₀₀ (Optical Density at 600 nm) of 0.05 with at least 50ml volume.

5. Let the bacteria grow for approximately 3-4 hours, monitoring OD₆₀₀ to make sure it does not grow above 0.5. It usually takes about 3 hours for the bacteria to grow to the desired OD₆₀₀ value.

6. When the OD₆₀₀ of the samples reaches 0.5, distribute samples into 50ml centrifuge tubes with 10ml per tube.

7. Add 100 μL pre-diluted autoinducer concentration gradient working solution to all 5 tubes with robotic liquid handling system.

8. Set the sampling program of robotic liquid handling system. Every 2 hours after adding autoinducer until 8 hours stimulation, take 20μL sample from all 5 centrifuge tubes, 3 parallel samples at a time. The samples should be added to a 96-well plate from well A4 to well D12.

9. Dilute all the signal receiver cultures to OD₆₀₀ of 0.05 with at least 20ml volume.

10. Let the bacteria to grow for approximately 3-4 hours, monitor OD₆₀₀ to make sure it does not grow above 0.5.

 

Note:

• When adding autoinducer concentration gradient working solution to 50ml centrifuge tubes, the order should be adding the lower concentrated working solution first to minimize the interference of leftover solution.

11. When the OD₆₀₀ of the signal receiver culture reaches 0.5, distribute samples into a 96-well plate used in step 8, 200μL per well.

12. Add pre-diluted autoinducer concentration gradient working solution to first three columns as the positive control to prove that the Las signal molecules exist in the liquid that induce LasR-GFP .

13. Put the plate in a multifunctional microplate reader and take regular measurements of OD₆₀₀ and fluorescence.

⁃For GFP use excitation frequency of 485nm and an emission frequency of 528nm