Team:Shanghaitech/Protocol
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
