Difference between revisions of "Team:ZJU-China/Experiments"

Protocol for chemical inducible expression of GFP
Materials:
    1、4 groups of induce solution with a concentration gradient of 10^-7, 10^-5, 10^-3, 10^-2;
    2、 Overnight bacterial culture or bacterial colonies;
    3、Phosphate Buffered Solution (PBS).

Procedure:
    1. Add 20 μ l of the overnight bacterial culture or pick a colony to 5 ml of LB antibiotic medium, Incubate at 37 degree in a shaker till the OD600 value reaches 0.4-0.6.
    2. Add 0.5 mL of the fresh bacterial culture and appropriate volume of inducer solution to prepare induction system with the concentration gradient of 10^-9, 10^-8, 10^-7, 10^-6, 10^-5,10^-4, 10^-3, 10^-2.
    3. Place the induction system at 37 degree for 2 hours.
    4. Pellet bacterial cells by 4 min centrifugation at 4000 rpm, discard the supernatant.
    5. Resuspend the pelleted cells in 500 μ l of PBS.
    6. Transfer 100 uL of bacterial resuspention into each well of 96-well plate to test the expression of GFP by flow cytometry or Microplate Reader.


    1. Late in the day, start a 37 °C, shaking overnight culture from a −80 °C stock in a tube containing 3 mL LB medium and the appropriate antibiotics (50 µg/mL kanamycin, 100 µg/mL spectinomycin and 34 µg/mL chloramphenicol for the CcaS-CcaR system, and the same antibiotics for the Cph8-OmpR system).
    2. After the overnight culture has grown for 10–12 h, prepare 200 mL LB medium. Add appropriate antibiotics to medium. Shake/stir the container to ensure the antibiotics are mixed well in the medium.
    3. Measure the OD 600 of the overnight culture.
    4. Dilute the overnight culture into the LB + antibiotics, bringing the OD 600 to 0.0001. Shake/stir the container to ensure the cells are mixed well in the medium.
    5. Distribute 10 mL of inoculated medium into each of15ml tubes.
    6. Place tubes in the shaker and grow at 37 °C with shaking at180r.p.m. for 8 h. Expose the tubes under light of specific wavelength.
    7. After 8 h of growth, harvest all test tubes by immediately transferring them into an ice-water bath. Wait 10 min for the cultures to equilibrate to the cold temperature and for gene expression to stop.
    8. Approximately 1.5 h before stopping the experimental cultures, begin preparing a solution of phosphate-buffered saline(PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mMKH2PO4 , pH to 7.4) + 500 µg/mL of the transcription inhibitorrifampicin .Rif dissolves slowly, so allow at least 45 min of stirring. Also at this time, begin preparing a 37 °C water bath.
    9. Filter the dissolved solution of PBS + Rif through a0.22-µm 20-mL syringe filter.
    10.6000r.p.mcentrifuge the culture for 3 minutes and refloat with PBS+Rif
    11. 37 °C water bath for 1 h.
    12. Transfer the samples back into ice-water bath.
    13. Wait 15 min, and then begin measuring each tube on a multiple plate reader.


Protocol for measuring and modeling of promoter pluxR
Materials
    1.Mutated plasmid Ptd103sfGFP
    2.Competent cell of MG1655 strain of E·coil
    3.LB media
    4.Phosphate Buffered Solution (PBS).
    5.Kanamycin
    6.N-Hexanoyl-L-homoserine lactone

Procedure:
    1.mutate pTD103luxI-sfGFP with knocking gene luxI out with PCR. We named the new plasmid pTD103sfGFP.
    2.Transform MG1655 strain of E·coil with pTD103sfGFP, inoculated positive colony in 5ml LB with antibiotic 100μg/ml kanamycin and cultured it over night.
    3.Inoculate over night culture with a 1:1000 dilution in 1ml LB and added AHL according a concentration range:10-6~10-11M when the bacteria solution reached an A600nm 0.1,. Culture it in 37℃ for 8h.
    4.Spin it down and concentrate in PBS (pH=7.2), measure the fluorescence intensity.

Verifying the single oscillation circuit
Materials：
    1.Plasmids pTD103luxI-sfGFP and pTD103aiiA-luxS、pSB1C3 plsr-YFP
    2.Competent cell of MG1655 strain of E·coil
    3.M9 media
    4.Kanamycin、Ampicillin、Chloromycetin

Procedure：
    1.Co-transform the MG1655 strain with pTD103aiiA and pTD103luxI-sfGFP.
    2.Screen the positive colony and inoculate in 50ml M9 media with antibiotic 100μg/ml ampicillin (Amp) and 50 mgml21 kanamycin (Kan).
    3.spin it down and concentrate it in 5ml M9 media when the bacteria solution reach an A600nm of 0.05~0.1.
    4.inoculate re-suspension with a 1:1000 dilution in 100ml fresh M9 media, when A600nm 0.1, add to 96 wells plate and measure the fluorescence intencity in 37℃.


Protocol for verifying the single oscillation circuit in microfluidics device
Materials:
    1.Plasmids pTD103luxI-sfGFP and pTD103aiiA
    2.Competent cell of MG1655 strain of E·coil
    3.LB media
    4.Kanamycin、Ampicillin
    5.Tween 20

Procedure:
    1.Co-transform the MG1655 strain with pTD103aiiA and pTD103luxI-sfGFP.
    2.Screen the positive colony and inoculate in 50ml LB with antibiotic 100μg/ml ampicillin (Amp) and 50 mg/ml kanamycin (Kan).
    3.Spin it down and concentrate it in 5ml of fresh media with surfactant concentration of 0.075% Tween20 when the bacteria solution reach an A600nm of 0.05~0.1.
    4.Load the sample from the cell port while keeping the media port at sufficiently higher pressure than the waste port below to prevent contamination. manually applied pressure pulses to line to induce a momentary flow change. The flow was then reversed and allow for cell to receive fresh media with 0.075% Tween20 which prevented cells from adhering to the main channels and waste ports.
    5.Take pictures of microfluidics in fluorescence microscope every 5 mins


Verifying the double oscillation circuit
Materials:
    1.Plasmids pTD103luxI-sfGFP and pTD103aiiA-luxS、pSB1C3 plsr-YFP
    2.Competent cell of MG1655 strain of E·coil
    3.M9 media
    4.Kanamycin、Ampicillin、Chloromycetin

Procedure:
    1.Co-transform the MG1655 strain with pTD103 aiiA-luxS、pTD103 luxI-sfGFP and pSB1C3 plsr-YFP.
    2.Screene the positive colony and inoculate it in 50ml M9 media with antibiotic 100μg/ml ampicillin (Amp) and 50 mg/ml kanamycin (Kan)and Chloromycetin(C).
    3.Spin it down and concentrate it in 5ml M9 media when the bacteria solution reach an A600nm of 0.05~0.1.
    4.Inoculate re-suspension with a 1:1000 dilution in 100ml fresh M9 media. When the bacteria solution reache A600nm 0.1， add to 96 wells plate and measured the fluorescence intensity every 3min.