Team:XMU-China/Experiments

2017.igem.org/Team:XMU-China/Experiments

  • ---* Molecular Cloning *---

  • Requirements:
    —TransGen® Trans5α Chemically Competetent Cell
    —LB broth
    —iGEM DNA Distribution Kit Plates, Plasmid DNA or DNA ligation mix
    —LB agar plates containing 15-100μg/mL antibiotic of choice
    —Nuclease-free 1.5mL microcentrifuge tubes
    —Water bath of 42°C
    —Shaking incubator of 37°C


    Before Starting (if you need plasmids from iGEM DNA Distribution Kit Plates):
    1. Punch a hole with a pipette tip through the foil cover into the corresponding well of the desired BioBrick part.
    2. Add 10μL sterile deionized water, pipette up and down several times.
    3. Transfer liquid from Step 2 into a 1.5mL microcentrifuge tube.
    4. Repeat Steps 2-3 twice.
    5. Store liquid (BioBrick plasmid) at -20°C


    Protocol:
    1. Add 5-10μL plasmid or ligation system into 50μL fresh competent cells, which is contained in 1.5mL centrifuge tube. Then mix gently.
    2. Incubate the tube on ice for 30 minutes.
    3. Heat shock in the water bath at 42°C for 45 seconds.
    4. Incubate on ice for 2 min.
    5. Add 450μL fresh LB broth into the tube.
    6. Incubate for 1 hour under the condition of 37°C, 200rpm using a shaking incubator.
    7. Spread 250μL liquid from Step6 on a LB agar plate, which contains appropriate antibiotics.
    8. Incubate overnight at 37°C (about 12 hours, no more than 18 hours).


  • Requirements:
    —LB agar plate containing transformed bacterial colonies incubated overnight
    —LB broth
    —Antibiotics
    —50mL centrifuge tubes
    —Shaking incubator of 37°C


    Protocol:
    1. Add 10mL LB broth into a centrifuge tube, then add the appropriate antibiotic needed.
    2. Select the single colony using the pipette tip from the agar plate, which contains the bacterial cells. Then put the pipette tip into the tube from Step 1.
    3. Incubate overnight at 37°C (about 12 hours, no more than 18 hours).


  • Materials and Equipments:
    —Bacterial culture
    —80% Glycerol
    —1.5mL cryogenic microtubes


    Protocol:
    1. Add 200µL glycerol into a cryogenic microtube.
    2. Pipet 800µL bacterial culture into glycerol in the cryogenic microtube from step 1 and mix by pipetting, save in -80°C freezer.
    3. This glycerol stock can be used whenever required, by just adding 10µL glycerol stock into 10mL LB broth and incubating overnight.


  • Requirements:
    —Omega E.Z.N.A.® Plasmid Mini Kit II
    —100% ethanol
    —Isopropanol
    —Microcentrifuge capable of at least 13,000 x g
    —Nuclease-free 1.5mL microcentrifuge tubes
    —Sterile deionized water
    —Electric dry oven of 65°C
    —Water bath of 65°C


    Before Starting:
    —Heat sterile deionized water to 65°C using water bath
    —Add the vial of RNase A to the bottle of Solution I if there's no mark on the bottle and store at 4°C
    —Add 100% ethanol to the bottle of DNA Wash Buffer if there's no mark on the bottle and store at room temperature
    —Add isopropanol to the bottle of HBC Buffer if there's no mark on the bottle and store at room temperature
    (The volume of ethanol and isopropanol is showed on the tag of the bottle)


    Protocol:
    1. Pellet 1.5mL bacteria in a clean 1.5mL microcentrifuge tube by centrifugation at 10,000 x g for 1 minute at room temperature. Decant or aspirate medium and discard.
    2. Add 250μL Solution I/RNase, pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.
    3. Add 250µL Solution II and gently mix by inverting and rotating the tube several times to obtain a clear lysate. A 2 minutes incubation is necessary. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity.
    4. Add 350µL Solution III and mix immediately by inverting the tube several times until a flocculent white precipitate forms. Incubate for 2 minutes.
    5. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes at room temperature. A compact white pellet will form. Promptly proceed to the next step.
    6. Insert a HiBind® DNA Mini Column into a 2mL Collection Tube.
    7. Transfer 700µL cleared lysate from Step 5 CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column.
    8. Centrifuge at maximum speed for 1 minute.
    9. Discard the filtrate and reuse the collection tube.
    10. Repeat Steps 7-9 until all cleared lysate has been transferred to the HiBind® DNA Mini Column.
    11. Add 500µL HBC Buffer.
    12. Centrifuge at maximum speed for 1 minute.
    13. Discard the filtrate and reuse the collection tube.
    14. Add 700µL DNA Wash Buffer.
    15. Centrifuge at maximum speed for 1 minute.
    16. Discard the filtrate and reuse the collection tube.
    17. Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
    18. Transfer HiBind® DNA Mini Column to a clean 1.5mL microcentrifuge tube. Open the lid and put it in the electric dry oven for 10 minutes to volatilize alcohol.
    19. Add 60µL sterile deionized water directly to the center of the column membrane.
    20. Let sit at room temperature for 2 minutes.
    21. Centrifuge at maximum speed for 1 minute.
    22. Store DNA at -20°C.


  • Requirements:
    —Plasmid DNA or PCR product
    —Restriction enzymes and buffers (produced by Takara Bio®)
    —Nuclease-free 0.2mL PCR tubes
    —Water bath at 37°C


    Protocol:
    1. Prepare reaction systems in a 0.2mL PCR tube according to the following table:



    Choose the buffer according to the following table:



    2. Incubate in the water bath at 37°C for 6-8 hours.


  • Requirements
    —50× TAE concentrate Solution (produced by Solarbio®)
    —Agarose (produced by Biowest®)
    —DNA dye (TransGen® GelStain)
    —100mL Erlenmeyer flask
    —Distilled water
    —Microwave oven
    —DNA samples
    —10× Loading buffer (produced by Takara®)
    —DNA marker (produced by TranGen®)
    —Electrophoresis instrument


    Before Starting:
    —Dilute 50× TAE concentrate Solution to 1× TAE buffer with distilled water
    —Add 10× loading buffer into marker and DNA samples. Loading buffer should occupy 10% of total volume


    Protocol:
    1. Weigh 0.36g agarose in an Erlenmeyer flask.
    2. Add 30mL 1× TAE buffer into the flask from Step 1.
    3. Make agarose melt by microwave oven (medium-high heat, about 3 minutes).
    4. Add 3μL TransGen® GelStain, mix by shocking.
    5. Assemble gel pouring apparatus by inserting gate into slots.
    6. Pour agarose gel into the gel tray.
    7. Cool for 40 minutes to solidify the DNA agarose gel.
    8. Remove the pouring apparatus, put the gel into an electrophoresis instrument.
    9. Pipet marker and DNA samples which have been mixed with loading buffer into the slots.
    10. Turn on the electrophoresis instrument, set the working electric current at 95-125mA.
    11. Electrophoresis for 45-90 minutes.
    12. Turn off the instrument, take the gel into the gel formatter to take and save photos.


  • Requirements:
    —Omega E.Z.N.A.® Gel Extraction Kit
    —DNA agarose gel sliced from the electrophoresed gel
    —100% ethanol
    —Microcentrifuge capable of at least 13,000 x g
    —Nuclease-free 1.5 mL microcentrifuge tubes
    —Sterile deionized water
    —Electric dry oven of 55°C and 65°C
    —Water bath of 65°C


    Before starting:
    —Heat sterile deionized water to 65°C using water bath
    —Add 100% ethanol to the bottle of SPW Wash Buffer if there's no mark on the bottle, store at room temperature
    (The volume of ethanol is showed on the tag of the bottle)


    Protocol:
    1. Put the gel slice in a clean 1.5mL microcentrifuge tube.
    2. Add Binding Buffer to fill the microcentrifuge tube from Step 1.
    3. Incubate at 55°C in a water bath, until the gel has completely melted. Shake the tube every 2-3 minutes.
    4. Insert a HiBind® DNA Mini Column in a 2mL Collection Tube.
    5. Add 700µL solution from Step 3 to the HiBind® DNA Mini Column.
    6. Centrifuge at 10,000 × g for 1 minute at room temperature.
    7. Discard the filtrate and reuse collection tube.
    8. Repeat Steps 5-7 until all of the sample has been transferred to the column.
    9. Add 300µL Binding Buffer.
    10. Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature.
    11. Discard the filtrate and reuse collection tube.
    12. Add 700µL SPW Wash Buffer.
    13. Centrifuge at maximum speed for 1 minute at room temperature.
    14. Discard the filtrate and reuse collection tube.
    μL 15. Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
    16. Transfer the HiBind® DNA Mini Column to a clean 1.5mL microcentrifuge tube. Open the lid and put it in the electric dry oven for 10 minutes to volatilize alcohol.
    17. Add 35µL sterile deionized water directly to the center of the column membrane.
    18. Let sit at room temperature for 2 minutes.
    19. Centrifuge at maximum speed for 1 minute.
    20. Store DNA at -20°C.


  • Requirements:
    —Takara PrimeSTAR® Max DNA Polymerase
    —DNA template
    —Primers (synthetized by Sangon Biotech®)
    —Nuclease-free 0.2mL PCR tubes
    —PCR instrument


    Protocol:
    1. The choice of reaction system:



    2. The choice of PCR program::



    Note: x is the annealing temperature of the reaction, usually 5°C to 10°C lower than Tm of the primer.
    The calculation of Tm: Tm=4(G+C)+2(A+T)°C


  • Requirements:
    —Omega E.Z.N.A.® Cycle Pure Kit
    —PCR product
    —100% ethanol
    —Microcentrifuge capable of at least 13,000 x g
    —Nuclease-free 1.5mL microcentrifuge tubes
    —Sterile deionized water
    —Electric dry oven of 65°C
    —Water bath of 65°C


    Before Starting:
    —Heat sterile deionized water to 65°C using water bath
    —Add 100% ethanol to the bottle of DNA Wash Buffer if there's no mark on the bottle and store at room temperature
    (The volume of ethanol is showed on the tag of the bottle)


    Protocol:
    1. Determine the volume of PCR product, and transfer the product into a clean 1.5mL microcentrifuge tube.
    2. Add 4-5 volumes CP Buffer. For PCR products smaller than 200bp, add 6 volumes CP Buffer.
    3. Vortex to mix thoroughly.
    4. Insert a HiBind® DNA Mini Column into a 2mL Collection Tube.
    5. Add the sample from Step 3 to the HiBind® DNA Mini Column.
    6. Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature.
    7. Discard the filtrate and reuse collection tube.
    8. Add 700µL DNA Wash Buffer.
    9. Centrifuge at maximum speed for 1 minute.
    10. Discard the filtrate and reuse collection minute.
    11. Centrifuge the empty HiBind® DNA Mini Column at maximum speed for 2 minutes to dry the column.
    12. Transfer the HiBind® DNA Mini Column into a clean 1.5mL microcentrifuge tube. Open the lid and put it in the electric dry oven for 10 minutes to volatilize alcohol.
    13. Add 40µL sterile deionized water directly to the center of column matrix.
    14. Let sit at room temperature for 2 minutes.
    15. Centrifuge at maximum speed for 1 minute.
    16. Store DNA at -20°C.


  • Requirements:
    —Digested DNA
    —T4 ligase and buffer (produced by Takara Bio®)
    —Nuclease-free 0.2mL PCR tubes
    —Water bath at 16°C or fridge at 4°C


    Protocol:
    1. Prepare reaction systems in a 0.2mL PCR tube according to the following table:





    V1: insert
    V2: vector
    M: molar weight (size)
    C: conc. (ng/mL)
    2. Incubate in the water bath at 16°C for 6 hours or fridge at 4°C for 12 hours.

  • ---* Characterization *---

  • Requirements:
    —TransGen® BL21(DE3) Chemically Competetent Cell
    —LB broth
    —Expression plasmid containing coding sequence
    —LB agar plates containing 15-100μg/mL antibiotic of choice
    —Nuclease-free 1.5mL microcentrifuge tubes
    —Water bath of 42°C
    —Shaking incubator of 37°C


    Protocol:
    1. Add 5-10μL plasmid into 50μL fresh competent cells, which is contained in 1.5mL centrifuge tube. Then mix gently.
    2. Incubate the tube on ice for 30 minutes.
    3. Heat shock in the water bath at 42°C for 45 seconds.
    4. Incubate on ice for 2 min.
    5. Add 450μL fresh LB broth into the tube.
    6. Incubate for 1 hour under the condition of 37°C, 200rpm using a shaking incubator.
    7. Spread 250μL liquid from Step6 on a LB agar plate, which contains appropriate antibiotics.
    8. Incubate overnight at 37°C (about 12 hours, no more than 16 hours).
    9. Growing the Single Colonies from Agar Plates (the same protocol as Molecular Cloning).


  • Requirements:
    —TransGen® BL21(DE3) Chemically Competetent Cell
    —LB broth
    —Plasmid DNA
    —LB agar plates containing 15-100μg/mL antibiotic of choice
    —Antibiotics
    —Nuclease-free 1.5mL microcentrifuge tubes
    —Shaking incubator of 37°C
    —50mL centrifuge tubes
    —96-well plates
    —Tecan® Infinite M200pro microplate reader
    —Isopropyl β-D-1-thiogalactopyranoside
    —Decanal


    Protocol-Expression Temperature:
    1. Resuscitate the bacteria which contains T7+luxAB plasmid. Add 10μl-20μl bacteria to 4ml LB.
    2. Incubate at 37°C until the OD is measured between 0.6 and 0.8.
    3. Add 4μl IPTG inductor to each tubes. Incubate respectively at 30°C, 33°C and 37°C.
    4. After incubation, take out 1ml of each tube as background and measure the OD.
    5. Take out 1ml bacteria from each tube and add 10μl 1% decanal.
    6. Take out 100μl bacteria from each tube and put it in a white light-proof 96 hole for measuring.


    Protocol-Influence of the number of bacteria and the substrate quantity:
    1. Resuscitate the bacteria which contains T7+luxAB plasmid. Add 2ml bacteria to 300ml LB.
    2. Measure the OD value at different time, take out 4ml bacteria to a tube when the OD value is 0.6, 1.2, 1.8, 2.4, 3.0 and 3.6. Each condition is in quadruplicate.
    3. Add 4μl IPTG inductor to each tubes and incubate for 3 hours.
    4. After incubation, take out 1ml of each tube as background and measure the OD.
    5. Take out 1ml bacteria from each tube and add decanal as shown in the table below:



    6. Take out 100μl bacteria from each tube and put it in a white light-proof 96 hole for measuring.


    Protocol-The influence of induction time:
    1. Resuscitate the bacteria which contains T7+luxAB plasmid. Add 2ml bacteria to 300ml LB.
    2. Measure the OD value at different time, take out 4ml bacteria to a tube when the OD value is 0.6, 1.2, 1.8, 2.4, 3.0 and 3.6. Each condition is in triplicate.
    3. Add 4μl IPTG inductor to each tubes and incubate at 30℃ for 1 hour, 2 hours,3 hours.
    4. After incubation, take out 1ml of each tube as background and measure the OD.
    5. Take out 1ml bacteria from each tube and add 10μl 1% decanal.
    6. Take out 100μl bacteria from each tube and put it in a white light-proof 96 hole for measuring.


    Protocol-The influence of IPTG concentration:
    1. Resuscitate the bacteria which contains T7+luxAB plasmid. Add 2ml bacteria to 300ml LB.
    2. Measure the OD value at different time, take out 4ml bacteria to a tube when the OD value is 0.6, 1.2, 1.8, 2.4, 3.0 and 3.6. Each condition is in quintuplicate.
    3. Add respectively 4μl,8μl,12μl,16μl and 20μl IPTG inductor to each tubes and incubate at 30℃ for 3 hours.
    4. After incubation, take out 1ml of each tube as background and measure the OD.
    5. Take out 1ml bacteria from each tube and add 10μl 1% decanal.
    6. Take out 100μl bacteria from each tube and put it in a white light-proof 96 hole for measuring.


  • Requirements:
    —TransGen® BL21(DE3) Chemically Competetent Cell
    —LB broth
    —Plasmid DNA
    —LB agar plates containing 15-100μg/mL antibiotic of choice
    —Antibiotics
    —Arsenic standard solution
    —Nuclease-free 1.5mL microcentrifuge tubes
    —Nuclease-free 0.2mL PCR tubes
    —Water bath of 42°C
    —Shaking incubator of 37°C
    —50mL centrifuge tubes
    —96-well plates
    —Tecan® Infinite M200pro microplate reader


    Protocol:
    1. Mix the metal ion sensing promoter + T7 RNA polymerase plasmid and T7 promoter + GFP plasmid together in the ratio of 1:1, transform the mixed plasmid on a LB agar plate which contains AMP and CM antibiotics.
    2. Incubate overnight at 37°C for 12 hours.
    3. Grow the Single Colonies from Agar Plates.
    4. Do a 1/750 dilution of cells into fresh media with AMP and CM antibiotics.
    5. Incubate at 37°C for 3 hours. Add arsenic standard solution by the concentration of 0 mmol/mL 、2.6mmol/ml、3.9mmol/ml、100mmol/ml. Each condition is in triplicate.
    6. Add 100 μl into each well into a 96 Well plate which is laid on the ice every thirty minutes.
    7. Continue sampling for 900 minutes.
    8. Measurements of absorbance and fluorescence:
    (1) OD 600
    Device: Plate Reader(Tecan Infinite M200pro) Wavelengths: 600 nm absorption.
    (2) Fluorescence (CFP and GFP)
    Device: Plate Reader(Tecan Infinite M200pro), 96-well plates.
    Wavelengths: CFP:439 nm excitation, 476 nm emission; GFP: 485 nm excitation, 520 nm emission.


  • Requirements:
    —TransGen® BL21(DE3) Chemically Competetent Cell
    —LB broth
    —Plasmid DNA
    —LB agar plates containing 15-100μg/mL antibiotic of choice
    —Antibiotics
    —Arsenic standard solution
    —Nuclease-free 1.5mL microcentrifuge tubes
    —Nuclease-free 0.2mL PCR tubes
    —Water bath of 42°C
    —Shaking incubator of 37°C
    —50mL centrifuge tubes
    —96-well plates
    —PAPG


    Protocol:
    1. Mix the metal ion sensing promoter + T7 RNA polymerase plasmid and T7 promoter + GFP plasmid together in the ratio of 1:1, transform the mixed plasmid on a LB agar plate which contains AMP and CM antibiotics.
    2. Incubate overnight at 37°C for 12 hours.
    3. Grow the Single Colonies from Agar Plates.
    4. Do a 1/750 dilution of cells into fresh media with AMP and CM antibiotics.
    5. Incubate at 37°C for 3 hours. Add arsenic standard solution by the concentration of 0 mmol/mL 、2.6mmol/ml、3.9mmol/ml、100mmol/ml. Each condition is in triplicate.
    6. Take out 1ml bacteria from tubes , add 4mg PAPG and incubate for 5 minutes each hour.
    7. Measure the electrical signal.


  • Protocol:
    1. Measurement of OD: after shaking cultivation to some concentration, measure OD600 of bacterial solution with ultraviolet spectrophotometer. It should be 1.1~1.2.
    2. Centrifugation: 1 mL solution of bacteria, 12000r for 2 min.
    3. Resuspension: add 500 L protective agents what we used are 5% skimmed milk, 10% skimmed milk, 20% skimmed milk, Luria-Bertani broth, 10% sucrose solution and 10% sucrose + Luria-Bertani to each centrifuge tube and then resuspend them.
    4. Pre-freezing: put the centrifuge tubes into the refrigerator to freeze in three different condition: gradient freeze(4℃ for 30 min、-20℃ for 30 min、-80℃ for 1h), -20℃ for 2h, -80 ℃ for 1h.
    5. Operate the lyophilizer: start the lyophilizer to allow the chamber temperature to drop to -50℃.
    6. Freeze-drying: after pre-freezing of the sample, put it into the lyophilizer. Then sublimation for 12 hours under vacuum condition which called Primary drying; Lastly, increase shelf temperature to about 30 ℃ to desorbe the residual water under vacuum condition.
    7. Resuscitation: dissolve the sample by adding 1 mL LB or ultra-pure water. Then use the shaking machine to cultivate for 1h under 37℃. After that, add 2 ‰ IPTG to induce gene expression.
    8. Cultured on the plate: dilute 10L solution of bacteria by 10000 times. Add 250L of the bacteria solution to plate and culture it for 12h.
    9. Observe and Analyze the Results.

  • ---* Addenda *---



  • Note: All of these antibiotics are produced by SolarBio®.






  • Primers:
    VF2 and VR
    VF2: TGCCACCTGACGTCTAAGAA
    VR: ATTACCGCCTTTGAGTGAGC
    Note: Our primers are synthetized by Sangon Biotech®, Shanghai

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