Protocols

1. Plasmid Construction

In our experimental design, plasmid construction is the basis of all work.In the construction of plasmid, we mainly did the following work:

Obtain the desired plasmid from the distribution kits:

1.With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that we want.

2.Pipette 10 µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye.

3.Transform 3 µL of the resuspended DNA into competent cells, plate the transformation with the appropriate antibiotic* and grow overnight.

4.Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.

5.Use the resulting culture to miniprep the DNA and make our own glycerol stock.

Using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

Glycerol stock

1.Add 0.1 ml of 80% glycerol in H2O to a cryogenic vial.

2.Add 0.4 ml sample from the culture of bacteria to be stored.

3.Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.

4.Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.

5.On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.

6.Store in a freezer box in a -80 °C freezer.

Make Competent Cells

(Most of the time we use commercialized competencies DH5α)

1.Add 200 μL E.coli DH5α to LB medium and row the cells on a shaker at 37 °C for 4 hours.

2.Take 1.5 mL of the culture product and add it to the EP tube. Centrifuge at 4000 rpm for five minutes 4 °C . Discard the liquid and add 0.6ml of CaCl2 solution. Ice bath After 30 minutes.

3.Centrifuge at 4000 rpm for five minutes 4 °C. Discard the liquid and add 80 μL of CaCl2 solution.

Single Tube Transformation

1.Thaw competent cells on ice.

2.Pipette 3 µl of resuspended DNA into 100 µl thawed cells. Gently pipette up and down a few times. Keep all tubes on ice.

3.Incubate on ice for 25 minutes.

4.Heat shock tubes at 42 °C in water bath for 45 seconds.

5.Incubate on ice for 5 minutes. Return transformation tubes to ice bucket.

6.Pipette 500 µl LB media to the transformation.

7.Incubate at 37 °C for 1 hours, shaking at 220 rpm.

8. Spin down cells at 6000 rpm for 1 minutes and discard 400 µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette the transformation onto petri plates Spread with sterilized spreader.

9.Incubate transformations overnight (14-18hours) at 37 °C: Incubate the plates upside down (agar side up).

Colony PCR

1.Prepare several 0.2 mL Eppendorf tubes with 20 μL ddH2O (double-distilled water) in each.

2.Using pipette tips, pick several single colonies from the agar gel medium into individual tubes. Fully vortex the mixture to suspend the bacterium.

3.Make up a 10 μL PCR system in PCR tubes(5 μL PCR Mix Master, 3 μL ddH2O, 1 μL suspension and 1 μL of primer mix)

4.Set the PCR protocol to “Colony PCR” *, place the tubes into the PCR machine and lock the heat cover. Start the protocol.

*Protocol of Colony PCR: 94 °C preheating for 5 minutes; 30 replicates of these steps: 94 °C heating for 30 seconds, 55 °C annealing for 30 seconds and 72 °C extending for 1 minute (at a speed of about 1 kbp/min); keep the products at the temperature of 16 °C .

Plasmid DNA extraction （lifefeng® ）

1.Transfer 2 mL of the culture to a Eppendorf tube. Centrifuge at 13000 rpm for 1 minute. Discard the supernatant. Add 250 μL of resuspension solution (P1 buffer). Completely resuspent cell pellet.

2.Add 250 μL of lysis solution (Buffer P2) and mix gently. Set aside for 1 minute.

3.Add 350 μL of neutralizing solution (P3 buffer) and mix by inverting the tubes for 5-10 times. Buried in the ice for 10 minutes.

4.Centrifuge at 12000 rpm for 10 minutes and carefully transfer the supernatant to a adsorption column. Centrifuge at 10000 rpm for 30 seconds, and remove the supernatant. Add 500 μL of W1 Buffer and centrifuge at 10000 rpm for 30seconds.

5.Add 500 μL of W2 Buffer and centrifuge at 10000 rpm for 30seconds. Repeat twice.

6.Centrifuge for 1 minute and open the lid for 1 minute to make the alcohol evaporate completely. Move the adsorption column to a new 1.5 ml Eppendorf tube and add 50 μL of H2O to obtain the plasmid DNA.

Gel extraction

1.Cut the DNA stripes from the gel under the Blue LED light （ keep the gel thin when you make it. ）

2.Use lifefeng® Gel Extraction Kit to extract target sequence from the gel just cut off.

Restriction endonuclease digestion

NEB restriction endonuclease kit was used and the following components were added to the 0.2 ml centrifugal tube:

1-2 μg Plasmid, 2 μL 10 *NEBuffer, 1 μL Restriction endonuclease 1 (20 units), 1 μL Restriction endonuclease 2 (20 units), add ddH2O to 20 μL. Mix well and centrifuge briefly, react at 37 ℃ for 30 min or longer, inactivate at 80 ℃ for 20 min.

DNA ligation

The following components were added to the 0.2 ml centrifugal tube:

1 μL TAKARA T4 DNA ligase，X μL Enzyme digested vector ，Y μL Enzyme digested DNA , 10 μL 2 x Quick Ligase Buffer, add ddH2O to 20 μL. Mix well and centrifuge for a short time, react at room temprature (25℃) for more than 30 minutes.

LB Solution (Liquid/Solid)

Luria-Bertani (LB) broth: 10g/L Tryptone, 5g/L Yeast extract, 10g/L NaCl, pH 7.4. LB plates: add 15-20g/L agar to the LB broth. Cool down the agar solution to 50 celcius degree before adding antibiotics.

2. Protein purification

Use BeyoGold™ His-tag Purification Kit to purify protein blaCMY10.

1. Bacteria was collected by centrifugation at 10000 rpm for 1 minute.

2. Discard the liquid and add 100 μL Lysis Solution.

3. Gently vortex the tube to avoid forming foam.

4. Add lysozyme (final concentration 1 mg/mL) (Add 2 μL) and incubate on ice for 30 minutes.

5. Ultrasound cracking bacteria on ice.

6. Centrifuge at 1000 rpm 4 ℃ for 10 minutes .

7. Pipette 10 μL supernatant and then add 20μL 50% BeyoGold His-tag Purification Resin and use the shaker at 4 ℃ for 30 minutes .

8. Centrifuge at 4 ℃ for 10 seconds to deposit the gel.

9. Pipette 20 μL as for further examination.

10. Add 40 μL non-denaturing wash buffer to resuspend the gel and centrifuge at 1000×g, 4 ℃ for 10 seconds. Pipette 20 μL for future examination.

11. Repeat step 10 and pipette 20 μL for future examination.

12. Add 20 μL non-denaturing wash buffer to resuspend the gel and centrifuge at 1000×g, 4 ℃ for 10 seconds. Collect the supernatant (Cmy10 His-tag protein).

13. Repeat the step 12 twice to get total 60μL purified protein (Cmy10 His-tag protein).

3. Function test for β-lactams detective system

Antibiotic concentration gradient test

Pipette 800 μL LB solution into 1.5 ml centrifugal tube, adding different concentration of ampicillin (0，0.05，0.5，2，5，15，20，30 μg/ml). Adding sample bacteria and incubating it at 37 ℃ overnight.

Antibiotic time gradient test

Diluting the overnight suspension culture to OD600nm = 0.1，adding ampicillin，cephalothin and cefoxitin to final concentration of 5 μg/mL. Detecting red fluorescence values for 0-22 hours.

4. Function test for β-lactams degradation system

Disc difusion assay

1. Coating the E.coli BL21 (DE3) suspension culture on LB plate and keep it in clean bench for drying. Puting small filter paper discs (bacteria-free) onto the LB plates and waiting for follow-up step.

2. 5 μL enzyme extract (0.24 mg/ml) is incubated with antibiotic substrates (1 mg/mL ampicillin, 0.5 mg/mL cephalothin, 0.5 mg/mL cefoxitin) in 50 mM Tris–HCl (pH 8.0) for 2 h at 35℃ and then reaction mixtures are loaded onto small filter paper discs.

2. Incubate the plate at 37 ℃, the diameters of the inhibition zones around the small paper discs were recorded.

Wastewater color test

1.Prepare the alkaline cupric tartrate solution: 0.01 mol/L CuSO4: 0.02 mol/L potassium sodium tertrate solution: 0.02 mol/L NaOH (2:2:1 v/v/v).

2.Add 5 μL antibiotics (1 mg/mL) into 50 μL alkaline cupric tartrate solution .

3. Add 5 μL Cmy protein (0.24 mg/mL) into the mixture, then add ddH2O upto 100 μL.

4.Incubate at 35 ℃ for 2 hours, then incubate at 70 ℃ for 15 minutes.

5.Cool on ice and measure the absorbance at OD310nm.

Team:ASTWS-China/Protocol