Team:TJUSLS China/Experiments

demonstrate

Experiments

Overview

This year, we selected 20 beta-lactamase genes and constructed all of them successfully. After working hard in this summer, finally, we have successfully attained four high-purity proteins including NDM-23, SPG-1, AFM-1 and ElBla II, and done enzyme activity determination, inhibitor high-throughput screening and UV-vis detection. In this experiment page, let’s take the protein NDM-23 we firstly purified as an example, and others are very similar to it.

Molecular Cloning of NDM-23

  • Preparation of NDM-23 gene

    • PCR

      pdfdx.doi.org/10.17504/protocols.io.4y7gxzn
      pdfDocument of primers
      1. Mix the ingredients according to the following.
        Template 50-1000ng
        Sense Primer（10μM） 2.5μl
        Anti-sense Primer（10μM） 2.5μl
        5x SuperStar Omni Buffer 5μl
        dNTP（each 2.5mM） 4μl
        SuperStar Omni DNA Polymerase 1μl
        10x Enhancer 5μl
        ddH2O up to 50μl

      2. Set the PCR instrument Procedure according to the following table
        Blog 1

    • Enzyme Digestion

      pdfdx.doi.org/10.17504/protocols.io.49egz3e
      1. Mix together:
        3μl Restriction endonuclease BamH I
        3μl Restriction endonuclease Xho I
        6μl 10x green buffer
        3000ng NDM-23 gene
        Add to 60μl ddH2O

      2. Heat in water bath or heat block at 37℃ for 1h.

    • Agarose Gel Electrophoresis

      pdfdx.doi.org/10.17504/protocols.io.48rgzv6
      1. Use 1×TAE buffer to prepare 1% Agarose mix in a flask, then put it in the microwave and heat it as long as it takes to completely dissolve the Agarose.
      2. Take out the conical flask, cool it in the wash basin to about 50°C. Add EB quickly, and then mix well. Pour the Agarose gel into gel tray and insert comb into slots. Let the gel solidify for 20min. Meanwhile, dilute the 10x green buffer to 1x and add to the DNA samples.
      3. Place the gel onto the electrophoresis apparatus ensuring that it is totally submerged in 1xTAE buffer. Carefully load each sample into its designated lane and 2μl DNA marker into a separate lane.
      4. Run at 120V for 20 min. If the sample have not completely separated, the time may be extended appropriately.
      5. Check the gel using a gel imager or under UV light, then take a photo with the Gel imager.

    • Gel purification

      pdfdx.doi.org/10.17504/protocols.io.48sgzwe
      1. Column balancing step: add 500μl buffer BL to the adsorption column CA2 (the adsorption column is put into the collection tube), centrifuge at 12,000 RPM (~13,400×g) for 1 min, dump the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
      2. Remove the single target DNA strip from the agarose gel (remove the excess as much as possible) and put it into a clean centrifuge tube, and weigh it.
      3. Add buffer PN to the glue block (if the gel weight is 0.1g, its volume can be considered as 100 μl, then add 100 μl PN), put in 50℃ water bath, during which gently turn up and down the centrifugal tube, to ensure that the glue block is fully dissolved. If there is any undissolved glue, continue to leave for a few minutes or add more PN until the glue is completely dissolved. Put the tube on ice.
      4. Add the solution obtained in the previous step to an adsorption column CA2 (the adsorption column was placed in the collection tube), place at room temperature for 2 min, centrifuge at 12,000 RPM (~13,400×g) for 30-60 SEC, and dump the waste liquid in the collection tube, place the adsorption column CA2 into the collection tube.
      5. Add 600μl bleach buffer PW to the adsorption column CA2 (check whether anhydrous ethanol has been added before use), centrifuge 30-60 SEC at 12,000rpm (~13,400×g), dump the waste liquid from the collection tube, and put the adsorption column CA2 into the collection tube.
      6. Repeat step 5.
      7. Put the adsorption column CA2 back into the collection tube and centrifuge at 12,000 rpm (~13,400×g) for 2 min to remove as much bleach as possible.Place the adsorption column CA2 at room temperature for several minutes to dry thoroughly to prevent the residual rinse fluid from affecting the next experiment.
      8. Put the adsorption column CA2 into a clean centrifugal tube, and drop an appropriate amount of ddH2O (about 30ul) onto the middle position of the adsorption film, and leave it at room temperature for 2 min. Final DNA solution was collected by centrifugation at 12,000 RPM (~13,400×g) for 2 min.

  • Preparation of empty vector

    • Transformation

      pdfdx.doi.org/10.17504/protocols.io.49ngz5e
      1. Take competent cells (E.coli DH5α)out of -80°C and thaw on ice (approximately 20-30 mins).
      2. Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
      3. Mix 1μl of DNA (usually 10 pg - 100 ng) into competent cells. Gently mix by flicking the bottom of the tube with your finger a few times. Incubate the competent cell/DNA mixture on ice for 20-30 mins.
      4. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 90 secs.
      5. Put the tubes back on ice for 2 min.
      6. Add 600μl LB media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.
      7. The bacterial liquid was centrifuged at 3500rpm for 3 minutes, 400 microliters of supernatant was discarded, and the bacterial liquid was suspended again.
      8. Plate the transformation onto a LB agar plate containing the appropriate antibiotic.
      9. Incubate plates at 37°C overnight.

    • Extraction of Plasmid

      pdfdx.doi.org/10.17504/protocols.io.489gzz6
      1. Collect the E. coli solution into the EP tube. Centrifuge at 12,000 rpm in a rotor for 1 minute. Remove the clear supernatant liquid.
      2. Add 250μL P1 (RNase A added, kept at 4 °C) to the EP tube to suspend bacterial precipitation.
      3. Add 250μL P2 to the EP tube, shake slightly up and down 6-8 times to lyse bacteria.
      4. Add 350μL P3 and invert the tube immediately and gently 6-8 times. Then centrifuge it at 12000rpm, 25℃ for 10 minutes.
      5. Regenerate column CP3 while centrifugation. Add 500μl Buffer BL. Centrifuge for 1 min at 12,000 rpm. Discard the flow-through.
      6. Move the clear supernatant liquid to CP3, at 12000rpm, 25℃ centrifuge for 30 seconds.
      7. Add 600μL PW to adsorption column CP3, 12000rpm, 25℃ centrifuge for 30 seconds.
      8. Repeat step 7.
      9. Move the adsorption column CP3 to new clean centrifuge tubes and then keep them opening for 5 minutes, so that the ethanol in the PW can be sufficiently volatilized.
      10. Drop 50μL 65°C double-distilled water into the middle of the adsorption membrane, static for 2min. Then centrifuge for 2 min at 12,000 rpm to collect DNA solution in EP tube.

    • Enzyme Digestion(Refer to the protocol above)


    • Agarose Gel Electrophoresis(Refer to the protocol above)


    • Gel purification(Refer to the protocol above)


  • Ligation

    pdfdx.doi.org/10.17504/protocols.io.49igz4e
    1. Mix together:
      5×T4 DNA Ligase Buffer 2μl
      T4 DNA Ligase 0.5—1μl
      Vector cut by enzyme
      NDM-23 gene cut by enzyme
      ddH2O Add to 10μl

    2. Heat in water bath or heat block at 22℃ for 30min.

  • Transformation

    (Refer to the protocol above, attention to that DH5α strain was changed into BL21(DE3) )

  • Strain Reservation

    In the clean bench, using Micro pipette tip pick a single colony and put it into a LB liquid medium. Put into shaking table 10-12 h. In a clean centrifuge tube, add 600μL bacterium liquid and 400μL glycerin, then put into -20℃ refrigerator.

Protein Expression of NDM-23

  • Exploration of expression condition

    pdfdx.doi.org/10.17504/protocols.io.5cfg2tn

    Set the gradient of condition to explore how to express it best. For example, we often use 0.5mM IPTG, 16°C/0.5mM IPTG, 37°C/1mM IPTG, 16°C/1mM IPTG, 37°C as different conditions.

    1. Transform the plasmid into E.coli(BL21 DE3) used to express NDM-23 protein.
    2. Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria.
    3. Test the OD600 number of bacteria, then pipe 5-10μl into each new 5 mL LB tube. Don’t forget to add antibiotic into tubes and mark them.
    4. Cultivate in shaking incubator for 3-4 hours until the OD600 of bacteria range from 0.6 to 0.8.
    5. Pipet 200μl bacterial liquid as uninduced sample, and take another 600μl to mix with 400μl 50% glycerol to store. Then add inducer IPTG into each tube in different concentration, and incubate at 16°C for 16 hours or at 37°C for 4 hours shaking at 200-300rpm.
    6. After cultivating, pipet 200μl for each as induced sample.(The method to make samples: 1) Centrifuge the taken bacterial liquid at 12,000rpm for 3 minutes
      2) Drop the supernatant and resuspend the precipitate using 100μl ddw.
      3) Pipet 50μl resuspending liquid to mix with 10μl 6XSDS Loading buffer
      4) Boil it in dry bath at 100°C for 10 minutes)
      Use SDS-PAGE to check whether the target protein express or not and what the most suitable condition for its expression is.

  • Pre-expression

    The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.

  • Massive expression

    pdfdx.doi.org/10.17504/protocols.io.49agz2e

    After taking samples, we transferred them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.

Protein purification of NDM-23

  • Affinity Chromatography

    pdfdx.doi.org/10.17504/protocols.io.5cgg2tw
    1. Lysis of the bacteria.
      1)Resuspend the frozen cell paste as best you can in the Lysis Buffer using a 10 mL pipet or whatever means necessary. Let this suspension incubate for 20 minutes at room temperature, or until the suspension becomes turbid and viscous due to release of the bacteria's genomic DNA.
      2)Smash the bacteria.
      3)Centrifuge at 18,000 rpm in a big rotor for 40 minutes at 4°C. Save the pellet and the supernatant.
    2. Affinity chromatography of glutathione transferase (take GST tag fused with NDM-23 protein as an example）
      1)Remove the GST column from the 4℃ refrigerator. Wash the column with GST-binding buffer for 10 minutes to balance the GST column.
      2)Add the protein solution to the column, let it flow naturally and bind to the column.
      3)Add GST-Washing buffer several times and let it flow. Take 5μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
      4)We do enzyme cutting in the column. Add 1mg PPase to the column, overnight restriction enzyme digestion under 4℃. 5)Add GST-Washing buffer several times. Check as above.
      5)Collect the eluted proteins for further operation. Recycle columns: Wash with 2×GSH buffer until Coomassie Brilliant Blue doesn’t turn blue when tested. Wash with 6M Guanidine hydrochloride , let stand for 10 min then drain. Wash with ddH2O for three times.
    3. Immobilized metal ion affinity chromatography (take the Sumo tag fused with SPG-1 protein as an example)
      1)Remove the Ni column from the 4℃ refrigerator, which contains 20% alcohol. Wash the column with water for one time. Change to Ni-binding buffer for another time and balance the Ni column.
      2)Add the protein solution to the column, let it flow naturally and bind to the column. Repeat until the medium turns gray ( usually twice ).
      3)Add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
      4)We do enzyme cutting in the column. Add 1mg ULPase to the column, overnight restriction enzyme digestion under 4℃.
      5)Add Ni-Washing buffer several times. Check as above.
      6)Collect the eluted proteins for further operation. Recycle columns: Wash with 0.2mM EDTA , let stand for 10 min then drain. Wash with 6M Guanidine hydrochloride, let stand for 10 min then drain. Wash with ddH2O for three times. Fill up with NiSO4, place on a shaker overnight at 4℃.

  • Ion Exchange of NDM-23

    pdfdx.doi.org/10.17504/protocols.io.5e5g3g6
    pdfdx.doi.org/10.17504/protocols.io.5e6g3he
    1. According to the predicted pI of the protein and the pH of the ion-exchange column buffer, firstly select the appropriate ion exchange column (anion exchange column or cation exchange column). The pH of buffer should deviate from the isoelectric point of the protein. After protein property prediction, we knew the NDM-23 protein’ pI was about 5. So select buffer pH7.5 and use the anion exchange column.
    2. The protein is concentrated with a 10KD concentration tube, and then the exchange buffer is used to exchange the protein to the ion-exchange liquid A. Finally, it is concentrated to less than 5ml by centrifuging at 4℃ and 3400rpm for 10 minutes in a high-speed centrifuge to remove insoluble substances and bubbles.
    3. Balance the selected column with liquid A.
    4. Through the AKTA pure protein purification system, the samples are loaded to the column at a flow rate of 2.0ml/min, and continue washing for 5min.
    5. Gradually increase the content of liquid B in the column, change the salt concentration and then change the interaction between the sample and the column, and collect the corresponding eluent according to the position of the peak.
    6. Use SDS-PAGE to check the result.

  • Gel filtration of NDM-23

    pdfdx.doi.org/10.17504/protocols.io.5fag3ie
    pdfdx.doi.org/10.17504/protocols.io.49qgz5w
    1. According to the size of the protein NDM-23(28.5kD), firstly select the superdex75 gel column to adapt to the size of protein.
    2. Connect the proper column which adapts to target protein to AKTA high pressure tomographic system, use buffer of gel filtration chromatography (buffer: 25mMTris， 150mMNaCl，pH7.5) to prebalance the column of gel filtration chromatography of about 1.5 columns.
    3. Concentrate the protein to less than 1mL by centrifuging at 4℃, 3400rpm for 10 minutes in a high-speed centrifuge to remove insoluble substances and bubbles.
    4. Add the protein to the column of gel filtration chromatography through a 1mL sample collecting loop. The protein is eluted at a flow rate of 0.5mL/min. The eluent was collected according to the peak position.
    5. Use SDS-PAGE to check the result.

Enzyme Activity Determination of NDM-23

pdfdx.doi.org/10.17504/protocols.io.6gthbwn
  1. Soak the 96-well plates in 75% ethanol and put the container in ultrasonic cleaner for 30min to 1 hour, then use ddH2O to wash these plates several times. Put these clean plates in drying oven at 55°C.
  2. Dilute the enzyme using its buffer. There we pipet 1 μL protein stock solution in 1mL buffer and mix gently. Then pipet 100 μL protein solution and mix with 400μL buffer each time, in order to dilute it as a 5-time gradient.
  3. Pipet 94μL protein solution into 9 wells in plate, usually choosing B2-D4 area, to set 3 parallel controls. Pipet 94 μL buffer without protein into 3 wells as negative controls. Then add 6μL fluorescent substrate into wells.
  4. Set up the program in Infinite M1000 Pro Automatic Microplate Reader. Shake for 10 sec at 654 rpm. Kinetic Cycle (to read fluorescent intensity each cycle)Fluorescent measure, 75 cycle, 10sec for each cycle.
  5. Put the plate in Microplate reader, and click Start button.
  6. When the facility ends testing, save data and import it into GraphPad Prism Software. Use “nonlinear fit” – “straight line” and compare R2 of lines under different concentrations to pick up the best linear fit one, whose R2 is most close to 1 .
  7. Take this concentration as standard value, then set up parallel gradient of its 2x, 0.5x, 0.25x, etc. Repeat step 3-5.
  8. Calculate the ratio of emission (rE = Q0/Qm, Q0 means the maximum fluorescent intensity of each reaction under different protein concentrations, Qm means the maximum fluorescent intensity of all reactions under different protein concentrations). Use GraphPad Prism Software to calculate EC80 value. Set log(concentration of protein) as X, the rate of emission as Y. Use “nonlinear fit” – “log(agonist) vs. response—Find ECanything”, input 80 as the value of F parameter.
  9. Usually we use the EC80 value as suitable protein concentration, and it can be adjusted according to the actual situation.
  10. Design experimental groups with the “N+(N-1) principle”.
  11. Since we use PBS as our protein buffer, and class B beta-lactamases are depend on Zn2+, so we choose the concentration of NaCl, the concentration of ZnCl2, and pH, as variables.
  12. Repeat step 3-5 to measure.
  13. When the facility ends testing, save data and import it into GraphPad Prism Software. Use “nonlinear fit” – “straight line” to calculate the initial velocity of each reaction a.k.a. its slope value. Choose the condition with higher initial velocity.
  14. Dilute protein again with the ensured most suitable solution into proper concentration.
  15. Dilute the fluorescent substrate as 2-time gradient for 8 groups.
  16. Repeat step 3-5 to measure.
  17. When the facility ends testing, save data and import it into GraphPad Prism Software. Use “nonlinear fit” – “straight line” to calculate the initial velocity of each reaction a.k.a. its slope value.
  18. Use “nonlinear fit” – “Michaelis-Menten” to fit Michaelis plot of this beta-lactamase. At the same time the software will calculate kinetic constants Km, Vmax automatically.
  19. Dilute protein as 2-time gradient for several groups. Repeat step 3-5 to measure. Take FI as Y, [S] as X, then use “nonlinear fit” – “straight line” to calculate fluorescent calibration value.
  20. Calculate kcat value. Kcat = Vmax/[E].

High Throughput Screening with Fluorescent Probe

pdfdx.doi.org/10.17504/protocols.io.6guhbww

  • Sample preparation

    1. Soak the 96-well plates in 75% ethanol and put the container in ultrasonic cleaner for 30min to 1 hour, then use ddH2O to wash these plates several times. Put clean plates in drying oven at 55°C.
    2. Dilute the enzyme using its buffer. There we pipet 1 μL protein stock solution in 1mL buffer and mix gently. Then pipet 200 μL protein solution then mix with 12.6mL buffer to reach our aiming concentration(1.51nM in reaction system).
    3. Dilute the substrate(fluorescent probe CDC-1) with DMSO to reach aiming concentration(10.2μM in reaction system).

  • Sample handling

    1. Pipet 94 μL protein solution into each well of 96-well plates using multi-channel pipette.
    2. Pipet 1μL compounds from FDA approved drug library into each well except for the first line of the plate. Pipet 1μL 100% DMSO into wells in the first line as negative controls. Then incubate the protein with compounds at room temperature for 5 min.
    3. Pipet 5 μL substrate into each well of 96-well plates using multi-channel pipette quickly.

  • Readouts and Data Acquisition

    1. Set up the program in Infinite M1000 Pro Automatic Microplate Reader.
      Shake for 10 sec at 654 rpm
      Kinetic Cycle (to read fluorescent intensity each cycle)
      Fluorescent measure, 25 cycle, 30sec for each cycle
    2. Put the plate in Microplate reader, click Start button.
    3. When the facility ends testing, save data and import it into GraphPad Prism Software. Use “nonlinear fit” – “straight line” to calculate the initial velocity of each reaction a.k.a. its slope value.
    4. Compare the values of wells which has added compounds with negative control. Calculate the residue activity(Ra = Vr/V0 *100%) and inhibition ratio(Ir = 1- Vr/V0 *100%). Choose the compounds with Ra <20%, that is to say, Ir80%, to screen again.

  • Repeat

    1. Repeat step 4-8 to screen again. Only to add chosen compounds and set three same wells as parallel experiments. Collect data and calculate Ir more precisely.
    2. Fluorescence quenching experiment: pipet 94μL protein and 5μL fluorescent substrate and mix them. Let it stand still for 30 mins. Then test its maximum fluorescent intensity(Q1). Pipet 1 μL positive compounds then test again(Q2). Calculate the fluorescence quenching rate Qr = (Q1 – Q2)/Q1 *100%.
    3. Ascertain the inhibitors, whose Ir is more than 80% while Qr is less than 20%. Then some inhibition kinetic constant can be measured.

Inhibition Kinetics Measurement

pdfdx.doi.org/10.17504/protocols.io.7y9hpz6
  1. Soak the 96-well plates in 75% ethanol and put the container in ultrasonic cleaner for 30min to 1 hour, then use ddH2O to wash these plates several times. Put these clean plates in drying oven at 55°C.
  2. Dilute inhibitors using 100%DMSO to 8mM (a.k.a. 80μM in reaction system) and dilute in 2-time gradients for 12 concentrations. Dilute the enzyme using its buffer to standard concentration (same as the determination of Km).
  3. Add the reaction system into 96-well plates. Pipet 94μL protein solution and 1μL inhibitor solution(diluted as 2-time gradients, at least 8 different concentrations), incubated at room temperature for 5 mins, then add substrate CDC-1 5μL.
  4. Set controls. Negative control: 94μL protein solution1μL DMSO, 5μL substrate Blank control: 94μL protein buffer1μL DMSO, 5μL substrate And there are 3 parallel holes for each system.
  5. Set up the program in Infinite M1000 Pro Automatic Microplate Reader. Shake for 10 sec at 654 rpm Kinetic Cycle (to read fluorescent intensity each cycle) Fluorescent measure, 20 cycle, 30sec for each cycle
  6. Put the plate in Microplate reader, and click Start button.
  7. When the facility ends testing, save data and import the data of 0-200s into GraphPad Prism Software. Use “nonlinear fit” – “straight line” and regulate the number of data to fit R2 .
  8. Move baseline of blank control. Calculate Ir= (1-Vr/V0)*100%. Take average Ir of each inhibitors’ concentrations as Y value, and take log[I] as X value. Then use “nonlinear fit” – “log(inhibitor) vs. normalized response – Variable slope” to fit IC50 curve, and its value would be calculated automatically.
  9. reversible/irreversible inhibition
    Set up gradient concentrations of protein(revolves around the value in screening system)
    As well as gradient concentrations of inhibitor(revolves around the value of IC50)
    Draw the plot of V0-[E] to see the movement of straight line.
    If they are parallel with each other, the mechanism is irreversible.
    If they are all through the origin, the mechanism is reversible.
  10. competitive/noncompetitive inhibition
    Set up gradient concentrations of substrate(revolves around the value in screening system)
    As well as gradient concentrations of inhibitor(revolves around the value of IC50).
    Draw the plot of 1/V0-1/[S] to see the movement of straight line (Lineweaver-Burk plot).
    If they gather at Y axis, the mechanism is competitive.
    If they pass the same point in X axis, the mechanism is noncompetitive.
    If they gather at another point in this dimension, it would be mixed type competition.(uncompetitive inhibition)
  11. Ki
    After decision of this inhibitor’s mechanism, a constant Ki can be calculated via different equations.
    For competitive inhibition,

    $$K_{\mathrm{i}}=\frac{I C_{\mathrm{so}}}{\left(S / K_{\mathrm{m}}+1\right)}\left\{\begin{array}{ll}{\ {if }\, S=K_{m},} & {K_{\mathrm{i}}=l C_{\mathrm50} / 2} \\\ {i f\, S>K_{m},} & {K_{\mathrm{i}} \ll l C_{\mathrm{50}}} \\ {\ {if }\, S < K_{m}}&{K_{\mathrm{i}}\cong l C_{\mathrm{50}}}\end{array}\right.$$


    For noncompetitive inhibition,

    $$K_{\mathrm{i}}=I C_{\mathrm{50}} \text { when } S=K_{\mathrm{m}} \text { or } S>>K_{\mathrm{m}} \text { or } S << K_{\mathrm{m}}$$


    For uncompetitive inhibition,

    $$K_{\mathrm{i}}=\frac{I C_{\mathrm{50}}}{K_{\mathrm{m}} / S+1}\left\{\begin{array}{ll}{\\ {if }\, S=K_{\mathrm{m}},} & {K_{\mathrm{i}}=I C_{50} / 2} \\ {i f \,S>>K_{\mathrm{m}},} & {K_{\mathrm{i}} \cong I C_{\mathrm{50}}} \\ {i f\, S << K_{\mathrm{m}}} & {K_{\mathrm{i}} \ll I C_{\mathrm{50}}}\end{array}\right.$$


    Also, we use a website tool
    (https://bioinfo-abcc.ncifcrf.gov/IC50_Ki_Converter/index.php)to calculate its value automatically.

UV-vis Detection of NDM-23

pdfdx.doi.org/10.17504/protocols.io.7y8hpzw
  1. Pipet 5μL NDM-23(used pET-28a vector) BL21(DE3) glycerol bacteria into 5ml LB medium, and 2.5μL kanamycin is added. Incubate aiming bacterial liquid at 37°C until its OD600 reach 0.5-0.6 then add inducer IPTG.
  2. Centrifuge bacterial liquid and add phosphate buffer to resuspend bacterial precipitation, then centrifuge again and discard phosphate buffer. Repeat 3 times to wash precipitate
  3. Mix bacterial precipitate in phosphate buffer in incubation, and dilute it. OD600 of the bacterial liquid used for next measurement is 0.15.
  4. UV-Vis test I. Test one experimental group together with 3 different controls. Record the absorption value every 300 seconds, 12 times in total.
    1)95μL bacterial liquid which express target protein, 5μL cefazolin(final concentration is 250μM);
    2)95μL beta-lactamase(final concentration is decided by characteristic of enzyme), 5μL cefazolin(final concentration is 250μM);
    3)95μL bacterial liquid which is transferred with blank vector, 5μL cefazolin(final concentration is 250μM);
    4)95μL phosphate buffer, 5μL cefazolin(final concentration is 250μM). Then plot the UV-vis spectroscopy with time.
  5. Establish a system for the determination of viable bacteria.95μL bacterial liquid with different induction time and OD value was mixed with 5μL cefazolin(final concentration is 250μM)to determine the optimal induction time and OD. Record the absorption value every 300 seconds, 24 times in total.
  6. UV-Vis test II. Test the UV absorption peak in 273nm(cefazolin), 307nm(meropenem), 300nm(faropenem), 360nm(tetracycline)
    1)95μL bacterial liquid which express target protein, 5μL cefazolin(final concentration is 250μM);
    2)95μL bacterial liquid which express target protein, 5μL meropenem(final concentration is 250μM);
    3)95μL bacterial liquid which express target protein, 5μL faropenem(final concentration is 250μM);
    4)95μL bacterial liquid which express target protein, 5μL tetracycline(final concentration is 250μM).
  7. UV-Vis test III.
    1)94μL bacterial liquid which express target protein, 5μL cefazolin(final concentration is 250μM), 1μL inhibitor;
    2)94μL bacterial liquid which express target protein, 5μL cefazolin(final concentration is 250μM), 1μL inhibitor’s solvent (100% DMSO);
    3)94μL phosphate buffer, 5μL cefazolin(final concentration is 250μM), 1μL inhibitor's solvent (100% DMSO);
    4)94μL phosphate buffer, 5μL cefazolin’s solvent, 1μL inhibitor's solvent (100% DMSO).
    Test a series of inhibitor’s concentration as a gradient and test 5 parallel control. Then calculate the inhibition rate for each concentration as equation 1, and plot IC50 curve.
    Equation 1: Inhibition rate% = 100*（1-（[St]-[Si]）/([St]-[So])）
    [St] = Initial absorption value of antibiotics
    [Si] = Terminated absorption value of antibiotics with the addition of inhibitors
    [So] = Terminated absorption value of antibiotics without the addition of inhibitors
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