iGEM 2019 — Team TJUSLS_China

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Dry Lab
Wet Lab

# Notebook

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##### CONTACT US

---

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# Team:TJUSLS China/Experiments

demonstrate

[UV-vis Detection](#news7)
[Inhibition Kinetics](#news6)
[HTS](#news5)
[Enzyme activity](#news4)
[Protein purification](#news3)
[Protein Expression](#news2)
[Molecular Cloning](#news1)

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# 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

![](https://static.igem.org/mediawiki/2019/1/11/T--TJUSLS_China--MolecularCloning.png)

* ### Preparation of NDM-23 gene

  + #### PCR

    ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.4y7gxzn](https://static.igem.org/mediawiki/2019/9/9d/T--TJUSLS_China--01PCR.pdf)

    ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)Document of primers](https://static.igem.org/mediawiki/2019/9/90/T--TJUSLS_China--Document_of_Primers.pdf)

    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](https://static.igem.org/mediawiki/2019/7/7c/T--TJUSLS_China--table4.png)

  + #### Enzyme Digestion

    ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.49egz3e](https://static.igem.org/mediawiki/2019/1/1f/T--TJUSLS_China--02_Enzyme_Digestion.pdf)

    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

    ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.48rgzv6](https://static.igem.org/mediawiki/2019/d/d6/T--TJUSLS_China--05Agarose_Gel_Electrophoresis.pdf)

    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

    ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.48sgzwe](https://static.igem.org/mediawiki/2019/b/b8/T--TJUSLS_China--06_Gel_purification.pdf)

    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

    ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.49ngz5e](https://static.igem.org/mediawiki/2019/0/0a/T--TJUSLS_China--04Transformation.pdf)

    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

    ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.489gzz6](https://static.igem.org/mediawiki/2019/8/8f/T--TJUSLS_China--07_Extraction_of_Plasmid.pdf)

    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

  ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.49igz4e](https://static.igem.org/mediawiki/2019/8/88/T--TJUSLS_China--04_Ligation.pdf)

  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

![](https://static.igem.org/mediawiki/2019/9/9d/T--TJUSLS_China--ProteinExpression.png)

* ### Exploration of expression condition

  ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.5cfg2tn](https://static.igem.org/mediawiki/2019/f/f6/T--TJUSLS_China--08_Exploration_of_expression_condition.pdf)

  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

  ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.49agz2e](https://static.igem.org/mediawiki/2019/6/6e/T--TJUSLS_China--09_Expression.pdf)

  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

![](https://static.igem.org/mediawiki/2019/9/95/T--TJUSLS_China--Proteinpurification.png)

* ### Affinity Chromatography

  ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.5cgg2tw](https://static.igem.org/mediawiki/2019/1/18/T--TJUSLS_China--10_Affinity_Chromatography.pdf)

  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

  ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.5e5g3g6](https://static.igem.org/mediawiki/2019/3/3b/T--TJUSLS_China--12_lon_Exchange.pdf)

  ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.5e6g3he](https://static.igem.org/mediawiki/2019/5/50/T--TJUSLS_China--11_Concentration_and_fluid_exchanging.pdf)

  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

  ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.5fag3ie](https://static.igem.org/mediawiki/2019/8/8b/T--TJUSLS_China--13_Gel_filtration.pdf)

  ###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.49qgz5w](https://static.igem.org/mediawiki/2019/4/48/T--TJUSLS_China--14_SDS-PAGE.pdf)

  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

###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.6gthbwn](https://static.igem.org/mediawiki/2019/1/13/T--TJUSLS_China--15_Determination_of_Enzyme_Activity.pdf)

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

![](https://static.igem.org/mediawiki/2019/7/79/T--TJUSLS_China--HTS.png)

###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.6guhbww](https://static.igem.org/mediawiki/2019/9/9a/T--TJUSLS_China--16_high_throughput_screening_with_fluorescent_probe.pdf)

* #### 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

###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.7y9hpz6](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--17_Inhibition_Kinetics_Measurement_V.2.pdf)

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

![](https://static.igem.org/mediawiki/2019/7/76/T--TJUSLS_China--UV-vs.png)

###### [![pdf](https://static.igem.org/mediawiki/2019/c/c7/T--TJUSLS_China--PDF.png)dx.doi.org/10.17504/protocols.io.7y8hpzw](https://static.igem.org/mediawiki/2019/e/e9/T--TJUSLS_China--18_Monitoring_in_living_bacterial_cells_by_UV-Vis_spectroscopy_V.2%282%29.pdf)

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

![](https://static.igem.org/mediawiki/2019/f/f6/T--TJUSLS_China--Attr-5%282%29.png)

![](https://static.igem.org/mediawiki/2019/9/98/T--TJUSLS_China--Attr-6_3.png)

##### CONTACT US

---

![](https://static.igem.org/mediawiki/2019/b/b8/T--TJUSLS_China--Attr-email.png)  EMAIL

tju2019igem@163.com

![](https://static.igem.org/mediawiki/2019/4/4c/T--TJUSLS_China--Attr-addressl.png)  ADDRESS

No.92 Weijin Road, Nankai District, Tianjin, China

↑

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# Team:TJUSLS China/Results

<!doctype html>

Results

References
Future Plans
Winding Roads
Data and Results
Overview

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# Results

## Overview

#### Build Responsory Parts

We totally submitted more than 200 parts. After testing the circuits with our engineering expression system,
we finally confirmed 4 of them that fit the system well and showed us satisfactory results. They are NDM-23
with GST tag([BBa\_K2933161](http://parts.igem.org/Part%3ABBa_K2933161)), SPG-1 with
SUMO
tag([BBa\_K2933183](http://parts.igem.org/Part%3ABBa_K2933183)), ElBla II with SUMO
tag([BBa\_K2933184](http://parts.igem.org/Part%3ABBa_K2933184)) and AFM-1 with GST
tag([BBa\_K2933164](http://parts.igem.org/Part%3ABBa_K2933164)).

#### Screen Out Broad-spectrum Inhibitors

With our specific fluorescent probe CDC-1, we firstly explored the most suitable high-throughput screening
system, then measured related enzyme kinetics parameters. Then we screened out effective inhibitors for
these four metallo-beta-lactamases from FDA drug libraries and traditional Chinese medicine libraries. Each
beta-lactamase has at least 10 inhibitors.

#### Ensure Inhibitors’ Influence

After initial screening effective inhibitors, further than that, we tested the inhibitory mechanism type of
each inhibitors to corresponding beta-lactamase and measured their inhibitory constants, IC50 and
Ki. Then we
tested them in living bacteria to evaluate.

## Data and Results

#### Performance of Genetic Circuits

We successfully constructed our parts, expressed and purified four kinds of metallo-beta-lactamase using
parts constructed which are NDM-23, SPG-1, AFM-1 and ElBla II.Below shows our purification results.

![fig0](https://static.igem.org/mediawiki/2019/b/bb/T--TJUSLS_China--Results-fig1add.png)

Fig. 1 The picture of PCR, SDS page and gel filtration A. NDM-23 B. SPG-1 C. AFM-1 D.
ElblaII

#### Establishment of Fluorescent HTS System

Following the principles mentioned in [Design](https://2019.igem.org/Team%3ATJUSLS_China/Design) part, we completed establishment of HTS system of 4 MBLs. Each
kind of enzyme owns its unique characteristics, and performs activity highly under different conditions.

##### For NDM-23

![fig1](https://static.igem.org/mediawiki/2019/7/7c/T--TJUSLS_China--Results-fig1.jpg)

Fig.2 Determination of enzyme concentration.

We set gradients then decided the proper concentration in system of NDM-23 is 1.51nM, through comparing
R2
and EC80 value. And this number of concentration is less than usual in HTS system, which shows
our circuit
works well to provide beta-lactamase with high activity.

![fig2](https://static.igem.org/mediawiki/2019/a/a7/T--TJUSLS_China--Results-fig2.jpg)

Fig.3 Determination of buffer condition.

The first picture shows the effect of NaCl concentration to protein activity, and the second is about
Zn2+
concentration. These results indicate that this kind of metallo-beta-lactamase depends on low concentration
of zinc ion, which shows the same as reports.[1] And under the circumstance with lower pH, the
enzyme can be
inactivated. Its most suitable pH is around 8.5.

To explore the potential influence of substrate’s and compounds’ solvent, DMSO, we change the percentage of
DMSO in reaction system when diluting protein, without changing the final concentrations for each
components. Data shows that at the range of 2% to 10%, the effect of DMSO can be ignored, and RSD value is
less than 5%.

Chart.1 HTS system of NDM-23

| Components | Volume | Concentration |
| --- | --- | --- |
| NDM-23 | 94μL | 1.51nM |
| NaCl | 140mM |
| ZnCl2 | 25μM |
| pH | 8.0 |
| CDC-1 | 5μL | 8.5μM |
| Compound | 1μL | 20μM |
| Total | 100μL |  |

##### For SPG-1

![fig3](https://static.igem.org/mediawiki/2019/8/81/T--TJUSLS_China--Results-fig3.jpg)

Fig.4 Determination of enzyme concentration.

The proper concentration in system of SPG-1 is 24.72nM, which shows a bit lower activity compared with
NDM-23.

![fig4](https://static.igem.org/mediawiki/2019/8/8c/T--TJUSLS_China--Results-fig4.jpg)

Fig.5 Determination of buffer condition.

Experimental data shows that this kind of MBL gets higher activity under higher pH of 9.5.

Chart.2 HTS system of SPG-1

| Components | Volume | Concentration |
| --- | --- | --- |
| SPG-1 | 94μL | 24.72nM |
| NaCl | 35mM |
| ZnCl2 | 25μM |
| pH | 9.5 |
| CDC-1 | 5μL | 8.5μM |
| Compound | 1μL | 20μM |
| Total | 100μL |  |

##### For AFM-1

![fig5](https://static.igem.org/mediawiki/2019/4/43/T--TJUSLS_China--Results-fig5.jpg)

Fig.6 Determination of enzyme concentration.

The proper concentration in system of AFM-1 is 0.85nM.

![fig6](https://static.igem.org/mediawiki/2019/8/85/T--TJUSLS_China--Results-fig6.jpg)

Fig.7 Determination of buffer condition.

Compared with other 3 MBLs, AFM-1 fits higher salt concentration and higher zinc concentration.

Chart.3 HTS system of AFM-1

| Components | Volume | Concentration |
| --- | --- | --- |
| AFM-1 | 94μL | 0.85nM |
| NaCl | 280mM |
| ZnCl2 | 100μM |
| pH | 8.5 |
| CDC-1 | 5μL | 8.5μM |
| Compound | 1μL | 20μM |
| Total | 100μL |  |

##### For beta-lactamase II [Erythrobacter litoralis HTCC2594] (ElBla II)

![fig7](https://static.igem.org/mediawiki/2019/5/5c/T--TJUSLS_China--Results-fig7.jpg)

Fig.8 Determination of enzyme concentration.

The proper concentration in system of ElBla II is 1.887nM.

![fig8](https://static.igem.org/mediawiki/2019/b/be/T--TJUSLS_China--Results-fig8.jpg)

Fig.9 Determination of buffer condition.

These results indicate that this kind of metallo-beta-lactamase depends on low concentration of NaCl.

Chart.4 HTS system of beta-lactamase II [Erythrobacter litoralis HTCC2594]

| Components | Volume | Concentration |
| --- | --- | --- |
| ElBla II | 94μL | 1.887nM |
| NaCl | 35mM |
| ZnCl2 | 25μM |
| pH | 8.5 |
| CDC-1 | 5μL | 8.5μM |
| Compound | 1μL | 20μM |
| Total | 100μL |  |

#### Determination of Enzyme Activity

As a step of system establishment, Km and kcat of MBLs are required to determine in
order to decide the
concentration of substrate. These kinetic parameters are our brilliant achievements as well. We use
Michaelis-Menten and Lineweaver-Burk plotting the same data then choose the best fit one, comparing their
R2
to improve the accuracy of values of constants.

![fig9](https://static.igem.org/mediawiki/2019/b/b7/T--TJUSLS_China--Results-fig9.jpg)

Fig.10 Michaelis-Menten plot and Lineweaver-Burk plot for NDM-23

Chart.5 Kinetic parameters of NDM-23. As reports[2]NDM-1’s Km with the same substrates
is 17.6μM,
which is
very close to our results of NDM-1’s variant NDM-23.

| Constant | Value |
| --- | --- |
| Km | 1.50×10-5 mol·L-1 |
| Vmax | 18.9 Fluorescent unit·s-1 |
| kcat | 7.97 s-1 |
| kcat/Km | 5.30×105 mol-1·L·s-1 |

![fig10](https://static.igem.org/mediawiki/2019/d/d3/T--TJUSLS_China--Results-fig10.jpg)

Fig.11 Michaelis-Menten plot and Lineweaver-Burk plot for SPG-1

Chart.6 Kinetic parameters of SPG-1.

| Constant | Value |
| --- | --- |
| Km | 2.243×10-5 mol·L-1 |
| Vmax | 167.64 Fluorescent unit·s-1 |
| kcat | 4.31 s-1 |
| kcat/Km | 1.92×105mol-1·L·s-1 |

![fig11](https://static.igem.org/mediawiki/2019/a/ad/T--TJUSLS_China--Results-fig11.jpg)

Fig.12 Michaelis-Menten plot and Lineweaver-Burk plot for AFM-1

Chart.7 Kinetic parameters of AFM-1.

| Constant | Value |
| --- | --- |
| Km | 9.21×10-6 mol·L-1 |
| Vmax | 57.55  Fluorescent unit·s-1 |
| kcat | 43.06 s-1 |
| kcat/Km | 4.68×105  mol-1·L·s-1 |

![fig12](https://static.igem.org/mediawiki/2019/7/7a/T--TJUSLS_China--Results-fig12.jpg)

Fig.13 Michaelis-Menten plot and Lineweaver-Burk plot for ElBla II

Chart.8 Kinetic parameters of ElBla II.

| Constant | Value |
| --- | --- |
| Km | 1.00×10-5 mol·L-1 |
| Vmax | 86.79  Fluorescent unit·s-1 |
| kcat | 29.26 s-1 |
| kcat/Km | 2.93×106  mol-1·L·s-1 |

#### Effective Inhibitors We Screened Out

Through high-throughput screening with fluorescent probe CDC-1, we successfully found several possible
inhibitor molecules, from thousands of compounds. Among these results, the broad-spectrum inhibitor
towards
all four MBLs has emerged and several compounds are found to be inhibitors of a MBL. However, this is not
our destination. Some kinetic parameters should be measured to validate their effectiveness, half
inhibitory
concentration (IC50) and Ki.

Chart.9 Results of screened inhibitors to NDM-23, SPG-1, AFM-1, ElBla II

##### Tannic Acid: a broad-spectrum arrow

![fig13](https://static.igem.org/mediawiki/2019/e/e5/T--TJUSLS_China--Results-fig13.jpg)

Fig.14 IC50 of Tannic acid for NDM-23, SPG-1, AFM-1 and ElBla II

Tannic Acid is a kind of ingredient of Chinese medical and is widespread in rhubarb. And it belongs to
polyphenols, a class of Phenolic compounds (PCs). PCs is one of the most diverse groups of plant secondary
metabolite which contain aromatic rings with one or more hydroxyl or methoxyl groups. The antimicrobial
activity of PCs is dependent on their chemical structure, in particular, the substitution position in the
benzene ring and the saturated chain length. It has been reported that the antimicrobial action of PCs is
related to the inactivation of cellular enzymes, by changing membrane permeability[3].

With the emergence of multidrug resistant bacteria, there is a need to develop not only one kind of MBL
inhibitors, but compounds with dual MBLs inhibition properties for use in combination
therapies[5].
Therefore we highlight broad-spectrum inhibitors. What we have found is that tannic acid have inhibitory
effect on all of four MBLs we obtained. It is a special arrow targeting at different
metallo-beta-lactamases.

![fig14](https://static.igem.org/mediawiki/2019/1/17/T--TJUSLS_China--Results-fig14.jpg)

Fig.15 Inhibitory mechanism of tannic acid to NDM-23

With different concentrations of inhibitors, the fit lines passing the origin, which shows that its
mechanism should be reversible inhibition. Then with different concentrations of substrate, fit lines
gather
at Y axis, which shows that its mechanism is competitive inhibition.

In our measurement of kinetic constants and mechanisms, tannic acid has a IC50 value in
microlevel of four
MBLs, which is distinctly lower than other researchers’ results[3]. After ensuring its
inhibitory
mechanism
of reversible and competitive, we can calculate the Ki value of tannic acid to NDM-23 is 0.1075μM
according
to the equation between IC50 with Ki[4].

Binding to the active site of NDM-23, this result is in harmony with modeling prediction, more explanation
can be seen in their docking discussion.

##### Arrows from Chinese Medicine Library

From the 24 effective inhibitors of NDM-23 screened out before, we selected 8 of them to test. Four are
from
Chinese medicine library, tannic acid, corilagin, ginkolic acid C15:1, and (-)-Epigallocatechin
gallate(EGCG). There is a similarity of them that they all belongs to phenolic compounds mentioned above.
However, with distinctions on structure, their IC50 and inhibitory mechanisms are not
same.

![fig15](https://static.igem.org/mediawiki/2019/0/06/T--TJUSLS_China--Results-fig15.jpg)

Fig.16 IC50 and inhibitory mechanism of Corilagin for NDM-23

Corilagin also shows a good inhibitory effect via IC50 value which is at the level of
micrometer. When the
concentration of corilagin changes, its Vmax value changes while Km value is same,
which shows that its
mechanism is uncompetitive type.

![fig16](https://static.igem.org/mediawiki/2019/5/53/T--TJUSLS_China--Results-fig16.jpg)

Fig.17 IC50 and inhibitory mechanism of (-)-Epigallocatechin gallate(EGCG) for NDM-23.

Having highly similar structure with tannic acid, EGCG was tested to have same inhibitory mechanism with
it,
reversible and competitive inhibition. It can be seen as a useful inhibitor as well. And its Ki value is
8.36μM.

![fig17](https://static.igem.org/mediawiki/2019/7/79/T--TJUSLS_China--Results-fig17.jpg)

Fig.18 IC50 and inhibitory mechanism of (-)-Epigallocatechin gallate(EGCG) for NDM-23

##### Arrows from FDA Approved Libraries

From FDA approved drug libraries, we have screened out more potent inhibitor compounds.

![fig18](https://static.igem.org/mediawiki/2019/5/53/T--TJUSLS_China--Results-fig16.jpg)

Fig.19 IC50 and inhibitory mechanism of Adapalene for NDM-23, and
IC50
of Adapalene for AFM-1. The curve shows that it is irreversible inhibition.

![fig19](https://static.igem.org/mediawiki/2019/c/c4/T--TJUSLS_China--Results-fig19.jpg)

Fig.20 IC50 and inhibitory mechanism of Silver sulfadiazine for NDM-23.

![fig20](https://static.igem.org/mediawiki/2019/8/87/T--TJUSLS_China--Results-fig20.jpg)

Fig.21 IC50 and inhibitory mechanism of Fingolimod (FTY720) HCl for NDM-23.

![fig21](https://static.igem.org/mediawiki/2019/6/6f/T--TJUSLS_China--Results-fig21.jpg)

Fig.22 IC50 of other specific inhibitors we screened, including
Etravirine(TMC125) for NDM-23, Punicalagin for SPG-1 and Isoconazole nitrate for ElBla II.

Chart.10 Kinetic constants of inhibitors

| Protein | Inhibitor | IC50(μM) | Inhibitory mechanism | Ki(μM) |
| --- | --- | --- | --- | --- |
| NDM-23 | Tannic acid | 0.1687 | Reversible, competitive | 0.1075 |
| Corilagin | 0.8069 | Reversible, uncompetitive | 0.2903 |
| Adapalene | 8.307 | Irreversible | - |
| Ginkgolic acid C15:1 | 4.969 | Irreversible | - |
| (-)-Epigallocatechin gallate | 13.07 | Reversible, competitive | 8.36 |
| Etravirine (TMC125) | 3.352 | - | - |
| Silver sulfadiazine | 0.6774 | Reversible, uncompetitive | 0.2437 |
| Fingolimod (FTY720) HCl | 7.721 | Reversible, competitive | 4.94 |
| SPG-1 | Tannic acid | 0.1674 | Reversible, competitive | 0.1124 |
| Punicalagin | 0.7235 | - | - |
| AFM-1 | Tannic acid | 1.175 | Reversible, competitive | 0.6108 |
| Adapalene | 25.84 | Irreversible | - |
| ElBla II | Tannic acid | 2.561 | Reversible, competitive | 1.3838 |
| Isoconazole nitrate | 8.618 | - | - |

#### Monitoring in living bacterial cells with antibiotics

##### Experiments proving the accuracy of the system

Firstly, NDM-23 protein solution at 3.02nM was mixed with cefazolin at 250 μM, and the characteristic
absorption peak of cefazolin at 273nm obtained by our experiment was continuously measured.

![fig](https://static.igem.org/mediawiki/2019/a/a6/T--TJUSLS_China--Results-fig22.png)

Fig.23 The curve of specific absorption peak of antibiotics mixed with NDM-23 was
revealed.

According to the figure, we can clearly see that the specific absorption peak value of antibiotics
decreases
with time, which proves that we can choose the specific absorption peak of antibiotics as the criterion to
evaluate whether antibiotics are decomposed or not.

Secondly, after phosphate buffer treatment, E. coli strain transferred to NDM-23 beta-lactamase gene was
diluted to OD600=0.15, and the strain was mixed with cefazolin with a final concentration of
250
μM, and the
peak value of cefazolin at 273nm was continuously measured. The results are shown below.

![fig23](https://static.igem.org/mediawiki/2019/9/9c/T--TJUSLS_China--Results-fig23.png)

Fig.24 The curve of specific absorption peak of antibiotics mixed with E. coli with the NDM23
beta-lactamase
gene was revealed

This figure shows that the parts constructed by us can operate in E.coli and play its original functions,
which proves
the feasibility of our next step——to evaluate the therapeutic effect of inhibitors

At the same time, the phosphate buffer used in the preparation of bacterial solution and the E.coli
without
the transfer
of NDM-23 beta-lactamase gene were mixed with cefazolin with a final concentration of 250 μM respectively
as
blank
control and negative control, and the specific absorption peak value of cefazolin at 273nm was
continuously
determined.

![fig24](https://static.igem.org/mediawiki/2019/6/62/T--TJUSLS_China--Results-fig24.png)

Fig.25 The curve of specific absorption peak of antibiotics mixed with the phosphate buffer and the E.coli
without the
transfer of NDM23 beta-lactamase gene was revealed. The one up is antibiotic combined with phosphate
buffer
and the one
down is antibiotic combined with the E coli without the transfer of NDM-23 beta-lactamase gene.

##### Establishment of measurement system

Since NDM-23 beta-lactamase is metallo-beta-lactamase, it should be able to hydrolyze most kinds of
beta-lactam
antibiotics. We put the cefazolin, faropenem and e.faecalis respectively in the form of a final
concentration of 250
microns and mixed them with E. coli with the NDM-23 beta-lactamase gene. Through continuous determination
of
experiments
we get the specific absorption peak and peak value of the three kinds of antibiotics, in order to find the
optimum
substrate. We also measured the characteristic peak absorption of the nonbeta-lactam antibiotic
tetracycline
over time
as proof of experimental accuracy. The results are as follows:

![fig25](https://static.igem.org/mediawiki/2019/b/b1/T--TJUSLS_China--Results-fig25.jpg)

Fig.26 The curve of specific absorption peak of antibiotics mixed with E. coli with the NDM-23
beta-lactamase gene was
revealed. The peaks of faropenem, meropenem and tetracycline were measured at 307nm, 300nm and 360nm,
respectively

According to the figure, tetracycline was not broken down by E.coli that transferred the NDM-23
beta-lactamase gene,
proving the accuracy of the experiment. Faropenem and meropenem are well decomposed by E. coli too.

After determining the substrate of the method，we establish a system for the determination of viable
bacteria. We have
choose the best condition of E.coil for the measurement for different beta-lactamase. For NDM-23, the OD
of
E.coil is 0.02
and the induction time is 2h. For SPG-1, the OD is 0.4 and the induction time is 2h. For Elbla II, the OD
is
0.1 and the
induction time is 3h.For AFM-1, the OD is 0.1 and the induction time is 3h.

We used EDTA as a positive control for therapeutic efficacy evaluation. As a metal ion chelator, EDTA can
chelate the
zinc ion of metal beta-lactamase and inactivate the lactamase. The result are as follows.

![fig26](https://static.igem.org/mediawiki/2019/b/b4/T--TJUSLS_China--Results-fig26.jpg)

Fig.27 EDTA inhibition curve of NDM23. The top curve is the decomposition curve of cefazolin at different
concentration
of EDTA. The below curve shows the inhibition curve of EDTA according to the inhibition rate.

As a metal ion chelator, EDTA can inhibit metal beta-lactamase well. The inhibitory effect of EDTA
measured
by
our
evaluation system is close to the literature value (IC50 values from the literature is
1.63
μM), which also
proves that
our therapeutic effect evaluation system is feasible

##### Evaluation of therapeutic effect of NDM-23

After determining the reaction system, we diluted the concentration gradient of several inhibitors and
incubated them
with the prepared E.coli which has been transferred into NDM-23 beta-lactamase gene in dark for 1h, then
added
cefazolin
with a final concentration of 250 μM and continuously measured the light absorption value at 273nm. The
results are as
follows.

![fig27](https://static.igem.org/mediawiki/2019/8/8b/T--TJUSLS_China--Results-fig27.jpg)

Fig.28 Corilagin inhibition curve of NDM23. The top curve is the decomposition curve of cefazolin at
different
concentration of Corilagin. The below curve shows the inhibition curve of Corilagin according to the
inhibition rate.

Corilagin is a water-soluble tannin. It can be extracted from longan, and the content of underleaf pearl
and
Tibetan
sweet root corilla jing is also relatively high. We screened it from the Chinese medicine library and
showed
a good
inhibitory effect on the protein level of NDM-23 beta-lactamase. Corilagin maintained a stable inhibitory
effect in the
evaluation of therapeutic effect.

![fig28](https://static.igem.org/mediawiki/2019/d/df/T--TJUSLS_China--Results-fig28.jpg)

Fig.29 EGCG inhibition curve of NDM23. The top curve is the decomposition curve of cefazolin at different
concentration
of EGCG. The below curve shows the inhibition curve of EGCG according to the inhibition rate.

Epigallocatechin gallate is the main component of green tea polyphenols which is a catechin monomer
isolated
from tea
leaves. It stands out from our modeling work. Our modeling group screened potential inhibitors by
homologous
modeling
followed by molecular docking. EGCG scored highly in our modeling work, indicating that it binds well with
NDM-23.EGCG
also showed good inhibitory effect in our evaluation of therapeutic effect, and its IC50
was relatively low,
which could
be used in combination with cefazolin to achieve good bacteriostatic effect.

##### Evaluation the therapeutic effect of screened broad-spectrum beta-lactamase inhibitors

After high-throughput screening, tannic acid was screened as the inhibitor of four beta-lactamases which
are
NDM-23, SPG-1,
ELBLA2-1 and AFM-1. We have used the UV visible method to assess the effectiveness of the treatment. The
results are as
follows .

![fig29](https://static.igem.org/mediawiki/2019/9/95/T--TJUSLS_China--Results-fig29.jpg)

Fig.30 Tannic acid inhibition curve of NDM-23. The top curve is the decomposition curve of cefazolin at
different
concentration of Tannic acid. The below curve shows the inhibition curve of Tannic acid according to the
inhibition
rate.

![fig30](https://static.igem.org/mediawiki/2019/7/73/T--TJUSLS_China--Results-fig30.jpg)

Fig.31 Tannic acid inhibition curve of SPG. The top curve is the decomposition curve of cefazolin at
different
concentration of SPG. The below curve shows the inhibition curve of SPG according to the inhibition rate.

![fig31](https://static.igem.org/mediawiki/2019/e/e0/T--TJUSLS_China--Results-fig31.jpg)

Fig.32 Tannic acid inhibition curve of ElBla II. The top curve is the decomposition curve of cefazolin at
different
concentration of ElBla II. The below curve shows the inhibition curve of ElBla II according to the
inhibition rate.

![fig32](https://static.igem.org/mediawiki/2019/6/6a/T--TJUSLS_China--Results-fig32.jpg)

Fig.33 Tannic acid inhibition curve of AFM-1. The top curve is the decomposition curve of cefazolin at
different
concentration of AFM-1. The below curve shows the inhibition curve of AFM-1 according to the inhibition
rate.

Tannic acid is a tannic acid derived from gallnut. According to the outcome of Evaluation the therapeutic
effect, we can
see that Tannic acid has a broad spectrum of inhibitory effects on beta-lactamase. Tannic acid has
different
abilities to
inhibit different beta-lactamase in living bacteria.

#### Brief summary of the project

After our high-throughput screening of FDA approved drug libraries, we have screened out good inhibitors
and
evaluated
the therapeutic effect of inhibitors with better inhibition rate. We have identified several
representative
inhibitors.
We also found a broad spectrum of inhibition of metal beta-lactamase inhibitors - tannic acid. It is
expected that
tannic acid can be used as an inhibitor of metal
beta-lactamase in combination with beta-lactam antibiotics to treat drug-resistant bacteria.

Chart.11 Our Excellent inhibitors

| Protein | Inhibitor | IC50(μM) | Living bacterium IC50(μM) | Inhibitory mechanism |
| --- | --- | --- | --- | --- |
| NDM-23 | Corilagin | 0.8069 | 38.02 | Reversible, uncompetitive |
| (-)-Epigallocatechin gallate | 13.07 | 15.73 | Reversible, competitive |
| Tannic acid | 0.1687 | 31.62 | Reversible, competitive |
| SPG-1 | Tannic acid | 0.1674 | 42.66 | Reversible, competitive |
| AFM-1 | Tannic acid | 1.175 | 25.12 | Reversible, competitive |
| ElBla II | Tannic acid | 2.561 | 28.18 | Reversible, competitive |

## Winding Roads

![](https://static.igem.org/mediawiki/2019/6/68/T--TJUSLS_China--Results-WindingRoads.png)

## Future Plans

##### Co-crystallization of beta-lactamase and inhibitor complex

##### Clinical antibiotic-resistant bacteria test

##### Minimum Inhibitory Concentration

## References [1] Lin-Cheng Ju, Zishuo Cheng, Walter Fast, Robert A. Bonomo, Michael W. Crowder, The Continuing Challenge of Metallo-beta-Lactamase Inhibition: Mechanism Matters, Trends in Pharmacological Sciences, Volume 39, Issue 7, 2018, 635-647. [2] van Berkel SS, Brem J, Rydzik AM, et al. Assay platform for clinically relevant metallo-beta-lactamases. J Med Chem. 2013;56(17):6945–6953. [3]Mandal S M , Dias R O , Franco O L . Phenolic Compounds in Antimicrobial Therapy[J]. Journal of Medicinal Food, 2017:jmf.2017.0017. [4]Cer RZ, Mudunuri U, Stephens R, Lebeda FJ. IC50-to-Ki: a web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding. Nucleic Acids Res. 2009;37(Web Server issue):W441-W445. [5] Brem, Jürgen, Van Berkel S S , Aik W S , et al. Rhodanine hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition[J]. Nature Chemistry, 2014, 6(12):1084-1090.

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