Team:Mingdao/Measurement

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INTRODUCTION

    Our goal is to genetically modify (GM) probiotics capable of attacking pathogens in an area of the body. Therefore, we developed tests to examine the efficacy in the antagonism relationship and functionality of our prototype. This series of measurements are repeatable and useful with good controls. We provided a simple and reproducible methodology that could help iGEM teams to design and validate their data in a concrete way in related projects.

PRODUCT MEASUREMENT

  Goal

To measure the product concentration of a GM probiotic

  Materials & Instrument

  • Substrate, co-reacting the enzyme with the GM product to generate fluorescent substances: Infra-red Peroxidase Substrate, Cat. MAK166A, SIGMA-ALDRICH
  • Enzyme: Horseradish Peroxidase, Cat. MAK166D, SIGMA-ALDRICH
  • Purified product, making a calibration curve: Hydrogen Peroxide, 3% (0.88 M) solution, Cat. MAK166B, SIGMA-ALDRICH
  • Product and control: the supernatants of the GM or WT probiotic overnight culture
  • Assay buffer, diluting the samples: Cat. MAK166C, SIGMA-ALDRICH
  • A 96-well flat-bottom black plate with clear bottoms for fluorimetric assay.
  • A fluorescent microplate reader, reading signals at Ex/Em = 640/680 nm: Synergy H1 Hybrid Multi-Mode Reader

  Principle

    Given an enzyme reacts with a substrate and a GM product to generate a fluorescent (infra-red) substance, which can be detected in a fluorescent microplate reader.

  Reagent preparation

  Infra-red peroxidase substrate stock

↓Reconstitute vials with 250 µl of DMSO
↓Mix well by pipetting
↓Make aliquots protected from light
↓Store at –20 °C if necessary

  Horseradish peroxidase stock (20 U/ml)

↓Reconstitute with 1 ml of Assay Buffer
↓Mix well by pipetting
↓Aliquot as one single use and protect them from light
↓Store at –20°C if necessary

  Procedure

  Standards for calibration

↓Add 22.7 µl of 3% H2O2 solution to 977 µl of Assay Buffer to prepare a 20 µM H2O2 stock solution
↓Add 1 µl of the 20 µM stock solution to 1,999 µl of Assay buffer to get a 10 µM working solution
↓Perform serial dilution to prepare 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0 µM for standards
↓Add 50 µl of the prepared standards to the wells in the 96 well plate. (All standards should be run in triplicates at least)

  Samples

↓Collect GM bacterial overnight culture
↓Centrifuge at 15,000 x g for 2 min
↓Transfer the supernatants into a new eppendorf
↓Add 50 µl of samples to wells. (All standards should be run in triplicates at least)

  Controls

↓Collect WT bacterial overnight culture
↓Centrifuge at 15,000 x g for 2 min
↓Transfer the supernatants into a new eppendorf
↓Add 50 µl of controls to wells. (All standards should be run in triplicates at least)

  Master mix

↓Prepare the Master Mix by mixing of 50 µl of Infra-red Peroxidase Substrate Stock, 200 µl of Horseradish Peroxidase Stock, and 4.75 ml of Assay Buffer

  Assay reaction

↓Add 50 µl of the Master Mix to each of the wells for samples, standards, and controls
↓Mix well and incubate the plate at room temperature for 30 min
↓Protect the plate from light during the incubation
↓Measure the fluorescence intensity at Ex/Em = 640/680 nm with Synergy H1 Hybrid Multi-Mode Reader

  Calculation for the results

↓Correct readings by subtracting the blank values
↓Make a calibration curve and obtain an equation
↓Determine the GM product concentration with the equation

Fig. 1. The calibration curve of H2O2 concentration at 0, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 µM.

GROWTH INHIBITION

  Goal

To test the product effect on the growth inhibition of a pathogen

  Materials & Instrument

  • Pathogen: S. mutans
  • Purified product, making different concentrations by serial dilution: Hydrogen peroxide solution, 50% in H2O (17.5M), Cat. 516813, SIGMA-ALDRICH
  • BHI broth: Brain Heart Infusion Broth, Cat. 53286, SIGMA-ALDRICH
  • A 96-well flat-bottom plate for measuring at OD600
  • A microplate reader: Synergy H1 Hybrid Multi-Mode Reader

  Procedure

↓Culture S. mutans in BHI media at 37°C overnight
↓Measure at OD600 and adjust to an OD600 of 0.1 with BHI media
↓Prepare various concentrations of H2O2 stocks from 6M to 0.0006M by serial dilution in BHI media

↓Add 100 µl of S. mutans at an OD600 of 0.1 ↓Add equal volume (100 µl) of H2O2 stocks to the indicated wells (see the table below) with five repeats. Therefore, S. mutans starts growing at an OD600 of 0.05 in working H2O2 concentrations between 0.0003M and 3M

↓Set the microplate reader with a kinetic mode (for anaerobic bacteria): 37°C, Reed OD600, Run 24 hr, Interval 20 min, (if measuring an aerobic culture, set additional shaking status)

↓Analyze data and draw a growth curve
↓Calculate the growth inhibition rate

Fig. 2. The representative data of growth curve of S. mutans in H2O2 inhibition assay.

Fig. 3. The growth rate of S. mutans in H2O2 inhibition assay.

ANTAGONISM TEST

  Goal

To verify the efficacy of the GM probiotic against a pathogen

  Materials & Instrument

  • Pathogen: S. mutans, which causes dental caries
  • Positive control: S. sanguinis, which is a natural antagonizer of S. mutans
  • Negative control: WT E. coli Nissle, which is a recognized probiotic
  • GM probiotic: GM E. coli Nissle carrying the H2O2-producing device in our study
  • LB agar plates
  • 37°C incubator

  Procedure

  Day 1

↓Culture S. mutans and S. sanguinis in BHI media, E. coli Nissle (EcN) in LB media, and GM EcN with H2O2-generating device in LB broth supplemented with 20 µg/ml of chloramphenicol.
↓Incubate at 37°C overnight.

  Day 2

↓Measure at OD600
↓Adjust OD600 to 0.5 in BHI media for S. sanguinis and S. mutans or in LB media for EcN.
↓Drop 8 µl of S. mutans on a LB agar plate at six separated position (as shown in Fig. 1)
↓Drop 8 µl of other bacteria on the same plate beside S. mutans in different distances respectively. (as shown in Fig. 4)
↓Incubate at 37°C for 24 hr

  Day 3

↓Observe the shape and size of the bacterial zone.
↓Find the optimized distance for antagonism test.

Fig. 4. The distance between two colonies dropped on agar plate for antagonism

Fig. 5. Antagonism test between S. mutans and S. sanguinis (A), S. mutans and WT EcN (B), S. mutans and GM EcN (C). 8 µL of the overnight cultures adjusted to an OD600 of ~0.5 were inoculated on LB agar plates beside each other. The data showed two repeats observed after 24-hr culture.

REFERENCE

  • Feuerstein O, Moreinos D, Steinberg D. Synergic antibacterial effect between visible light and hydrogen peroxide on Streptococcus mutans. J Antimicrob Chemother. 2006 May;57(5):872-6. doi: 10.1093/jac/dkl070.
  • Kreth J, Merritt J, Shi W, Qi F. Competition and coexistence between Streptococcus mutans and Streptococcus sanguinis in the dental biofilm. J Bacteriol. 2005 Nov;187(21):7193-203. doi: 10.1128/JB.187.21.7193-7203.2005.
  • Kreth J, Zhang Y, Herzberg MC. Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans. J Bacteriol. 2008 Jul;190(13):4632-40. doi: 10.1128/JB.00276-08.
  • Spitzer AJ, Tian Q, Choudhary RK, Zhao FQ. Bacterial Endotoxin Induces Oxidative Stress and Reduces Milk Protein Expression and Hypoxia in the Mouse Mammary Gland. Oxid Med Cell Longev. 2020 Mar 24;2020:3894309. doi: 10.1155/2020/3894309.