Team:Grenoble-Alpes/protocols

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PROTOCOLS

In the following page, all the protocols used during this project are described. blablablablablablablablablablablablablablablablablablablablablablablablablablablablablablablabla

Antibiotic preparation

CHLORAMPHENICOL (25µg/mL) stock

Materials

  • 0.25g of Chloramphenicol (25mg/mL)
  • 10mL of ethanol

Methods

  • Add 0.25g of Cam per 10mL of ethanol
  • Homogenize by vortexing
  • Filter with a 22mm membrane filter
  • Conserve at -20°C in 1mL aliquot


AMPICILLIN (100 mg/mL) stock

Materials

  • 1g of Ampicillin
  • 10mL of ethanol

Methods

  • Add 1g of Amp per 10mL of ethanol
  • Homogenize by vortexing
  • Filter with a 22mm membrane filter
  • Store at -20°C in 1mL aliquot

Competent bacteria

Materials

  • 100mL bacteria culture (DO = 0,5-0,6)
  • 20mL MgCl2 (100mM)
  • 2mL CaCl2 (100mM)
  • Glycerol 15%

Methods

  • Centrifuge the culture at 5000 rpm for 10 minutes at 4°C
  • Remove the supernatant and resuspend the pellet in 20mL of cold MgCl2 100mM
  • Incubate 30 minutes on ice
  • Centrifuge at 4000 rpm for 10 minutes at 4°C
  • Remove the supernatant and resuspend the pellet in 2mL of CaCl2 (100mM), Glycerol 15%
  • Aliquot 50µL in Eppendorf previously cooled at -80°C
  • Store at -80°C

Bacterial transformation

Materials

  • 25µl of DH5alpha competent cells
  • 1 to 5µl of DNA (concentration > 20ng/µl)
  • 450µl of SOC medium
  • 2 Petri dishes

Methods

NB: work under microbiological safety bench and on ice
  • Add DNA in 25µl of competent bacteria
  • Gently invert the tube 4-5 times to mix cells and DNA. Do not vortex
  • Incubate 30 minutes on ice
  • Heat shock at 42°C for 1 minute. Do not mix
  • Place on ice for 2 minutes. Do not mix
  • Add 450µL of SOC medium.
  • Incubate at 37°C and 200rpm for 2h
  • Mix the cells thoroughly by inverting the tube
  • Deposit 50µl on the first plate
  • Centrifuge at 1000rpm for 3 minutes at Room Temperature
  • Remove a part of the supernatant and resuspend pellet with the rest
  • Deposit the mixing on the second plate
  • Incubate overnight at 37°C with plates upside down

Bacteriophages protocols

Bacteriophage amplification via liquid medium

Material

  • Liquid bacteriophage sample and its bacterial host grown in agar plate
  • Luria-Bertani (LB) broth medium
  • CaCl2 1M and MgCl2 1M solutions

Methods

  • Grow bacterial host in LB broth (e.g. 5mL) overnight at 37 °C with agitation (200rpm).
  • Prepare a bacterial culture with new LB broth adding 1/10 of overnight bacterial host (for example add 3,5mL of overnight
  • bacterial host in 35mL of new LB broth medium). Mix.
  • Incubate at 37°C with agitation (200rpm) a few hours or overnight.*
  • Add a final concentration of 2mM of CaCl2 and MgCl2 (for example add 70µL of CaCl2 1M and 70µL of MgCl2 1M in the 35mL culture). Mix.
  • Add 100µL of high titer bacteriophage stock (>108 pfu/mL) (the volume can be adapted according the titer).
  • Mix.
  • Incubate at 37°C with agitation until lysate clears (disappearance of the turbid) - approximately 5 hours -.
  • Store at 4°C.
  • The bacteriophage lysate titer can be measured to ensure the success of the amplification.

    • *It depends on the growth rate of the bacterial host

Bacteriophage purification

Material

  • Bacteriophage lysate sample
  • SM (Sodium chloride - Magnesium sulphate) Buffer [*]
  • PEG(20%)/NaCl(2.5M) solution
  • NaCl 5M solution

Methods

  • Add, to the bacteriophage lysate, a final concentration of 1M of NaCl (for example add 7mL of NaCl 5M solution in 35mL bacteriophage lysate). Mix.
  • Centrifuge for 15 min at ⩾4,000 x rpm.
  • Recover the supernatant.
  • Add a minimum of 0.25 volume of PEG(20%)/NaCl(2.5M) solution (PEG 6000 or 8000 can be used) to the supernatant (e.g. add a minimum of 10mL of PEG/NaCl to for 40mL of supernatant) . Mix (do no vortex).
  • Let mixed to the sample at room temperature for 20 min and overnight at 4°C. *
  • Centrifuge at ⩾4,000 x rpm for 20 min at 4°C.
  • Discard the supernatant.
  • Resuspend the pellet in SM Buffer with 0.01-0.02 initial culture volume (e.g. add around 500µL for an initial bacterial culture of 50mL).**
  • Let resuspend the pellet at 4°C for 1h minimum.*
  • Store at 4°C

*The bacteriophage lysate titer can be measured (by Plaque Assay) to ensure the success of this step.
**For better purification, add 10mL of SM Buffer, do the next step, and repeat the steps 4 to 9.

Plaque assay for determination of bacteriophage titer

Material

  • Bacteriophage sample requiring titering and its bacterial host grown in agar plate
  • LB broth medium
  • LB top agar
  • SM Buffer
  • Agar plate

Methods

  • Grow bacterial host in LB broth (e.g. 5mL) at 37 °C with agitation (200 rpm) for few hours.
  • Heat LB top agar in microwave until completely melted
  • Allow top agar to cool until it is possible to touch the container (between 42 and 56°C).
  • Do a serial dilution by diluting 10µL of bacteriophages stock in SM Buffer (90µL for 10-1 dilution or 990µL for 10-2 dilution) and repeat this step with the sample obtained until get the desired dilution.

Spot titration

  • Spot titration is used to get a rough idea of the bacteriophage titer.
  • Add 250µL of bacterial host for 5mL of LB top agar and mix.*
  • Dispense the LB top agar-bacteria mixture onto an agar plate.
  • Let gel.
  • Spot 10µL of a bacteriophage dilution in a part of the plate and do the same for other dilutions. Until 10 dilutions can be spotted in one plate.
  • Allow the liquid from the spots to absorb into the LB top agar (15 min).
  • Invert the plate and incubate at 37°C overnight.

Full titration

  • Full titration is used to get a accurate idea of the bacteriophage titer. It’s advisable to do a plate with the valid dilution (obtained with the spot titration) and the two nearest dilutions.
  • Add 250µL of overnight bacterial host and 250µL of bacteriophage dilution for 5mL of LB top agar.*
  • Mix and dispense the mixture onto an agar plate.
  • Let gel and invert the plate.
  • Incubate at 37°C overnight.

*Suggested quantities are valid for one plate.

Solutions preparation for bacteriophages protocols

SM buffer preparation (500mL)

Material

  • 2.9g of NaCl
  • 1g of MgSO4⋅7H2O
  • 25mL of Tris⋅HCl 1M pH7.4
  • Distilled water to 500mL

Methods

  • Add 2.9g of NaCl, 1g of MgSO4⋅7H2O and 25mL of Tris⋅HCl 1M pH7.4 in 500mL of distilled water.
  • Shake until dissolved.
  • Autoclave.

NaCl 5M preparation (200mL)

Material

  • 58.44 g of NaCl
  • Distilled water to 200mL

Methods

  • Add 58.44g of NaCl in 200mL of distilled water.
  • Shake until dissolved.
  • Autoclave.

PEG(20%)/NaCl(2.5M) preparation (200mL)

Materials

  • 29.22g of NaCl
  • 40g of PEG 6000 or PEG 8000
  • Distilled water to 200mL

Methods

  • Add 29.22g of NaCl, 40g of PEG 8000 (or PEG 6000) in 200mL of distilled water.
  • Allow the PEG to completely dissolve, mixing frequently.
  • Autoclave.

CaCl2 1M preparation (100mL)

Materials

  • 14.70g of CaCl2⋅2H2O
  • Distilled water to 100mL

Methods

  • Add 14.70g of CaCl2⋅2H2O in 100mL of distilled water.
  • Shake until dissolved.
  • Autoclave.

MgCl2 1M preparation (100mL)

Materials

  • 20.33g of MgCl2⋅6H2O
  • Distilled water to 100mL

Methods

  • Add 20.33g of MgCl2⋅6H2O in 100mL of distilled water.
  • Shake until dissolved.
  • Autoclave.

LB top agar preparation (500mL)

Materials

  • 4g of agar-agar
  • 1mL of CaCl2 1M
  • 1mL of MgCl2 1M
  • LB broth medium to 500mL

Methods

  • Add 4g of agar-agar, 1mL of CaCl2 1M, 1mL of MgCl2 1M in 500mL of LB broth.
  • Shake.
  • Autoclave.

Tris⋅HCl 1M pH 7.4 preparation (100mL)

Materials

  • 12.11g of Tris
  • Concentrated HCl solution
  • Distilled water to 100mL

Methods

  • Add 12.11g of Tris in 30mL of distilled water.
  • Shake until dissolved.
  • Adjust slowly pH to 7.4 with the appropriate volume of concentrated HCl (helping with a pH meter).
  • Add distilled water to 100mL
  • Shake.
  • Autoclave.

REFERENCES

Bonilla, N., Rojas, M. I., Netto Flores Cruz, G., Hung, S.-H., Rohwer, F., & Barr, J. J. (2016). Phage on tap–a quick and efficient protocol for the preparation of bacteriophage laboratory stocks. PeerJ, 4, e2261. doi:10.7717/peerj.2261

M. Poxleitner, W. Pope, D. Jacobs-Sera, V. Sivanathan & G. Hatful, 2017, Phage Discovery Guide.

DNA extraction (from lysis of bacteria by bacteriophages)

Materials

  • Lysis buffer
  • Extraction buffer
  • Elution buffer
  • Magnetic beads
  • Magnet
  • Luria-Bertani (LB) broth medium
  • CaCl2 1M and MgCl2 1M solutions
  • Liquid bacteriophage sample (>1025 pfu/mL) and its bacterial host grown in agar plate.

Methods

  • Grow bacterial host in LB broth (e.g. 5mL) overnight at 37 °C with agitation (200rpm).
  • Add a final concentration of 2mM of CaCl2 and MgCl2 (for example add 10µL of CaCl2 1M and 10µL of MgCl2 1M in the 5mL culture) and mix.
  • The next steps are realized in eppendorfs with a volume of 200µL of bacterial host.
  • Add 50µL of very high titer bacteriophage stock (>1025 pfu/mL) and mix.
  • Incubate at 37°C with agitation approximately 4.5 hours.
  • Add 20µL of magnetic beads and flush.
  • Incubate 10 minutes at room temperature.
  • Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
  • Wash by adding 500µL of Extraction Buffer and flush.
  • Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
  • Wash again by adding 500µL of Extraction Buffer. Flush
  • Remove the supernatant by keeping magnetic beads in the eppendorf using the magnet.
  • Wash one last time by adding 500µL of Elution Buffer. Flush
  • Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
  • Add 30µL of Elution Buffer and flush.
  • Incubate 5 minutes at 70°C.
  • Remove the magnetic beads by sliding them along the wall of the eppendorf using the magnet.
  • Retrieve the eluent containing the DNA using a pipette and deposit it in a new eppendorf.
  • The DNA can be stored at -20°C.

Positive control:

  • Add 400µL of Lysis Buffer in 200µL of bacterial host.
  • Incubate 10min at room temperature.
  • Add 20µL of magnetic beads
  • Incubate 10min at room temperature
  • Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
  • Wash by adding 500µL of Lysis Buffer and flush.
  • Remove the supernatant by keeping the magnetic beads in the eppendorf using the magnet.
  • Continu by repeating from step 8. of the DNA extraction of bacteria lysed by bacteriophages.

Negative control (1):

Do the same protocol of extraction from bacteria lysed by phages but add 50µL of SM Buffer instead of 50µL of bacteriophages (step 3).

Negative control (2):

Replace the 200µL of bacterial host (step 1 of original protocol) by 200µL of LB broth medium and do the same protocol for the next.