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.
Agarose gel electrophoresis
Materials
1g of agarose (powder)
100mL TAE 1X
Distilled water
25µL of GelRed
Methods (for a 1% agarose gel)
Gel preparation
Dissolve 1g of agarose in 100mL of TAE (1X)
Pour the solution into the gel mold
Let the solution gel (almost 15 minutes)
Preparation of the samples to deposit
Add loading dye 6X to dilute it until 1X (usually 2µL added to 10µL sample)
NB: maximum volume in the well is around 25µL and minimum quantity of DNA detectable is around 25µg (for plasmid > 3Kb)
The migration is done at 100V for 30 minutes
Revelation
Prepare 250mL of distilled water in a tank
Add 25µl of Gel Red. Do not forget to use gloves to manipulate the Gel Red
Incubate the gel in the solution for 10 minutes to 1h protected from light
Wash the gel in a tank of distilled water
Read under UV
Protocol Gibson Assembly Master Mix
Materials
Materials (out of the NEB kit*):
High-Fidelity DNA polymerase (Q5)
Lysogeny broth (LB)
SOC medium
DH5α bacteria
Methods
Gibson assembly reaction
NB: All the Gibson experiments have to be done into ice.
*X μl: Optimized cloning efficiency requires about 50 – 100 ng of vector and at least 2-fold excess inserts. Use 5X more insert if the size is less than 200 bps.
Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled. Following incubation, store samples on ice or at – 20°C for subsequent transformation.
Transform competent cells with 2μl of the assembly reaction, using the following the transformation protocol.
Transfer 50 μl of competent cells to a 1.5 ml microcentrifuge tube (if necessary).
If the chemically competent cells are from New England Biolabs, add 2 μl of assembled product to NEB competent cells.
Mix gently by pipetting up and down or flicking the tube 4-5 times. Do not vortex. Place the mixture on ice for 30min. Do not mix.
Heat shock at 42°C for 45 seconds. Do not mix.
Transfer tubes on ice for 2 minutes.
Add 950 μl of room temperature SOC media to tubes.
Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
Warm selection plates to 37°C.
Spread 100 μl of the cell onto the plates with appropriate antibiotics. Use Amp plates for a positive control sample.
Incubate plates overnight at 37°C.
*ref. of the NEB Kit
Solutions preparation
IPTG (4,5 mM)
Materials
0.95mg of IPTG
80µL of sterilized water
10mL LB
10uL Cam (25 µg/mL)
Methods
Add 10µl of Cam in 10mL of LB
Homogenize
Add 0.95mg of IPTG in 80µl of sterilized water
Homogenize
Remove 90µl of Cam/LB mix and add 80µl of IPTG
Conserve at -20°C
For a serial dilution
Materials
888.8µl of IPTG (4,5mM)
Methods
LB AGAR
Materials
32g of LB agar (powder)
1L of distilled water
Methods
Add 32g of LB agar per 1L of distilled water
Homogenize
Autoclave at 121°C for 15 minutes
LB BROTH
Materials
20g LB Broth Base (powder)
1L distilled water
Methods
Add 20g of LB Broth Base per 1L of distilled water
Homogenize
Autoclave at 121°C for 15 minutes
MINIPREP & MIDIPREP
Minipreps were carried out according to the NucleoSpin Plasmid Miniprep Kit (NEB).
Midipreps were carried out according to the NucleoBond XtraMidi (NEB).
