Protocols

Overview

This section collects the protocols performed during our 4 month internship in the laboratory. We present here all the media used for cultures, the molecular biology protocols, the experimental conditions to test cell agglutination, and we provide some hints about the high-throughput screening methods we performed.

Medium

LB agar medium preparation

The LB agar medium is used to grow our E. coli strains on Petri dishes. The protocol described here is for 1 L.

Materials

  • • 10 g peptone (given by our lab)
  • • 5 g yeast extract (Biokar diagnostics, ref : A1202 HA)
  • • 5 g NaCl (given by our lab)
  • • 15 g agar (Biokar diagnostics, ref : A1012 HA)
  • • Distilled water q.s. to 1 L

Procedure

  1. 1) Mix all the ingredients without specific order of addition (besides adding distilled water last).

  2. 2) Add water up to 1 L.

  3. 3) The medium should be transferred to a glass bottle before autoclaving.

Buffer preparation

Procedure to prepare a buffer and equalize its pH.

Materials

  • • Glassware (Schott falcon or volumetric flask of desired volume)
  • • Machinery
    • • balance
    • • pH-meter with acid and basic calibration solutions
    • • magnetic stirrer

Procedure

  1. 1) Pipette or weigh each product and place them first in a Schott falcon to insert the pH probe in it.

  2. 2) Complete with solvent (generally milli-Q water) at approximately 90% of final volume (to have a margin for the pH equalization).

  3. 3) Place the falcon under the pH-meter and agitate it with a magnetic stirrer.

  4. 4) Calibrate the pH-meter.

    • • Caution: before measuring any solution, the probe should be washed with distilled water.

    • -Dispose the pH-meter to measure mode.

    • -Calibrate the probe.

  5. 5) Measure the pH of the sample. If it is too low, add some drops of NaOH solution. If it is too high, add some drops of HCl solution.

  6. 6) When the pH is optimal, transfer the content into a volumetric flask, and complete to the final volume with solvent.

Preparation of the glycerol stocks

The aim of this protocol is to conserve bacterial strains at -80°C. They have to be kept in a medium containing glycerol. An overnight preculture has to be prepared to obtain a maximum of cells to conserve.

Materials

  • • Sterile LB medium
  • • Sterile glycerol at 50% (v/v) (given by our lab)
  • • 1.5 mL-Eppendorf tubes

Procedure

  1. 1) From a single colony, inoculate a culture in LB with appropriate antibiotics.

  2. 2) Grow overnight at 37°C under 200 rpm shaking.

  3. 3) To a 700 μL culture sample, add 300 μL 50% glycerol (final glycerol concentration: 15% v/v).

  4. 4) Stock at -80°C.

Electrophoresis gel

Materials

  • • 50X TAE Buffer (given by our lab)
  • • Milli-Q water
  • • Agarose (Ultra pure ™ Agarose de invitrogen by life technologies ™, Ref : 16500-100)

Procedure

Mix 1X TAE with agarose. The more agarose the gel contains, the longer it will take for ADN to migrate. Use the following quantities:


Average length DNA matrix (bp) Percentage of agar Weight of agarose for 100 mL TAE 1X

4000

0.6

1.2 g

1500

0.8

0.8 g

800

1

1 g

300

1.2

0.6 g

Tips : mix well in a Schott falcon and heat it in the microwave until the liquid becomes transparent. Pour the gel into a caster and let it solidify. The gel is ready-to-use. For later use, the gel can be stored at room temperature and re-heated with a microwave or directly stored at 55°C.

Molecular biology

Synthetic DNA pieces resuspension

This protocol describes how to resuspend the various synthesized DNA pieces (gBlocks®, primers, etc.).
Notes : DNA sequences are ordered from IDT and primers from Eurofin genomics.

Materials

  • • Lyophilized DNA piece
  • • Milli-Q water

Procedure

• For gBlocks® resuspension :

  1. 1) Prior to opening, centrifuge the tube at a minimum of 3000 g to ensure that the material is at the bottom of the tube.

  2. 2) Add milli-Q water to reach a final concentration of 10 ng/μL for gBlocks®.

  3. 3) Pipette 2-3 times to mix.

  4. 4) The gBlocks® are stored at -20°C.

•For primers resuspension :

  1. 1) Prior to opening, centrifuge the tube at a minimum of 3000 g to ensure that the material is at the bottom of the tube.

  2. 2) Add milli-Q water to reach a final concentration of 100 μM for the primer.

  3. 3) To prepare ready-to-use aliquots, add milli-Q water to reach a final concentration of 10 μM.

  4. 4) Pipette 2-3 times to mix.

  5. 5) The primers are stored at -20°C. The aliquots can be stored at 4°C.

PCR

Routine PCR protocol, each concentration is given for a 50 μL PCR mix.

Materials

  • • Milli-Q water
  • • dNTPs mix (New England BioLabs, ref : N0447S)
  • • Buffer adapted to the polymerase
  • • Forward primer (stock at 10 μM)
  • • Reverse primer (stock at 10 μM)
  • • Polymerase: Phusion High Fidelity if you want a high fidelity to avoid undesired mutations (New England BioLabs, ref : M0530S) or One Taq DNA polymerase for PCR screenings (New England BioLabs, ref : M0480S)
  • • DNA matrix
  • • Machinery :
    • -PCR machine
    • -Vortex
    • -Table-top centrifuge

Procedure

• Part 1: PCR Phusion polymerase:

Use this protocol if you need to reuse the amplified sequence for further experiment. The fusion polymerase is a high fidelity enzyme.

  1. 1) Set the PCR machine with the following parameters:

    1. • 30" - 98°C (pre-denaturation step)
    2. • 10" - 98°C (denaturation step)
    3. • 10" - at the relevant temperature, if you don't know, perform a temperature gradient between 50 to 65°C (annealing step)
    4. • X - 72°C (elongation step), to be adapted to the length of the fragment of interest, knowing that the Phusion DNA polymerase processes DNA at around 2kb/min
      Repeat 30 cycles of steps 2-3-4
    5. • 5' - 72°c (final elongation step)

  2. 2) Prepare the PCR premix in the following order, vortexing at each step before adding the polymerase. Quantities are given for one PCR tube containing a 50 μL reaction.
    Multiply each quantity by the number of reactions needed.

    1. • 31,5 μL Milli-Q water
    2. • 1 μL dNTP mix
    3. • 10 μL Buffer
    4. • 2,5 μL Forward primer (stock at 10 μM)
    5. • 2,5 μL Reverse primer (stock at 10 μM)
    6. • 0,5 μL Phusion DNA polymerase (when added, never vortex the mix, mix by smooth pipetting)

  3. 3) Centrifuge with a table-top centrifuge for a few seconds if needed.

  4. 4) Distribute 49 μL of premix in eac PCR tube.

  5. 5) Add 1 μL of the DNA matrix to reach 10-20 ng DNA (for fragments) or 2,5-5 ng (for plasmids) in the PCR mix.

  6. 6) Centrifuge with a table-top centrifuge for a few seconds if needed.

  7. 7) Place the PCR tubes in the thermocycler and start your run.

• Part 2 : PCR Taq polymerase

Use this protocol if you don't need to reuse the amplicon but just to verify its presence or size.

  1. 1) Set the PCR machine with the following parameters:

    1. • 30" - 94°C (pre-denaturation step)
    2. • 10" - 94°C (denaturation step)
    3. • 10" - at the relevant temperature, if you don't know, perform a temperature gradient between 50 to 68°C (annealing step)
    4. • X - 72°C (elongation step), to be adapted to the length of the fragment of interest, knowing that the Phusion DNA polymerase processes DNA at around 1kb/min
      Repeat 30 cycles of steps 2-3-4
    5. • 5' - 72°c (final elongation step)

  2. 2) Prepare the PCR premix in the following order, vortexing at each step before adding the polymerase. Quantities are given for one PCR tube containing a 50 μL reaction.
    Multiply each quantity by the number of reactions needed.

  3. 3) Run PCR with :
    1. • 31,5 μL Milli-Q water
    2. • 1 μL dNTP mix
    3. • 10 μL Buffer
    4. • 1 μL Forward primer (stock at 10 μM)
    5. • 1 μL Reverse primer (stock at 10 μM)
    6. • 0,5 μL Taq DNA polymerase (When added, never vortex the mix, mix by smooth pipetting)

  4. 4) Centrifuge with a table-top centrifuge for a few seconds if needed.

  5. 5) Distribute 45 μL of premix in eac PCR tube.

  6. 6) Add 5 μL of DNA matrix to have 10-20 ng DNA in the PCR mix.

  7. 7) Centrifuge with a table-top centrifuge for a few seconds if needed.

  8. 8) Place the tubes in the thermocycler and run the PCR.

Part 3 : Screening PCR

Notes : this colony PCR protocol is used to perform PCR from isolated colonies on solid LB medium, but also from purified DNA. For this PCR, we use a One Tag Quick Load 2X Master Mix with standard buffer (New England BioLabs, ref : M0482L)

  1. 1) Prepare the PCR premix in the following order. Quantities are given for one 25 μL reaction per PCR tube. Multiply each quantity by the number of tubes needed.
    1. • 11,5 μL Milli-Q water
    2. • 12,5 μL OneTaq Master Mix
    3. • 0,5 μL Forward screening primer (stock at 10 μM)
    4. • 0,5 μL Reverse screening primer (stock at 10 μM)

  2. 2) For each microtube, scratch one colony with a pipette tip, wipe it smoothly on a clean petri dish, and gently tap (20 times) the tip inside the microtube containing the PCR mix. Remember to keep one microtube for the negative control without a matrix.

  3. 3) Run PCR with:
    • • 5' - 94°C
    • • 30" - 94°C
    • • 30" - 58°C
    • • 1'/kb) - 68°C
    • Repeat 30 cycles of steps 2-3-4
    • • 5' - 68°C
    • • 2' - 20°C
    • Store the amplicons at 4°C before further analysis.

Electrophoresis

Procedure to migrate a DNA sample on an agarose gel.

Materials

  • • Migration of the sample
    • • Electrophoresis tank
    • • Microtubes
    • • Gel loading dye, purple 6X (New England BioLabs, ref : B7024S)
    • • Size ladder (Quick-Load Purple 1kb Plus DNA Ladder, New England BioLabs, ref : N0550S)
    • • Sample (generally PCR or digested sample)
    • • Agarose gel with 1X TAE

Procedure

  1. 1) Pour the gel into a mold with a plate and place the appropriate comb. Let it solidify for 5-10 min. When the gel is solid, remove the comb.
    Place the gel in the electrophoresis tank with the loading zone on the anode side (upper side). Cover the gel with 1X TAE.

  2. 2) Analytic gel :

    • • In a microtube, lay 5 μL of the sample, add 1 μL of gel loading dye and mix gently with a pipette avoiding bubbles.
    • • Add the preparation in a well, repeat for every sample.
    • • Add 3-4 μL of size ladder in the middle well.
    • • Adjust the voltage at 135 V and stop when the purple front of dye is near the bottom of the gel.

    Note : this is the same procedure for a preparatory gel.

  3. 3) ETB analysis
    Ethidium bromide is a carcinogenic substance and should be handled with care, in a dedicated ETB room with required protection equipment and following safety procedures.

    • • Incubate the gel for 10 min in an ETB solution bath (few drops of pure ETB in 200 mL milli-Q water).
    • • Rinse the gel once in milli-Q water.
    • • Place the gel in a dedicated detection setup to visualize fluorescence under UV illumination. Take a picture.

Gel extraction

Procedure to extract a DNA band from the agarose gel after electrophoresis

Materials

  • • UV bench and safety equipment
  • • Scalpel
  • • 2 mL Eppendorf tube
  • • NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, ref : 740609.50)

Procedure

  1. 1) After observing the gel under UV illumination and taking pictures, the bands of interest are cut with a scalpel.

  2. 2) The gel slices are placed in 2mL microtubes and weighed using an empty 2mL tube as a reference.

  3. 3) 200 μL of NTI solution is added per 100 mg of gel.

    Note: the NTI solution has to be stored in the fridge.

  4. 4) The tubes are placed at 50°C for 10 min.

  5. 5) The contents are transferred to the kit columns.

  6. 6) Centrifuge at 11,000 g for 30s and empty the tubes.

  7. 7) Add 700 μL of NT3 on column. Centrifuge at 11,000 g for 30s and empty the tubes.

  8. 8) Add another 700 μL of NT3 to the column. Centrifuge at 11,000 g for 30s and empty the tubes.

  9. 9) Centrifuge a last time at 11,000 g for 1 min without adding anything, in order to completely remove the NT3.

  10. 10) Discard the tube and place the column in a new 1.5 mL microtube.

  11. 11) Add 20μL of milliQ water to the Silica membrane without touching it or the edges.

  12. 12) Incubate the tube at room temperature for 1 min.

  13. 13) Centrifuge at 11 000 g for 1 min.

  14. 14) Discard the column.

  15. 15) To measure the concentration of DNA in the solution and check the purity, use a nanodrop.

  16. 16) The tubes can be stored at -20°C.

PCR clean-up

Procedure to purify the DNA directly without a gel.

Materials

  • • NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, ref : 740609.50)

Procedure

  1. 1) If the PCR reaction has a smaller volume than 100 μL,complete with Milli-Q water to get a volume of 100 μL. Add 200 μL of NTI Buffer, mix by pipetting.

  2. 2) The contents are transferred onto the columns from the kit.

  3. 3) Centrifuge at 11,000 g for 30s and empty the flow-through.

  4. 4) Add 700 μL of NT3 Buffer to the column. Centrifuge at 11,000 g for 30s and empty the flow-through.

    NB: Make sure that you have added ethanol to the NT3 Buffer before using it.

  5. 5) Add another 700 μL of NT3 Buffer to the column. Centrifuge at 11,000 g for 30s and empty the flow-through.

  6. 6) Centrifuge for 1 min at 11,000 g to completely remove the NT3 Buffer.

  7. 7) Discard the flow-through and place the column in a new 1.5 mL microtube.

  8. 8) Add 20 μL of milli-Q water to the Silica membrane without touching it or the edges.

  9. 9) Incubate the tube at room temperature for 1 min.

  10. 10) Centrifuge for 1 min at 11 000 g.

  11. 11) Discard the column. The tubes containing the flow-through are stored at -20°C.

In-Fusion®

This technique allows insertion of DNA fragments into our linearised plasmids.

Materials

  • • Linearized vector
  • • DNA fragment to be cloned (from the PCR clean-up or gel extraction protocol)
  • • 5X In-Fusion® Snap Assembly Master Mix (Takara Bio Europe SAS, ref : 638948)
  • • Linearized pUC19 Control Vector (50 ng/μl)
  • • 2 kb Control Insert (40 ng/μl)

Procedure

  1. 1) Thaw the vector and the PCR product (insert).
  2. 2) Plan the In-Fusion® cloning reaction. Use the table below for reaction recommendations.

    3. Component Cloning Negative Control Positive Control

    Purified PCR fragment

    10-200 ng

    0 μL

    1 μL of 2 kb control insert

    Linearized vector

    50-500 ng

    1 μL

    0,5 μL of pUC19 control vector

    5X In-Fusion® HD Enzyme Premix

    1 μL

    1 μL

    1 μL

    Deionized Water

    q.s 5 μL

    q.s 5 μL

    q.s 5 μL

    Note : we use a reaction volume of 5μL, with a insert:vector molar ratio of 2:1.
    Tips : the optimal total DNA amount (insert + vector) is 200 ng for a 10-μl In-Fusion® Cloning reaction, but it should not exceed 70% of the reaction volume.

  3. 3) Incubate the reaction for 15 min at 50 °C, then place it on ice.

  4. 4) Continue with the transformation procedure. You can store the In-Fusion reactions at –20°C until you are ready.

Transformation of E. coli Stellar competent cells

Procedure to transform chimio-competent E. coli cells

Materials

  • • Chimio-competent cells (Stellar Competent cells, ref : 636763)
  • • Milli-Q water
  • • In-Fusion mixture containing the assembled plasmids
  • • SOC medium (given by our lab)
  • • Autoclaved LB medium (for tuner transformation)
  • • LB agar plates containing ampicillin
  • • Water bath
  • • Incubator
  • • Glass beads
  • • Micropipettes

Procedure

  1. 1) Unfreeze the chemically competent cells on ice (stored at -80°C).

  2. 2) To a 50 μL cell aliquot, add 10 μL of the In-Fusion mix at most. Also include positive and negative controls as indicated on the In-Fusion manual.

  3. 3) Let the tubes rest on ice for 30 min.

  4. 4) Proceed to the heat shock step: incubate the tube in water at 42°C for 45 seconds. Be really precise with the timing.

  5. 5) Let the tubes on ice for 2 min.

  6. 6) Add 500 μL of SOC medium to each tube.

  7. 7) Process to the phenotypic expression : Incubate for 1h at 37°C under agitation (or at least 15 minutes if the antibiotic for selection is ampicillin).

  8. 8) Plate 100 μL of each tube on LB plates containing Amp or the appropriate antibiotic if different.

  9. 9) Centrifuge the rest of the solution 3 min at 4000g.

  10. 10) Remove 350 μL of the supernatant by pipetting.

  11. 11) Resuspend the cells with what is left of medium (about 100 μL).

  12. 12) Restreak it on LB plates containing Amp or the appropriate antibiotic.

  13. 13) Incubate overnight at 37°C.

  14. 14) Count the number of colonies that you get on the next day. The colonies can be analyzed by colony PCR screening.

Preparation of chimio-competent cells E. coli

The aim of this protocol is to prepare competent cells E. coli to perform bacterial transformations using the heat shock procedure.

Materials

  • • A plate of fresh E. coli colonies
  • • Cold and sterile LB medium
  • • Cold and sterile CaCl2 (50mM) (given by our lab)
  • • Cold and sterile CaCl2 (80mM) (given by our lab)
  • • Cold and sterile glycerol (15%) (given by our lab)
  • • Ice

Procedure

  • • Day 1

    1. 1) Inoculate the strain from a single colony in 5mL of LB medium.

    2. 2) Incubate at 37°C and 130 RPM overnight in an incubator.

  • • Day 2

    1. 1) Put the sterile CaCl2 (50mM and 80mM) and glycerol (50% v/v) solutions at 4°C.

    2. 2) Dilute the overnight culture at 1:50 (2mL of culture in 100 mL of LB medium). Place it back at 37°C 130 RPM.

    3. 3) Follow the optical density and let the culture grow until it reaches OD=0,6 at 600nm.

    4. 4) Transfer the culture into Falcon tubes that are kept on ice.

    5. 5) Centrifuge at 4000 RPM and 4°C for 20 minutes.

    6. 6) Eliminate the supernatant under BSC, on ice.

    7. 7) Resuspend the pellets in 50mL of sterile and cold CaCl2 (50mM), working on ice.

    8. 8) Incubate on ice for 5 minutes.

    9. 9) Centrifuge at 6000 RPM and 4°C for 20 minutes.

    10. 10) Eliminate cautiously the supernatant under BSC on ice.

    11. 11) Resuspend the pellets in 1/20 of the initial volume.

    12. 12) Add sterile glycerol from the 50% (v/v) stock to reach a final concentration of 15% (v/v).

    13. 13) Aliquot the competent cells quickly, distributing200 μL in each sterile eppendorf tube.

    14. 14) Freeze at -80°C.

Cultures

This protocol describes how to inoculate liquid cultures of E. coli from colonies (part 1) or from overnight (part 2).

Materials

  • • LB medium
  • • Ampicillin (stock concentration at 1000 mg/mL)
  • • Colonies on plate or on liquid medium

Procedure

  • • Part 1 : culture with cells on solid medium.

    1. 1) Resuspend one colony in 10 mL of LB containing the appropriate antibiotic.

    2. 2) Agitate for 12 hours at 37°C under 130 RPM shaking.

  • • Part 2 : culture with cells from liquid medium.
    Note: You need a liquid preculture to follow this protocol.

    1. 1) Dilute the preculture 1/100 into 50 mL of LB containing the appropriate antibiotic.

    2. 2) 2h after inoculation, follow the OD600 of the cultures.

    3. 3) Measure the OD600 every 30 minutes.

    4. 4) When OD600 ≈ 0,5-0,6, the culture has reached the exponential phase and is ready for further experiment, for example for an induction.

Miniprep

Extraction of plasmids from bacteria and purification of them.

Materials

  • • Centrifuge
  • • Plasmid extraction kit (Euromedex, ref : BI-BS614)

Procedure

  1. 1) From a liquid culture, the sample is centrifuged for 15 min at 4°C and 4000 ppm.

  2. 2) Discard the supernatant.

  3. 3) Add 100 μL of solution I and resuspend the pellet trying to avoid bubbles.

  4. 4) Transfer into microtubes.

  5. 5) Add 200 μL of solution II.

  6. 6) Mix by inverting 4-6 times.

  7. 7) Incubate for 1 min at room temperature.

  8. 8) Add 350 μL of solution III.

  9. 9) Mix by inverting 4-6 times.

  10. 10) Incubate for 1 min at room temperature.

  11. 11) Centrifuge for 5 min at 11,000 g.

  12. 12) Transfer the supernatant into the kit column.

  13. 13) Centrifuge for 2 min at 11,000 g.

  14. 14) Discard the flow through.

  15. 15) Add 750 μL of Wash Solution.

  16. 16) Centrifuge for 2 min at 11 000 g.

  17. 17) Discard the flow through.

  18. 18) Add another 750 μL of Wash Solution.

  19. 19) Centrifuge for 2 minutes at 11,000 g.

  20. 20) Discard the flow through, place the column back into the tube.

  21. 21) Centrifuge for 2 minutes at 11000 g.

  22. 22) Place the column in a new 1.5 mL tube.

  23. 23) Deposit 50 μL of elution buffer at the center of the silica membrane without touching it.

  24. 24) Incubate for 3 min at room temperature.

  25. 25) Centrifuge at 10,000 rpm for 2 min.

  26. 26) Store the purified DNA at -20°C.

Digestion by restriction enzymes

This protocol explains how to perform a diagnostic restriction digest to verify the correct assembly of a plasmid or any other DNA part.

Materials

  • • Restriction enzyme (New England BioLabs)
  • • 10X rCutsmart Buffer (New England BioLabs, ref : B6004S)
  • • Milli-Q water
  • • Purified DNA (at least 100 ng)
  • • Agarose gel
  • • Ladder
  • • Loading buffer

Procedure

  1. 1) Mix 2 μL of 10X Cutsmart Buffer with a volume corresponding to 100 ng of the DNA sample.

  2. 2) Add 1 μL of the restriction enzyme and milli-Q water q.s. 20 μL.

  3. 3) Incubate 1h at 37°C.

    Note : The enzyme volume should not exceed 5% of the final volume.

  4. 4) Deposit the samples on an agarose gel and perform an electrophoresis to separate the fragments.

Plasmid digestion with DpnI

The DpnI enzyme cuts methylated DNA only. It is used to remove the remaining plasmid DNA template after a PCR: the plasmid DNA template will be cut but not the generated PCR amplicon.

Materials

  • • 10X rCutSmart Buffer
  • • DpnI enzyme (New England BioLabs, ref : R0176S)
  • • PCR product (amplified from a plasmid or methylated DNA part)

Procedure

  1. 1) Prepare the pre-mix for one 50 μL tube. For that, we add 5 μL rCutSmart Buffer and 0,4 μL DpnI.

  2. 2) Add 45 μL of the PCR product.

  3. 3) Incubate in the thermocycler with the following program :

    • • 30' - 37°C
    • • 18' - 80°C (heat inactivation)

Induction with IPTG in liquid medium

This protocol describes how to induce the expression of our genes of interest under the control of the T7 promoter, which is inducible by IPTG.

Materials

  • • Culture at OD600 = 0,5
  • • IPTG (initial concentration = 500mM) (Euromedex, ref : EU0008-B)
  • • Micropipette

Procedure

  1. 1) Add the desired IPTG concentration (range: 1 μM to 1 mM) to the culture.

  2. 2) Incubate the culture at 37°C over the desired period of time.

  3. 3) At the end of the induction, centrifuge the tube (5000 g for 3 min).

  4. 4) Aspirate the supernatant.

  5. 5) Add HEPES buffer (the volume and the type of buffer depends on the further experiments).

  6. 6) Store at -20°C (depending on the further experiments).

Agglutination test

Aggregation

This protocol describes how to test the aggregation of strains A and D mediated by IgE. To conduct this experiment, you must have grown bacteria in an (at least) 50 mL culture and induced the expression of the gene of interest with IPTG.

Materials

  • • 15 mL Falcon tubes
  • • 1X PBS (given by our lab)
  • • IPTG-induced culture of strains A (E. coli tuner transformed with pET21-OmpA-Arah2)
  • • IPTG-induced culture of strains D (E. coli tuner transformed with pET21-OmpA-DARPin)
  • • Anti-Ara h 2 IgE (Absolute antibody, ref : AB01493-14.0) or Anti-Derp1 IgE (Absolute antibody, ref : AB00132-14.0)

Procedure

Note : The stock concentration of IgE is 1 mg/mL. As the molecular weight of IgE in general is 190 kDa, their initial molar concentration is approximately 5e-6 mol/L. As the concentration of IgE in human serum is 1e-9 mol/L, we diluted them in order to get closer to this value. To be sure for the experiment to be sensitive enough, we used the following IgE dilutions: 1:100, 1:250, 1:1000.

  1. 1) Transfer 3 mL of each culture (strain A and strain D) in 15 mL tubes.

  2. 2) Centrifuge at 5000 g for 3 minutes.

  3. 3) Remove the supernatant.

  4. 4) Resuspend each pellet in 1200 μL 1X PBS.

  5. 5) Mix according to the following protocol to get the appropriate dilution range.

    6. A + D + IgE
    (1:100)         		A + D + IgE
    (1:250)         		A + D + IgE
    (1:1000)         		A + D

    Volume of A (now in PBS)

    10 μL

    10 μL

    10 μL

    10 μL

    Volume of D (now in PBS)

    10 μL

    10 μL

    10 μL

    10 μL

    Volume of IgE

    0,2 μL

    Pre-dilution: 0,2 μL IgE + 1,8 μL 1X PBS

    0 μL

    0,8 μL of pre-dilution

    0,2 μL of pre-dilution

    Exact dilution rate of IgE

    1:101

    1:260

    1:1010

    0

  6. 6) Incubate at 4°C for the desired period of time (generally overnight, so approximately 18 hours).

  7. 7) Observe 2 μL of the sample under the microscope to look for the presence of aggregates.

Allergen detection and recognition on the surface by immunofluorescence

This protocol describes how to detect the binding of IgE at the surface of the bacteria, with the help of an anti-IgE IgG antibody. This is a way to determine if the protein of interest is indeed expressed at the surface and if the allergen displays correctly its epitope. To conduct this experiment, you must have grown bacteria in a (at least) 50 mL culture and induce the expression of the gene of interest with IPTG. Once the OD is 0.5-0.6, you can start the experiment.

Materials

  • • TEV protease solution (1 mg/mL)
  • • 1X PBS
  • • Marked anti-IgE IgG (Absolute antibody, ref : AB00497-23.0, fluorescently labeled)
  • • Anti-Ara h 2 IgE (Absolute antibody, ref : AB01493-14.0) or Anti-Der p1 IgE (Absolute antibody, ref : AB00132-14.0)
  • • Cultures: pET21b(+)_OmpA_DARPin and pET21b(+)_OmpA_Arah2, pET21b(+) in Tuner cells; induction at 25 μM of IPTG in LB with ampicillin

Procedure

  • • Preparation of TEV solution 1:10

    1. 1) Pipet 1800 μL of PBS.

    2. 2) Add 200 μL of TEV at 1 mg/mL.

  • • Preparation of the cultures

    1. 1) Transfer 2mL from the culture flask into a 15mL Falcon tube.

    2. 2) Centrifuge at 4000 g for 6 minutes.

    3. 3) Remove the supernatant.

    4. 4) Resuspend the pellet in 400 μL of sterile 1X PBS.

    5. 5) Distribute 100 μL of the mixture in three Eppendorf tubes. We'll use these tubes for the antibody dilutions.

  • • IgE (primary antibodies) binding (the three IgE dilutions tested are: 1:101, 1:260 and 1:1010)

    1. 1) For the 1:101 dilution: add 1 μL of IgE commercial solution to the 100 μL.

    2. 2) For the 1:260 dilution: prepare an 1:10 IgE solution (diluted in PBS), and add 4 μL of this solution to the 100 μL.

    3. 3) For the 1:1010 dilution: prepare an 1:10 IgE solution (diluted in PBS), and add 1 μL of this solution to the 100 μL.

    4. 4) Perform these three steps for pET21b(+)_OmpA_DARPin and pET21b(+)_OmpA_Arah2 (so 6 samples are prepared).

    5. 5) For the negative control with pET21b(+), perform the first dilution (1:101) in one tube, and do not add antibodies to the second tube.

    6. 6) You obtain 8 tubes (3 pET21b(+)_OmpA_DARPin, 3 pET21b(+)_OmpA_Arah2 and 2 pET21b(+)). The tubes must rest at 4°C for 1 hour.

  • • Anti-IgE IgG (secondary antibodies) binding

    1. 1) Centrifuge each tube at 4000 g for 3 minutes.

    2. 2) Remove the supernatant.

    3. 3) Resuspend the pellet in 100 μL of PBS.

      4. Note : Every manipulation must be done in the dark from now.

    4. 4) Add IgG to the tube, following the same dilution as for IgE (1:101, 1:260 or 1:1010). No IgG must be added to the negative control (pET12b(+)).

    5. 5) The 8 tubes must rest at 4°C for 1h in the dark

  • • TEV treatment

    1. 1) Split 50 μL of each sample into two separate tubes.

    2. 2) Centrifuge at 4000 g for 3 minutes.

    3. 3) Remove the supernatant.

    4. 4) For the first tube : resuspend the pellet in 50 μL of the diluted TEV solution (100 μg/mL in 1X PBS).

    5. 5) For the second tube: resuspend the pellet in 50 μL of 1X PBS.

    6. 6) The tubes must rest for 30 minutes at room temperature.

    7. 7) Centrifuge at 4000 g for 3 minutes.

    8. 8) Remove the supernatant.

    9. 9) Resuspend the pellet in 50 μL of PBS.

  • • Observation

    1. 1) 2 μL are needed for the observation.

    2. 2) Prepare the microscope slides and observe them with a fluorescence microscope.

Cell fractionation and surface control

This protocol is divided into two phases. The first phase consists in expressing the DARPin-sfGFP construct and looking at its subcellular localization. The second phase consists in performing an experiment using the Der p 1 producing strain, IgE and partially purified DARPin-sfGFP.

Materials

  • • Culture and induction
    • • DARPin-sfGFP Tuner strain
    • • Der p 1 Tuner strain
    • • LB medium
    • • Ampicillin (stock at 100 mg/mL)
    • • IPTG (500 mM)
  • • Cell fractionation
    • • Resuspension buffer : HEPES buffer (50 mM pH8)
    • • Fractionation buffer : HEPES (50 mM pH8) + MgCl2 (10 μM) + Triton X-100 (2 % = 2 g for 100 mL)
    • • TEV (recombinant homemade), 0.1 mg/mL
  • • Surface control
    • • IgE anti-Der p 1 (1 mg/mL)
    • • 1X PBS

Procedure

  • • Day 1 : Preculture of DARPin-sfGFP and DARPin Tuner

    1. 1) Inoculate the DARPin-sfGFP and DARPin Tuner strains in 10 mL LB + 10 μL Ampicillin each.

    2. 2) Incubate at 37°C under 130 rpm overnight.

  • • Day 2: Cultures and IPTG induction

    1. 1) Inoculate 2 mL of each preculture in 200 mL LB + 200 μL Ampicillin (1L baffled erlenmeyer flask).

    2. 2) Follow the OD until reaching OD 0.6.

    3. 3) Then add 25 μM IPTG (the initial concentration of IPTG is 500 mM, so add 10 μL).

    4. 4) Incubate for 4h at 37°C to perform the induction. Measure the OD at the end of the induction step (it should equals 1.8 to 2, if it is not the case adjust the volume).

    5. 5) Transfer the total culture into 50 mL Falcon tubes (so 4 tubes each) and centrifuge at 5000 g for 6 min.

    6. 6) Remove the supernatant and resuspend the pellet with 5 mL of resuspension buffer for each Falcon (so the total volume for each strain is 20 mL).

    7. 7) Collect and pool the resulting resuspensions of each culture in one 30 mL tube per strain (so two tubes: DARPin and DARPin-sfGFP). Resuspensions are stored overnight at -20°C.

  • • Day 3 : Cell fractionation and treatment with TEV

    1. 1) Cells are defrost quickly in a water bath at 37°C and placed on ice, samples have to stay hereafter on ice.

    2. 2) Cells are disrupted by sonication, at 25 % for 1 min.

    3. 3) Samples are transferred into tubes capable of resisting strong centrifugation.

    4. 4) Centrifuge at 8°C, at 10 000 g for 15 min.

    5. 5) Collect the supernatant (C) and store it on ice.

      6. Note: The supernatant contains the total soluble fraction (cytoplasmic and periplasmic) while the pellet contains the insoluble fraction (membrane and inclusion bodies).

    6. 6) The pellet is resuspended with 20 mL of fractionation buffer (exactly the same volume as the previous resuspension buffer) and incubated for 20 min at room temperature.

    7. 7) Centrifuge at 8°C, at 50 000 g for 30 min.

    8. 8) Collect the supernatant (hereafter referred to as supernatant M) and store it on ice.

    9. 9) Store the pellet on ice, just in case.

      10. Note : the supernatant contains all the membrane proteins while the pellet contains the inclusion bodies and aggregates.

    10. 10) Supernatants C and M are aliquoted (900 μL in 15 mL Falcon tubes) in two series : TEV and No-TEV. Remaining supernatants are stored at 4 °C.

    11. 11) Tubes from series TEV are supplemented with 100 μL of TEV (final concentration: 0,01 mg/mL) whereas tubes from series No-TEV are supplemented with 100 μL of 1X PBS.

    12. 12) Tubes from series TEV and No-TEV are incubated 30 min at 30°C in a water bath.

    13. 13) 200 μL of each tube are dispensed in a 96-well microplate with a dilution series of 1/2. Fluorescence in each well is measured at Exc 488 nm/ Em530 nm in a microplate reader.

  • • Always day 3 : Der p 1 preculture (if microplate lecture show fluorescence)

    1. 1) Inoculate the Der p 1 Tuner strain in 10mL LB + 10 μL Ampicillin.

    2. 2) Inoculate at 37°C under 130 rpm shaking overnight.

  • Day 4 : Der p 1 culture and IPTG induction

    1. 1) Inoculate 500 μL Der p 1 preculture in 50 mL LB + 50 μL Ampicillin (250 mL baffled erlenmeyer flask).

    2. 2) Follow the OD of the culture until it reaches OD 0.6.

    3. 3) Then add 25 μM IPTG (the initial concentration of IPTG is 500 mM, so add 2.5 μL).

    4. 4) Incubate for 4h at 37°C to perform the induction. Measure the OD at the end of the induction step (it should equals 1.8 to 2, if it is not the case adjust the volume).

  • Always day 4 : Surface control

    1. 1) Transfer 3 mL of culture into 15 mL Falcon tubes.

    2. 2) Centrifuge at 5 000 g for 6 minutes.

    3. 3) Remove the supernatant.

    4. 4) Resuspend the pellet in 600 μL of 1X PBS.

    5. 5) Split 100 μL of Der p 1 sample in six separate Eppendorf tubes.

    6. 6) Here is what to add to each tube :

      • 1. Nothing
      • 2. 1 μL of IgE anti-Der p 1
      • 3. 100 μL supernatant M (containing membrane)
      • 4. 1 μL of IgE anti-Der p 1 + 100 μL supernatant M (containing membrane)
      • 5. 100 μL supernatant C (containing cytoplasm)
      • 6. 1 μL of IgE anti-Der p 1 + 100 μL supernatant C (containing cytoplasm)

      Note : after adding a supernatant, wash: centrifuge at 3000 g for 5 min, remove supernatant, resuspend in 100 μL PBS. If there is no pellet, try again.

    7. 7) Keep at room temperature for 1 hour.

    8. 8) Observe under the fluorescence microscope.

    9. 9) To concentrate the samples : centrifugate 3 000 g for 5 minutes and discard the majority of the supernatant.

    10. 10) Resuspend the pellet in the remaining volume and observe this on a microscope.

HIS-tag detection by immunofluorescence

This protocol describes how to detect a histidine tagged protein at the surface of the bacteria, with the help of an anti-histag antibody. This is a way to determine if the protein of interest is indeed expressed at the surface. To conduct this experiment, you must have grown bacteria in an (at least) 50 mL culture and induce the expression of the gene of interest with IPTG.

Materials

  • • TEV
  • • 1 X PBS
  • • Anti-histag IgG (Absolute antibody, ref : AB02419-23.0, fluorescently labeled)
  • • LB medium
  • • Ampicilline

Procedure

Treat the samples and control (tuner with empty pET21) with anti-HIS-tag IgG. Test three different dilutions of IgG (1:100, 1:250, 1:1000).
Caution: from the moment when antibodies are added, the experiment has to be conducted in the darkest conditions possible, to avoid bleaching of the fluorochrome attached to the antibodies.

  • • For the control (empty pET21).

    1. 1) Transfer 2 mL of the control culture in a 15 mL Falcon tube.

    2. 2) Centrifuge at 5000 g for 3 minutes.

    3. 3) Remove the supernatant.

    4. 4) Resuspend the pellet in 800 μL PBS.

    5. 5) Do the following protocol.

      6. Step

      Volume of each tube

      Separate in 3 microtubes

      200 μL culture

      200 μL culture

      200 μL culture

      Centrifuge and remove supernatant

      Pellet

      Pellet

      Pellet

      Resuspend in PBS buffer with anti-HIS-tag IgG if needed

      200 μL PBS

      200 μL PBS + IgG (1:100)

      200 μL PBS + IgG (1:100)

      Leave at 4°C for 1h

      Centrifuge and remove supernatant

      Pellet

      Pellet

      Pellet

      Resuspend in PBS buffer with TEV solution if needed

      200 μL PBS

      200 μL PBS

      200 μL PBS + TEV

      Leave at rest for 30 min

      Prepare the microscope slides

      Expected result

      No fluorescence

      No fluorescence

      No fluorescence

  • • For the sample
    1. 1) Transfer 6 mL of the culture in a 15 mL Falcon tube, repeat three times.

      2. Note : you should have transferred 18 mL in total (in 3 different Falcon tubes).

    2. 2) Centrifuge at 5000 g for 3 minutes.

    3. 3) Remove the supernatant.

    4. 4) Resuspend each pellet in 800 μL PBS.

    5. 5) Do the following protocol.

      6. Step

      Volume of each tube

      Tubes (1:100)

      Tubes (1:250)

      Tubes (1:1000)

      Separate in 3 (or 2) microtubes

      200 μL culture

      200 μL culture

      200 μL culture

      200 μL culture

      200 μL culture

      200 μL culture

      200 μL culture

      Centrifuge and remove supernatant

      Pellet

      Pellet

      Pellet

      Pellet

      Pellet

      Pellet

      Pellet

      Resuspend in PBS buffer with anti-HIS-tag IgG if needed

      200 μL PBS

      200 μL PBS + IgG (1:100)

      200 μL PBS + IgG (1:100)

      200 μL PBS + IgG (1:250)

      200 μL PBS + IgG (1:250)

      200 μL PBS + IgG (1:1000)

      200 μL PBS + IgG (1:1000)

      Leave at 4°C for 1h

      Centrifuge and remove supernatant

      Pellet

      Pellet

      Pellet

      Pellet

      Pellet

      Pellet

      Pellet

      Resuspend in PBS buffer with TEV solution if needed

      200 μL PBS

      200 μL PBS

      200 μL PBS + TEV

      200 μL PBS

      200 μL PBS + TEV

      200 μL PBS

      200 μL PBS + TEV

      Leave at 4°C for 30 min

      Prepare the microscope slides

      Expected result

      No fluorescence

      Fluorescence

      No fluorescence

      Fluorescence

      No fluorescence

      Fluorescence

      No fluorescence

      Note : the first column (without any antibody) is a negative control, to consider the possible auto-fluorescence of the cells.
      Note : the antibody dilutions are trivial (for a 1:100 dilution, add 200 μL PBS and 2 μL IgG).

High-throughput screening methods

Flow cytometry and FACS

Flow cytometry was used to sort the aggregates.

Materials

  • • All the material from the aggregation protocol
  • • 1X PBS
  • • LB ampicillin medium
  • • Infors incubator with hygrometry control
  • • Spectral FACS (machine used is from Cytek Biosciences)

Procedure

  1. 1) The aggregation protocol was performed with different strains of allergens. Optical Density of each culture is assessed before mixing strain A and strain D.

  2. 2) After mixing, samples are diluted in PBS to a concentration of 2.10^6 cells/mL.

  3. 3) Filter your sample with 200 micrometers filters to remove the bigger aggregates in order not to clog the machine.

  4. 4) Strain A alone, strain D alone and a mix of both without IgE were used as negative control and the assay was done with different strain A, strain D and IgE.

  5. 5) For the utilization of the FACS machine, the help of a specialist is required. They will calibrate the software. We thank Chloe Habouzit and Delphine Lestrade from TWB for their help. Briefly, after a pre-adapted calibration for E. coli. Then, samples are run with only strain A (and the red signal from mScarlet-1) and the DARPin strain D (with the BFP signal). Negative controls are used to identify A or D signals on the graphs. Then, the sizes of bacteria to be selected as aggregates is defined with a high stringency (double red-blue signals). Laser channel V3-A was used for blue signals and YG3-A for red signals.

  6. 6) 96 aggregates are sorted by FACS on a 96-well microplate with 150 microliters LB-ampicillin medium. The plate is grown for 48 to 72 h with agitation and hygrometry control.

  7. 7) After 72 h, 150 microliters are centrifuged and colony PCR are performed from this pellet with one couple of primers for each allergen that needs to be detected.



Droplets

This protocol describes how to test the aggregation of strains A and D mediated by IgE in microdroplets. To conduct this experiment, you must have grown bacteria in an 50 mL culture, induced the expression of the gene of interest with IPTG and resuspend the cells in PBS.

Materials

  • • 15 mL Falcon tubes
  • • 1X PBS
  • • IPTG-induced culture of strains A (E. coli tuner transformed with pET21-OmpA-Arah2)
  • • IPTG-induced culture of strains D (E. coli tuner transformed with pET21-OmpA-DARPin)
  • • Fluosurf surfactant
  • • Anti-Ara h 2 IgE (Absolute antibody, ref : AB01493-14.0)
  • • Syringes
  • • Microtubes

Procedure

Note: The stock concentration of IgE is 1 mg/mL. As the molecular weight of IgE in general is 190 kDa, their initial molar concentration is approximately 5e-6 mol/L. As there is a risk of saturating the IgE binding sites at this concentration, the IgE solution must be diluted 100 00 times to obtain a concentration of a few hundreds IgE per droplet.
The cell density must be calculated in order to control the average number of cells per droplet, called λ.

  • OD = number of cell per droplet / (droplet volume x number of cells per mL per uDO)

For E. coli, the equation is :

  • OD = λ / (3.1 x 10^-9 x 8.6 x 10^8)

    • The advantage of encapsulating the cells in droplet before the aggregation is to isolate each strain A displaying a different allergen. There must not be more than one strain A per droplet. To do so, the cells will be diluted a lot so most of the droplet does not contain any strain A. The optimal λ for this purpose is 0,26.

    1. 1) Resuspend the strain A and strain D in PBS to obtain the chosen λ.

    2. 2) Add IgE to the strain D solution to obtain a IgE dilution factor of 1:100 000(to have a margin for the pH equalization).

    3. 3) Sample 200μL of each cell suspension and of Fluosurf with syringes.

    4. 4) Place the syringes in the microfluidic platform from Cetoni and connect them to the input pits of the microfluidic chip.

    5. 5) Control the flow rate with the software QmixElements.

    6. 6) Observe the droplet generation with an optic microscope. When the generated droplets have a uniform size, collect them in a microtube.

    7. 7) Incubate overnight at room temperature without agitation.

    8. 8) Observe the cells in the droplet under a microscope.

    This method was developed by Sophie Lajus and Gabrielle Potocki and the IA and labeling parts are patented. We encourage anyone willing to know more to contact Sophie Lajus (lajus@insa-toulouse.fr) for further information.



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