Team:Aboa/Experiments

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Experiments

This page describes what we did in our research project and all the protocols we used. If you want to read more about our project, please read our project description .

This page contains only the succesful part of the project. If you want to know exactly what we did, please read our notebook .

Building plasmids

When we recieved our ordered DNA from the iGEM sponsors, we digested them and the plasmids we recieved from University of Turku with SfiI and HindIII restriction enzymes (ThermoFisher). Digestion was done according to the manufacturer's protocol. After digestion, DNA was purified with agarose gel electrophoresis. We used the Macherey-Nagel Gel and PCR cleanup-kit to extract the right DNA bands from the agarose gel.

The next step was to ligate the purified genes and plasmid backbones together. We used T4 ligase (ThermoFisher), and followed the manufacturer’s protocol. Following ligation, we used electroporation to transform the plasmid to E.coli XL-1 Blue -strain, which we used for cloning our plasmids.

Several colonies of transformed cells were picked from plates and grown overnight for plasmid extraction. Plasmids were extracted with the Macherey-Nagel NucleoSpin Plasmid EasyPure -kit and sent to Eurofins Genomics for sequencing.

When the sequencing results came, we picked the verified plasmids and continued our work with them.

Protocols used are described below.

Here is a protocol for 60 mL agarose gel. The protocol is fully scalable for larger gels.

  1. 0.6 g Agarose or 1.2 g for a 2% gel
  2. Add TBE-buffer to a final volume of 60 ml
  3. Heat the mix until the agarose fully dissolves in the TBE buffer. DO NOT LET IT BOIL OVER!
  4. Optional step: Wait until the solution has cooled down to 50 C and add the DNA stain to the solution.
  5. Cast the solution into a casting tray and put the well comb in its place.
  6. Wait until the solution solidifies. (It takes about 15 min in +4 C and 30 min in RT
  1. Place the gel into the gel box.
  2. Cover the gel with TBE-buffer.
  3. Add loading buffer to your samples. (We used Nippon Genetics Midori Green Direct stain + loading buffer)
  4. Place your sample carefully in the gel well. Do not let air bubles enter the well.
  5. Connect the gel box to a voltage source.
  6. Set the voltage to 70 V - 100 V and run the gel until the loading buffer has reached the bottom fourth of your gel.
  7. Turn the power off and disconnect the voltage source from the gel box.
  8. Remove the gel from the gel box and visualize the DNA bands according to the manufacturer’s instructions for your particular DNA stain.

This guide is for 2 x 250 mL cultures

  1. Inoculate 1 colony from an o/n culture of the strain to be made into electrocompetent cells into 10 mL of SB in a 125 mL flask. Incubate for 16-18 hours at 37 oC and 250 rpm.
  2. Have ready two 1 L flasks containing 250 mL each of SB medium pre-warmed to 37°C. Add two drops of the o/n culture to each of the flasks.
  3. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Place 1 L of 10% glycerol on ice.
  4. Place the cultures on ice for 15 minutes. From this point on, the cultures must be kept ice cold. Pour each of the 250 mL culture into chilled 500 mL (or 1000 mL) centrifuge bottles.
  5. Centrifuge at 5000 rpm for 10 minutes. Discard the supernatant and aspirate any residual broth.
  6. Add 250 mL of glycerol to each of the centrifuge bottles and completely resuspend the cells by pipetting up and down.
  7. Centrifuge at 5000 rpm for 10 minutes. Discard the supernatant (it is not necessary to aspirate). Completely resuspend the cells in 250 mL glycerol and centrifuge again for 10 minutes at 5000 rpm.
  8. Discard the supernatant and resuspend the cells in the residual glycerol by pipetting up and down.
  9. At this point, you can electroporate or freeze the cells. To freeze them, add 100 µl of the culture to a microcentrifuge tube on ice. Once you have used all of the culture, transfer the tubes to dry ice or liquid nitrogen for 10 minutes. Once the cultures are frozen, transfer them to a -80°C freezer. The cultures should be good for >6 months.
  1. 4 g NaCl
  2. 4 g Tryptone
  3. 2 g Yeast extract
  4. Add dH2O to a final volume of 400 mL. Autoclave to sterilize.

SB medium can be used as an alternative to LB medium.

  1. 30 g Tryptone
  2. 20 g Yeast extract
  3. 10 g MOPS (3-[N-morpholino]-propanesulfonic acid)
  4. Add 1 L dH2O. Autoclave to sterilize.
  1. Prepare 14 mL round-bottom culture tubes at RT. Place LB medium in a 37°C water bath. Pre-warm selective plates at 37°C for 1 hour.
  2. Place the electroporation cuvettes (1 mm) and the microcentrifuge tubes on ice.
  3. Thaw the electrocompetent cells on ice for 10 - 20 minutes and mix the cells by flicking the tube gently. Transfer 25 μl of the cell suspension (or the amount specified for the cuvettes) to a chilled microcentrifuge tube. Add 1 μl of the DNA solution.
  4. Carefully transfer the cell/DNA mixture into a chilled cuvette, without introducing bubbles, and make sure that the cells deposit across the empty crevasse of the cuvette. Electroporate using the following conditions for Bio-Rad GenePulser electroporators: 1,8 kV.
  5. Record time constants.
  6. Immediately add 1 ml of 37°C LB to the cuvette, gently mix up and down twice, then transfer to the round-bottom culture tube.
  7. Shake at 250 rpm at 37°C for 45 min.
  8. Dilute the cells as appropriate (1/10, 1/100) then spread 100-200 μl onto pre- warmed selective plates.
  9. Incubate the plates for 8 hours to overnight at 37°C.

Cell culturing & Protein production

Once we had confirmed that our plasmids contained the right genes, we continued to produce antibodies. We transformed the E.coli 321.deltaA -strain (C321.ΔA; a gift from George Church (Addgene plasmid # 48998)), which we used as a production strain, with plasmids containing the antibody gene and a plasmid containing a gene for p-azidophenylalanine tRNA synthetase (pEVOL-pAzF (Addgene plasmid # 31186 ; http://n2t.net/addgene:31186 ; RRID:Addgene_31186)).

First, we made a small scale production, just to confirm that our plasmids and E.coli strain work as they're supposed to. Then, we moved on to large scale productions.

Protocols used are below.

Materials

  • E. coli XL1 Blue cells containing the appropriate vector (e.g. pEB06H-scFv or pLK06H-scFv)
  • LA plate (Tryptone 10 g/l, Yeast extract 5 g/l, NaCl 5 g/l, Agar 15 g/l) with with 0.5% glucose and appropriate antibiotics
  • SB medium
  • D-Glucose, stock solution 20% (w/v) in H2O, autoclaved
  • Appropriate antibiotic
  • IPTG: isopropyl-b-D-thio-galactoside, stock solution 100 mM in H2O, sterilized by filtering Ø0.22 µm
  • Benzonase
  • Lysotsyme

    • Preculture

  • Inoculate cells from a plate or glycerol prep to 4 ml (test production) or 20 ml (big production) SB medium with 0.5% glucose and appropriate antibiotics.
  • Incubate at 37°C o/n.

    • Main culture

  • In the morning dilute the preculture into OD600 of 0.1 in SB with 0.05 % glucose and appropriate antibiotics, i.e. 2 ml of o/n culture in 200 ml of medium in a 1L Erlenmeyer flask.
  • Incubate at 37°C, 300 rpm, until OD600 is about 0.8 (0.6–1.0).

    • Protein production

  • Induce tRNA production by addition of 0.5 % arabinose and 1 mM pAzF.
  • After 10 min induce Fab production by addition of 500 µM IPTG.
  • Culture at 26°C, 250 rpm, o/n.

    • The antibiotics we used in our work were zeocin, tetracycline, ampicillin and chloramphenicol. Their working concentrations in growth mediums and plates are listed below:

     

    working concentration (µg/ml)

    Zeocin

    50

    Tetracycline

    15

    Ampicillin

    100

    Chloramphenicol

    25

    MW of pAzF is 206,1 g/mol
    50 ml of 100 mM stock, where 100 mmol/l is 0,1 mol/l:
    the amount of pAzF is 0,1 mol/l x 0,05 l = 0,005 mol.
    0,005 mol is 206,1 g/mol x 0,005 mol= 1,0305 g of pAzF.

    Made 50mM pAzF stock solution, 2 g weighed.
    100 ml of 0,1 M NaOH
    100 ml of NaOH

    Protein purification and analysis

    After profuction, antibodies need to be purified and it needs to be verified that p-azidophenylalanine has been incorporated into the antibody. Purifying was done with Ni-NTA. Fab0 (antibody with no p-azidophenylalanine) was also purified with Size Exclusion Chromatography.

    Purified antibody yield was measured with an immunoassay. We used Goat Anti-Human IgG coated plates (University of Turku) as a capture surface and Europium-labeled 2A11-IgG (HyTest) as a label.

    To verify that p-azidophenylalanine has been incorporated into our antibody, we labeled antibodies with DBCO-Cy5.5 (Jena Biosciences). As a negative contol we used the same method to label Fab0. Labeled proteins were run with SDS-PAGE.

    Protocols used are described below.

    2 L of modified NPI-10 was made by adding 20 ml of 1 M imidazole in 2 L of PBS.
    1 L of modified NPI-20 was made by adding 20 ml of 1 M imidazole in 1 L of PBS.
    1 L of modified NPI-20 was made by adding 250 ml of 1 M imidazole in 500 ml of 2xPBS and 250 ml MQ.
    10 ml of 1 M MgCl2 was added to 1 L of NPI-10 to make the lysis buffer.
    1 L of NPI-10, NPI-20 and NPI-250 were adjusted to pH 8 with HCl.

    60 µl of benzonase (stock 25 U/µl) was added.

    NPI-500 was made to test if there is Fabs that are bound more strongly.

    NPI-500:
    10 ml 10 x PBS
    50 ml 1 M imidazole -> 500 mM
    40 ml MQ
    pH 8

    Cell collection

  • Take a 1 ml sample of cell culture (label properly: Fab, vector, T/C, culture, date).
  • Centrifuge cellt 4,500–6,000 x g for 15 min at 4 °C. Remove supernatant.
  • To wash the cells resuspend in NPI-10 and centrifuge again. Remove supernatant.
  • Cell pellets may be stored at -20 °C or -80 °C until needed.

    • Purification of scFv-ALP clones

    Buffers

  • NPI-10/Equilibrium buffer: PBS: 50 mM NaH2PO4 sodium phosphate, 300 mM sodium chloride, 10 mM imidazol; pH 8
  • Lysis buffer: Equilibrium buffer with 10 mM MgCl2, 25 U/ml benzonase, 0.4 mg/ml lysozyme, pH 8
  • Lysozyme from chicken egg white, L6876-10G, Sigma-Aldrich, USA
  • MgCl2 1M -
  • Benzonase, Merck, Germany, 250 U/µl
  • NPI-20/Binding/Wash buffer - PBS with 20 mM imidazole, pH 8
  • NPI-250/Elution buffer - PBS with 250 mM imidazole, pH 8
  • (Regeneration: if necessary, check Macherey-Nagel's protocol for this step)

    • Cell lysis

  • Thaw the cell pellet from an E. coli expression culture on ice (if frozen).
  • Resuspend 1 g of pelleted, wet cells in 2–5 mL lysis buffer. Pipette up and down, or stir until complete resuspension without visible cell aggregates. Perform this step on ice.
  • optional: Incubate at room temperature (RT) in rotation for 30 min.
  • Freeze and thaw cells three times (20 min in -70 °C → 10 min +40 °C water bath).
  • Centrifuge (Avanti J-26 XP Centrifuge, JS-25.50, Beckman Coulter) 13000 rpm, 45 min, +4°C.
  • Take a 50 µl sample of the supernatant and store it at -20 °C (label properly: Fab, vector, T/C, lysate, date). Proceed to protein purification with the rest of supernatant.

    • Protein purification, Ni-NTA and buffer exchange

  • more info here
  • EQUILIBRIUM
  • Ni-NTA, x mL resin bed. Do this in a dark room!
    • Do not expose to light or it is ruined for ever and ever and ever! Samples lost.
  • Equilibrate column(s) to working temperature. Perform purifications at room temperature or covered with ice.
  • Resuspend Protino® Ni-NTA Agarose by mixing thoroughly to achieve a homogeneous suspension.
  • Immediately transfer an appropriate amount of suspension to an appropriate chromatography column, which allows slow flow rates of 0.5–1 mL/min. Pipette 2 mL of the original 50 % suspension per 1 mL of bed volume required.
  • Allow the column to drain by gravity.
  • Add 10 bed volumes of NPI-10 to equilibrate the gel.
  • Allow the column to drain by gravity.
  • BINDING
  • Add the clarified E.coli lysate or protein extract to the equilibrated gel.
  • Allow the column to drain by gravity using a flow rate of 0.5–1 mL/min.
  • Note: If the flow rate is too high polyhistidine-tagged proteins may not bind to the column efficiently. Reduce the flow rate or re-apply the flow-through to improve binding.
  • WASH
  • Wash the gel by adding 10 bed volumes of NPI-20.
  • Allow the column to drain by gravity.
  • Repeat the washing step (total wash 2 x 10 bed volumes of NPI-20).
  • Take a 50 µł sample from the flowthrough (label properly: Fab, vector, T/C, wash, date).
  • ELUTION
  • Add 5–10 bed volumes of NPI-250 to the gel.
  • Allow the column to drain by gravity and collect the eluate in fractions (manual).
  • Store eluted protein on ice.
  • Analyze fractions for the presence of the target protein. To determine the protein concentration use a Bradford protein assay (quick and easy) or measure the absorbance at 280 nm. Note that imidazole will also absorb at 280 nm.
  • Pool fractions containing the majority of the eluted polyhistidine-tagged protein and remove an aliquot for SDS-PAGE analysis.
  • Store protein at -70 °C or -20 °C. Note that many proteins irreversibly precipitate out of solution in the presence of imidazole. In this case remove imidazole prior to freezing. For proper storage it is recommended to remove the imidazole by ultrafiltration or dialysis.
  • Remove imidazole by changing the buffer with a buffer exchange column into PBS. Store at -20 °C.
  • Note: the proteins won't last long in PBS.
  • CLEANING THE Ni-NTA RESIN
  • Wash Protino® Ni-NTA Agarose with 15 CV of 0.5 M NaOH for 30 min to solubilize and desorb contaminants. When using columns adjust the flow rate accordingly. For example, wash a Protino® Ni-NTA Columns 1 mL by using a flow rate of 0.5 mL/min for 30 min, corresponding to a total volume of 15 mL.
  • Remove the NaOH solution by washing with 10 bed volumes of de-ionized water.
  • If you are reusing the resin directly, wash with 10 bed volumes of NPI-10/equilibrium buffer to equilibrate the resin.
  • For storage wash with 2 CV of 30 % ethanol. Resuspend the resin in 30 % ethanol and store at 2–8 °C.

    • Column used: Superdex 200 Increase 10/300 GL

    1. Equilibriate the column with PBS, pH 7.4 according to manufacturer’s guide.
    2. Concentrate the sample with 10 000 MWCO ultrafiltration spin column, 4000g, 3 min, +4 C.
    3. Inject samples into the column
    4. Use a flow rate of 0,2 ml/min, fraction size 0,5 ml.
    5. Use 280 nm light absorbance for detecting proteins.
    6. Measure Fab concentration with nanodrop and pool contiguous fractions. Use a Fab activity assay to determine the correct fractions.
    1. Add 13 pmol DBCO-Cy5.5 to every µg of protein.
    2. Incubate in shaker. Reaction should happen in 15 min, but we incubated o/n at +4 C

      3. Yep, that's how easy it really is!

    Separation gel
    1. up to 5 ml ddH2O
    2. x ml 30% acrylamide/Bis (calculate amount needed for protein size)
    3. 1.25 mL 1.5 M Tris (pH 8.8)
    4. 25 μl 20% SDS
    5. 25 μl 10% ammonium persulfate (make it fresh and store at 4 °C up to a month)
    6. 2.5 μl TEMED (add it last)
    7. Pipet the separation gel into the casting chamber and fill up to ~ 2.5 cm from the top. Add a small layer of isopropanol to the top of the gel prior to polymerization to straighten the level of the gel.
    Stacking gel
    1. 2.08 ml dH2O
    2. 0.506 ml 30% acrylamide/Bis
    3. 0.375 ml 1 M Tris (pH 6.8)
    4. 15 μl 20% (w/v) SDS
    5. 15 μl 10% ammonium persulfate
    6. Remove the isopropanol layer by using a filter paper. Rinse the top layer of the gel with ddH2O and dry off as much of the water as possible by using a filter paper. Add 1.5 µl of TEMED into the stacking gel and mix the solution well. Add the stacking gel and insert the comb.
    Sample buffer (10 mL, 4x)
    1. 2.5 ml 1 M Tris-HCl pH 6.8
    2. 0.5 ml of ddH20
    3. 1.0 g SDS
    4. 0.8 ml 0.1% Bromophenol Blue
    5. 4 ml 100% glycerol
    6. 2 ml 14.3 M β-mercaptoethanol (100% stock)
    7. Adjust the final volume to 10 ml with ddH20

    more detailed recipes and instructions for preparing the protein samples can be found at: https://bio-protocol.org/bio101/e80 and at: https://openwetware.org/wiki/SDS-PAGE_sample_buffer_(Morris_formulation)

    1. Dilute cell lysates e.g. 1/100, 1/500. 1/5000 ETC.

    2. Incubate 200 µL of lysates in Goat Anti-Human plate's wells for 30 min in shaking.

    3. Wash the wells twice to remove unbound Ab's.

    4. Add 50 ng of Eu-2A11 mAb dilution to each well in the volume of 200 µL. Filter the Eu-2A11 mAb solution before use.

    5. Incubate 30 minutes on shaking.

    6. Wash the wells four times to remove unbound Ab's.

    7. Add 200 µL of DELFIA Enhancement solution to each well.

    8. Incubate for 10 minutes in shaking.

    9. Measure time-resolved fluorescence with Hidex or Victor Multilabel Counter

    Testing Site-specific immobilization

    To test the function of the antibodies and the effect of site-specific immobilization, the antibodies were conjugated to DBCO-coated magnetic beads (Jena Biosciences). Before conjugating the antibodies to the beads, an azide group was added to the control antibody (Fab0) with NHS-Azide (ThermoFisher) to simulate unspecific conjugation with biotin. Amount of succesfully coated antibodies were measured with Eu labeled 226A2 antibodies.

    Site-specifically and unspecifically coated beads were saturated with Cy5 labeled Digoxigenin (University of Turku). Cy5 signal intensity and variation was measured with flow-cytometer.

    Protocols are described bellow.

    1. Prepare a standard series (e.g. 10, 30, 60 and 100 ng/per reaction) of azide-reactive-group-containing Fabs in the volume of 100 µL.

    2. Add 1.08 pmol of DBCO beads to the reactions to coat the beads with Fabs. Incubate overnight at + 4 °C.
    Note: If Fab-coated beads are stored for longer, a preservative, such as DMSO, is recommended.

    3. Wash the beads twice on a magnetic rack.

    4. Add 50 ng of Eu-226A2 mAb in the volume of 100 µL. Filter the Eu-226A2 solution before use.
    Note: A non-Fab-coated bead control should be used.

    5. Incubate for 1 hour at RT.

    6. Wash the beads twice on a magnetic rack.

    7. Add 200 µl of DELFIA Enhancement solution into each well.

    8. Incubate 10 minutes in shaking.

    9. Measure time-resolved fluorescence with Hidex or Victor Multilabel Counter.

    1. Vortex all bead samples
    2. Transfer similar amout of coated beads to reaction (we used 3 million beads/reaction).
    3. Add PBS + 0,5% Tween -buffer to final volume of 496 µL
    4. Add 4 µl 25µM Cy5 labeled Digoxigeninto each reaction
    5. Incubate in orbital shaker for 1h
    6. Wash beads 1x with PBS + 0,5% Tween(remember to immobilize beads with a magnet!)
    7. Add PBS 0,5% Tween to a final volume on 500 µL
    8. Analyze beads with flow cytometer, settings as follows:
      • Number of counts: 100 000
      • Flow rate 14 µL/min
      • Core size 10 µm
      • Excitation: 650 nm
      • Emission: 670 nm

    1. Prepare a standard series (e.g. 0, 10, 30, 60 and 100 ng/per reaction) of biotinylated Fabs and add 200 µL of each dilution to wells of a streptavidin-coated plate.

    2. Incubate the biotin-streptavidin reaction for one hour in shaking.

    3. Wash the wells four times with Kaivogen Wash Buffer.

    4. Add 50 ng of Eu-226A2 mAb in the volume of 200 µL. Filter the Eu-226A2 solution before use.

    5. Incubate for one hour in shaking.

    6. Wash the wells four times with Kaivogen Wash Buffer.

    7. Add 200 µl of DELFIA Enhancement soltuion into each well.

    8. Incubate 10 minutes in shaking.

    9. Measure Time-resolved fluorescence with Hidex or Victor Multilabel Counter.