Team:Nantes/Experiments

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Experiments
Gibson Assembly Cloning Protocol Mini preparation of DNA XL1 Blue cells and E.coli Expression Protocol and Dialysis Chemical Lysis BugBuster (Merck) Purification Control FACE Protocol HPAEC-PAD Protocol Claus Protocol Contact Process
Gibson Assembly Cloning Protocol


Cloning our genes of interest from cloning vectors (pUC) to expression vectors (pET11)
Source : New England BioLabs
Reagents: 
  • Agarose gel
  • Restriction enzymes: NdeI et BamHI
  • Gibson Cloning Master Mix:
    • T5 Exonuclease 
    • Phusion DNA Polymerase 
    • Taq DNA Ligase
 Equipment:
  • PCR tubes
  • Thermal cyler
  • Electrophoresis
  • Ice
Design
  1. Design your plasmid and order primers.
  2. Adjacent segments should have identical sequences on the ends. These identical sequences can be created via PCR with primers that contain a 5′ end that is identical to an adjacent segment and a 3′ end that anneals to the target sequence.
    Avoid strong secondary structures in the homology region.
    3. You can find the details in the Design Section on our wiki.

    Protocol
  3. Generate DNA segments by PCR
  4. Run PCR product on an agarose gel
  5. Purify DNA segments (Monarch® DNA Cleanup Kit - by NewEnglands BioLabs)
  6. Set up the following reaction on ice: 
    7. Reagents 2-3 Fragment Assembly Positive control
    Total Amount of Fragments 0.02 - 0.5 pmols  X µL 10µL
    Gibson Assembly Master Mix (2X) 10µL 10µL
    Deionized H20 10-XµL 0
    Total Volume 

    20µL

    		20µL
  7. 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.
  8. Transform the DNA into bacteria with 2 µL of the assembly reaction (XL1 Blue E. coli cells).
  9. For Minipreparation of DNA see below
  10. Screen for the correct plasmid product by restriction digest (NdeI and BamHI).
  11. Sequence the PCR product of your final plasmid.


Mini preparation of DNA


Minipreparation of DNA : Extraction of DNA from transformed XL1-Blue cells
Source : McKnight Brain Institute - University of Florida

Reagents: 
  • XL1-Blue E. coli cells 
  • Antibiotic
  • LB medium
  • Solution A : 25mM Tris-HCl, pH 8.0, 10mM EDTA
  • RNAse A
  • Lysis Buffer : 50 µL 400mM NaOH, 50 uL 2% SDS
  • Solution K (neutralization buffer) : 5M potassium acetate, pH 5.5
  • Isopropanol
  • 70% ethanol 
 Equipment:
  • Falcon Tubes
  • Microcentrifuge tube

  1. Inoculate 3 mL LB + ampicillin medium with XL1-Blue cells, culture with vigorous shaking at 37°C for 14-16 hrs.
  2. Aliquot 2 x 750 µL culture into a 1.5 mL microcentrifuge tube, harvest the bacteria by spinning at 12000rpm for 1 min. Aspirate supernatant and keep pellet.
  3. Resuspend bacterial pellet by complete vortexing in 100 µL Solution A resuspension buffer.
    4. To prepare the working solution A, add 3uL of RNAse A to 1 mL solution A and mix well. The bacteria should be completely resuspended - no clumps should be visible.
  4. Add 100 µL freshly prepared lysis buffer and mix gently by inverting 5-6 times at room temperature. Do not allow the lysis to occur for longer than 5 min.
  5. Add 120 µL of solution K and mix gently by inverting 5-6 times.
    6. Incubate the solution at room temperature for 3 min. The mixture should contain flocculent white precipitate at this point that consists of bacterial DNA and protein
  6. Remove bacterial/protein debris by centrifugation at 12000rpm for 15 min. Transfer the supernatant to a fresh microcentrifuge tube.
  7. Add 200 µL isopropanol to precipitate plasmid DNA from supernatant.
    8. Mix thoroughly by inverting 10 times.  Incubate at room temperature for 1 min.
  8. Collect DNA by centrifugation at 14000 rpm for 1 min. Immediately pour off isopropanol into beaker, shaking tube after the pour to remove a bit of the excess isopropanol.
  9. Add 500 µL 70% ethanol to the tube, mix by inverting or brief vortexing. Re-spin the DNA down at 14000 rpm (full speed) for 1 min.
  10. Respin for 1 min (immediately pour off ethanol).
    11. Take off any excess ethanol. Air-dry DNA for 10 min with caps open on the lab bench.
  11. Dissolve DNA in 20-35 µL water (volume used depending on whether you saw pellets). The final concentration of DNA should be 0.5 – 2.0 μg /μL and the OD260/280 value should be above 1.8.


XL1 Blue cells and E.coli BL21 (DE3) Transformation Protocol


Transformation of XL1 blue cells and then, E.coli BL21 (DE3) competent cells with pEVOL and pET plasmids or pET plasmid only
Source : UFIP (UMR 6286) Nantes
Reagents: 
  • Culture of XL1 Blue cells or E.coli BL21 (DE3) competent cells 
  • LB medium
  • DNA (pEVOL and pET plasmids)
  • Ampicillin
  • Chloramphenicol
 Equipment:
  • Ice
  • LB medium plate 
  1. Add pEVOL and pET or pET plasmids only to the E.coli BL21 competent cells : 2 µL per 100 µL tube.
    2. OR
    Add pEVOL or pET plasmids to the XL1 Blue competent cells: 2 µL per 100 µL tube.
  2. Incubate  30 min at 4°C (in ice)
  3. Heat-shock: Incubate 45 sec at 42°C.
  4. Put back on ice for 2 min.
  5. Add 0.5mL of LB medium.
  6. Incubate 1h at 37°C under shaking with the tube in horizontal position.
  7. Spread out 100 µL of the culture in a LB  + Ampicillin (pET alone) or Ampicillin and Chloramphenicol (pET and pEVOL) plate.
    8. OR
    Spread out 100 µL of the culture in a LB  + Ampicillin (pET) or Chloramphenicol (pEVOL) plate.
  8. Centrifugation of the rest of the culture 4 min at 4000 rpm.
  9. Resuspend the pellet in 100 µL of supernatant.
  10. Spread on a LB + Ampicillin or Ampicillin and Chloramphenicol plate.
    11. OR
    Spread on a LB + Ampicillin or Chloramphenicol plate.
  11. Incubate O/N at 37°C.


Expression Protocol and Dialysis


Expression of our protein in E. coli BL21 (DE3)
Source : Reisky and al, 2019
Reagents: 
  • Culture of E.coli BL21 (DE3) transformed cells
  • LB medium
  • Ampicillin and Chloramphenicol
  • IPTG
  • Arabinose
  • TB medium
  • HEPES buffer 
  • NaCl
 Equipment:
  • Ice
  • LB medium plate 
Degradation enzymes   
Day 0 :
  • E. coli BL21 (DE3) was previously transformed with pET plasmids containing enzymes de dégradation genes
  1. Inoculate 1 mL of TB medium with 100µg.mL-1 Ampicillin from an O/N culture in a LB medium containing 50µg.mL-1 of ampicillin

Day 2 :
  1. Centrifuge at 30°C and 180 rpm until the DO600nm = 1 
  2. Incubate at 20°C for 48h 

Day 4 :
  1. Add 0,5 mM IPTG 
  2. Incubate at 18°C O/N
Co-expression of FGE and Sulfatases
Day 0 :
  • E. coli BL21 (DE3) was previously transformed with pET11 and pEVOL plasmid containing sulfatases and FGE genes, respectively.  

Day 1 :
  1. Inoculate LB medium with 100µL.mL-1 ampicillin and 50µg.mL-1 chloramphenicol on a O/N culture in LB medium with 50µg.mL-1 ampicillin

Day 2 :
  1. Incubate at 37°C and 180 rpm until DO600nm= 0,3 - 0,5
  2. Add 1,5mM L-Arabinose 
  3. Incubate 90 min at 37°C
  4. Incubate the culture 2h at 18°C  
  5. Add 0,5 mM IPTG
  6. Incubate O/N at 18°C
  1. Harvest cells by centrifugation at 10 000g at 4°C for 20min and store at -20°C.
  2. Resuspend the pellet with  500µL 50nM HEPES and 100mM NaCl (pH 7,4).
  3. For Chemical lysis Protocol see below.
  4. Remove cellular debris by centrifugation at 13 000g à 4°C for 10min.
  5. Dialysis 
    1. Put phosphate buffer (PBS) in a beaker.
    2. Dialyze for 2h to 3h  at room temperature.
    3. Change the buffer.
    4. Dialyse O/N à 4°C.
  6. For Purification Protocol see below. 



Chemical Lysis BugBuster (Merck)


Extraction for obtaining the soluble fraction
Source : Merck
Reagents: 
  • Transformed E. coli BL21 (DE3) cells 
  • BugBuster 10X Protein Extraction Reagent kit
    • BugBUster reagent
    • Benzonase®Nuclease : 1 µL
    • rLysozyme™ Solution
    • rLysozyme Dilution Buffer : 100 mM NaCl, 50 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton® X-100, pH 7.5
  • SDS Sample Buffer (Cat. No. 70607-3)
  • Electrophoresis gel
 Equipment:
  • Ice
  • Electrophoresis
  • 1.5 mL tubes
  1. Harvest cells from liquid culture by centrifugation at 10 000g for 10 min. For small scale extractions (1.5 mL or less), centrifugation can be performed in a 1.5-mL tube at 14000-16000g. Decant and allow the pellet to drain, removing as much liquid as possible. Determine the wet weight of the pellet.

    2. Note: If using BugBuster 10X Protein Extraction Reagent, dilute to 1X in desired buffer. 

  2. Resuspend the cell pellet in room temperature BugBuster reagent by pipetting or gentle vortexing, using 5 mL reagent/ g of wet cell paste
  3. To reduce viscosity of the lysate, add 1 μL (25 units) Benzonase®Nuclease/ 1 mL BugBuster reagent (125 Units/gram cell paste) used for resuspension.
  4. To improve protein extraction efficiency in non-pLysS and -pLysE hosts, add 1 KU rLysozyme™ Solution per 1 mL BugBuster reagent (5 KU/gram cell paste). rLysoyzme Solution can be diluted using rLysozyme Dilution Buffer (100 mM NaCl, 50 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton® X-100, pH 7.5). Dilutions should be stored on ice or at 4°C.

    5. Experiments are performed on ice.

  5. Incubate the cell suspension on a shaking platform or rotating mixer at a slow setting for 10-20 min at room temperature.
  6. Remove insoluble cell debris by centrifugation at 16.000g for 20 min at 4°C. 
  7. Transfer the supernatant to a fresh tube. 
  8. For SDS-PAGE analysis, remove a small sample (25-50 μL) and combine with equal volume of 4X SDS Sample Buffer. Immediately heat for 3 min at 85°C to denature proteins and then store at -20°C until SDS-PAGE analysis.
  9. For Purification Protocol see below.


Purification Protocol


Purification of protein with a Nickel-NTA (NiNTA) column
Source : UFIP (UMR 6286) Nantes
Reagents: 
  • NiNTA beads
  • Cold lysis buffer : NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 10mM
  • Washing buffer : NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 20mM
  • Elution buffer : NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 250mM
  • Bradford reagent
  • Loading buffer
  • β-mercaptoethanol
  • SDS-PAGE 10%
 Equipment:
  • Ice
  • Column
  • Electrophoresis
  1. Add NiNTA beads previously rinsed with the cell lysate and incubate about 1h at 4°C on a wheel.
  2. Load the mix on a column. Collect the unbound fraction and keep at 4°C.
  3. Wash the column 3 times with 5 to 10 volumes of washing buffer  (NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 20mM) solution per gel volume  by rinsing the sides of the column and then with 10 mL of washing buffer. 
    4. Keep an aliquot of the washing step to load on the SDS PAGE.
  4. Elute the protein with 4 times 1 mL of elution buffer (NaH2PO4 50mM, NaCl 150mM, pH8, imidazole 250mM) and collect 4 fractions of 1 mL.
  5. Perform a protein assay on the different elution fractions with the Bradford reagent :
    1. 10µL of elution fraction + 790µL water + 200µL of Bradford reagent.
  6. Read OD595nm and Compare with a standard curve of BSA.
  7. Load 15µL of purified sample (+5µL of loading buffer + β-mercaptoethanol) on a 1% SDS PAGE.



FACE (Fluorophore Assisted Carbohydrate Electrophoresis) Protocol


Polysaccharide detection following the degradation of ulvan.
Source :MyBioSource
Reagents: 
  • 100 mM Ammonium Acetate, pH 7.0
  • Tris-Acetate Gel Solution
  • AMAC (2-aminoacridone) Solution
  • 10x Proteinase K (PK)
  • Hyaluronidase SD
  • Chondroitinase ABC
  • Heparinase (Flavobacterium heparinum)
  • Heparitinase I (Flavobacterium heparinum)
  • Heparitinase II (Flavobacterium heparinum)
  • 1X TBE (electrophoresis buffer)
  • Ammonium Persulfate (10 g) 
  • TEMED 
 Equipment:
  • Electrophoresis 
Protocol
    1. Proteinase K Digestion: 
      1. Tissues: Add 250 μl 10X PK per 100 mg wet weight of tissue. Incubate the samples at 60°C for 2 hours or until the tissue is digested. For tissue samples, vortex the samples every 30 min until the tissue is completely dissociated. 
    2. First Ethanol Precipitation: 
      1. Add 4 volumes of pre-chilled (-20°C) absolute ethanol to each sample and proceed to the next step or incubate at -20°C O/N. 
      2. Next day, centrifuge samples at 14000g for 10 min at RT. 
      3. Discard the supernatant by dumping it into a beaker. 
      4. Wash pellet by adding 4 volumes of pre-chilled (-20°C) 75% EtOH and vortex. 
      5. Centrifuge samples at 14,000 g for 10 minutes as before. 
      6. Discard wash as before, but this time you will need to remove residual ethanol with a pipette. 
      7. Air-dry the pellet at RT for 20 minutes (do not use a vacuum and do not over dry; this will make the pellet more difficult to re-dissolve). 
      8. Add 35 μL 100 mM ammonium acetate to each sample. Vortex/Spin. 
      9. Incubate at RT for 20 minutes. Vortex/Spin. 
      10. Heat inactivate the PK by incubating the samples at 1000 C for 5 min, then chill them on ice for 5 min.
    3. Digestion of Chondroitin Sulfate and Hyaluronan:
      1. Add enzymes of interest 1 μL of chondroitinase ABC and/or 1μL hyaluronidase SD to each 35 μL sample. 
      2. Vortex/Spin. 
      3. Incubate at 37°C O/N (at least 18 hrs).
    4. Second Ethanol Precipitation: 
      1. Add 160 μL of pre-chilled (–20°C) 200 proof ethanol to each sample. 
      2. Vortex/Spin. 
      3. Proceed to next step or incubate O/N at –20°C. 
      4. Centrifuge samples at 14,000 g for 10 minutes at RT. 
      5. Save supernatant in a separate 1.5 mL tube 
      6. Wash the pellet with 100 μL of pre-chilled (-20°C) 75% ethanol and centrifuge as before. 
      7. Pool this wash with the previous supernatant for each sample. 
      8. Proceed to step 7 if uninterested in heparan sulfate analysis. Air-dry the pellet for 20 min at RT. 
      9. Resuspend the pellet in 20 μL of 100 mM ammonium acetate and incubate at RT for 20 min. Vortex/spin the pellet. 
      10. Heat inactivate the enzymes by incubating the samples at 100o C for 5 min, then chill them on ice for 5 min. Then, proceed to step 5. For the supernatants, lyophilize them (using a “Speed-Vac” or equivalent) and proceed to step 7.
    5. Digestion of Heparan Sulfate: 
      1. Mix equal portions of Heparinase, Heparitinase I and Heparitinase II together and add 1 μL of this mixture to each 20 μL sample. 
      2. Incubate at 37°C O/N. 
    6. Third Ethanol Precipitation: 
      1. Add 80 μL of pre-chilled (–20°C) 200 proof ethanol to each sample. Vortex/Spin. 
      2. Proceed to the next step or incubate overnight at -20°C. 
      3. Centrifuge samples at 14000g for 10 min at RT. 
      4. Save supernatant in a separate 1.5 mL tube.
      5. Wash the pellet with 100 μL of pre-chilled (-20°C) 75% ethanol and centrifuge as before. 
      6. Pool this wash with the previous supernatant for each sample. 
      7. Air dry the pellet for 20 min at RT. Resuspend the pellet in 20 μL of 100 mM ammonium acetate and incubate at RT for 20 min. 
      8. Vortex/spin the pellet. 
      9. Heat inactivate the enzymes by incubating the samples at 100°C for 5 min, then chill them on ice for 5 min. If desired, DNA levels can be measured from this resuspended pellet.

    7. Labeling with AMAC: Lyophilize supernatants

      1. Add 1 μL of the AMAC solution per cm2 of culturing area (for both cell layer and conditioned media), 
      2. Add 1 μL of the AMAC solution for every 2.5 mg of tissue wet weight. 
      3. Add less AMAC to dilute down later since concentration is not an option.
      4. Incubate the sample at 37°C for 18 hrs in the dark. 
Gel preparation
  1. Gel Casting :
    1. Transfer the amount of gel solution you need to a clean tube
    2. You will need about 5 mL/gel for a gel with 0.75 mm spacers
    3. For each 5 mL, add 25 μL ammonium persulfate (10%) and 5 μL TEMED
    4. Mix by inversion and add to your pre-assembled casting plates. 
  2. Chilling the Gels: 
    1. Place the gels you just cast into their gel apparatus. 
    2. Add electrophoresis buffer to the inner/outer chamber 
    3. Place the gel box in a secondary container. 
    4. Surround the gel apparatus with ice up to the top of the apparatus. 
    5. Pour water into the ice to create a slurry until the water level reaches about halfway up the gel apparatus.
    6. Cool to 4°C before running the gel.  
  3. Loading the Samples:
    1. Add 2-5 μL of sample per lane.
    2. Add them directly to the well. 
    3. Taking the ratio of the IOD of the sample disaccharides to the standard disaccharides will enable an estimation of the concentration of disaccharides in your samples. 
    4. Add an AMAC “blank” to one of your wells. 
  4. Electrophoresis
    1. Run the gel at 500 V constant voltage.


HPAEC-PAD Protocol


Characterization of the ulvan chemical composition
Source : Elsevier Ltd (2020). Composition and structure of cell wall ulvans recovered from Ulva spp. along the Swedish west coast. Carbohydrate Polymers, Volume 233, 1 April 2020, 115852.
Reagents: 
  • 5 mM Tris-HCl
  • 0.1M NaCl
  • 0.5 mM CaCl2
  • Eluent : NaOH
  • Standard solution : Rhamnose, xylose, glucuronic acid and iduronic acid
  • TFA
 Equipment:
  • HPAEC-PAD : ICS-3000 Dionex
  • CarboPac PA1 column : 4 x 250 mm

HPAEC-PAD
  1. The eluent was pumped at 1.5 mL/min using a gradient program starting with 0.10 M NaOH and increasing to 0.16 M NaOH and 0.19 M sodium acetate during the run.
  2. A mixture of arabinose, rhamnose, galactose, glucose, xylose, mannose, glucuronic acid and iduronic acid was used as the reference.
  3. The data were processed and analyzed.
Sulfate content
  1. Sulfate groups on the ulvan backbones were released by hydrolysis in 2M Trifluoroacetic acid (TFA) at 3.3−4 mg/mL for 3h at 100 °C.
  2. Released sulfate was measured by a turbidimetric sulfate assay kit (product number MAK132, Sigma-Aldrich) with 0.2 M TFA in the standard curve to fit the dilution of samples.
  3. Absorbances at 600 nm were measured with a SPECTROstarNano plate reader (BMG LABTECH).
  4. Free sulfate in the solid material was determined by dissolving 3.3−4 mg ulvan in 1 mL Milli-Q water and heating as for samples, 3h at 100 °C.
  5. Aliquots of the samples were centrifuged at 20 000g for 10 min and sulfate was analyzed on the supernatants by the Sigma kit above (standards without TFA).
  6. The degree of sulfation was calculated by adjusting the sulfate determined after TFA hydrolysis with the free sulfate level.


Claus Protocol


Oxidation of H2S to SO2
Source : ScienceDirect- Claus Process
Reagents: 
  • H2S
  • O2 from the air 
 Equipment:
  • Burner
  • Reactor
  • Boiler
  • Condenser
Thermal step :
  1. Adjust the reactor at a pressure between 130 and 180 kPa and Temperature between 975 and 1300°C.
  2. O2 from the air in the reactor in stoichiometric quantities.
  3. Inject H2S in the reactor.
  4. A third of the gas is burned to produce SO2.
  5. Cooling of the gas in the boiler up to 250°C.
  6. Sulfur condensation in liquid forms in the condenser.
  7. Ejection of the water vapor created.


Contact Process


Production of liquid sulfuric acid
Source : L’Elémentarium - Sulfuric Acid
Reagents: 
  • SO2
  • Vanadium pentoxide : V2O5
  • Water
 Equipment:
  • Purification unit
  • Converter consisting of an alumina support (4 catalytic beds)
  1. Purifying the sulfur dioxide in a purification unit.
  2. SO2 is washed with water and dried by sulfuric acid.
  3. Adding an excess of oxygen to sulfur dioxide in the presence of the catalyst vanadium pentoxide at 440 °C and 100-200 kPa in the converter.
  4. The hot sulfur trioxide formed is dissolved in concentrated H2SO4 in the absorption tower to form oleum.
H2SO4 (l) + SO3 (g) → H2S2O7 (l)
  1. The oleum is then added to water to form sulfuric acid about 98% concentrated.
H2S2O7 (l) + H2O(l) → 2 H2SO4 (l)


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