Team:Pasteur Paris/Protocols/CellBio

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DNA Assembly and Microbiology

Agarose Gel Preparation

Bacterial Stock

Gel Extraction

Ligation

Liquid Culture

Midiprep for plasmid extraction

Miniprep for plasmid extraction

Transformation of E. coli BL21 (DE3) pLys

Transformation of E. coli DH5-alpha

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Aim

Prepare an 8% agarose gel for the electrophoresis of DNA samples.


Materials

  • UltraPure™ Agarose (Invitrogen, 16500-100)
  • UltraPure™ 10X TAE Buffer (Invitrogen, 15558)
  • Gel Green Nucleic Acid Stain (Biotium, 41005)
  • Scale
  • Microwave
  • Spatula
  • Erlenmeyer (250 mL)
  • Measuring cylinder
  • PowerPac™ Basic (Bio-Rad, 1645050)
  • Owl Scientific (Thermofisher) B2 Series gel block (plus Biorad powerpack adaptor)

Procedure


  1. Prepare 600 mL of TAE 1X by diluting 60 mL of 10X buffer in 540 mL of deionized water.
  2. Weigh 0.6 g of agarose on a scale.
  3. Place the agarose in an Erlenmeyer.
  4. Fill the Erlenmeyer with 75 mL of TAE 1X.
  5. Heat the Erlenmeyer for 2 min 30 s at 350W in the microwave oven.
  6. Stir and place it again in the microwave for an additional minute.
  7. Let the mixture cool down until it's cold enough to touch the glass without hurting your hand and add 5 μL of Gel Green.
  8. Pour the agarose in the horizontal electrophoresis system. Don’t forget to place the comb before!
  9. Let the gel cool down for 20-30 minutes before deposing the samples.



Aim

Store a bacterial culture at -80 °C.


Materials

  • Desired bacterial cultures on petri dish
  • Sterile LB media
  • Appropriate antibiotics: Carbenicillin (50 mg/mL) or Chloramphenicol (25 mg/mL)
  • Glycerol 50% v/v sterile
  • Dry Ice
  • Falcon 15 mL and 50 mL
  • Erlenmeyer (125 mL)
  • Sterile cryotube
  • Inoculation loop
  • Pipette p200 + p20 and associated cones
  • Plastic graduated pipette (25 mL)
  • Electric Pipetman (propipet)

Procedure

In advance:

  • Prepare a stock solution of LB + desired antibiotic in 50 mL falcon tube depending on how many strains you want to stock in glycerol.
  • Prepare a sterile stock solution of glycerol 50 %.
  1. In a 15 ml sterile falcon, add 5 mL of LB medium.
  2. Vortex the stock solution of antibiotic and add 5 μL to the LB.
  3. Using an inoculation loop, gently touch a colony of transformed bacteria from the petri dish, plastic side facing you.
  4. Immerse and dip the inoculation loop in the liquid media and stir.
  5. Place the liquid culture in the incubator at 37˚C and 180 rpm for 16 h.
  6. After 16 h, centrifuge the tubes 5 minutes at 3000 rpm.
  7. Discard supernatant.
  8. Resuspend the pellet in 5 mL of fresh sterile LB medium.
  9. Centrifuge the tubes for 5 minutes at 3000 rpm.
  10. Discard supernatant.
  11. Resuspend the pellet in 1 mL of fresh sterile LB medium.
  12. In a 125 mL Erlenmeyer, add 1 mL of bacterial culture in 24 mL of LB + desired antibiotic.
  13. Incubate the culture at 37°C and 180 rpm.
  14. Measure the optical density at 600 nm (OD600) every hour for the first 3 h and then every 20 minutes.
  15. When the OD600 reaches 0.6 to 0.7, withdraw 5 mL of the bacterial liquid culture and add 5 mL of glycerol 50%.
  16. Vortex the tube.
  17. Aliquot the 10 mL into 1 mL volumes in the sterile cryotubes.
  18. Place into dry ice and freeze at -80°C.



Aim

Extract a specific DNA band from an agarose electrophoresis gel.


Materials

  • QIAquick Gel Extraction Kit (Qiagen, 28706)
  • Scale
  • Scalpel
  • Heating block
  • Water bath
  • UV light box, equipped with blue diodes (long wavelength)

Procedure


According to the QIAquick Gel Extraction Kit's manual

  1. Using a UV light, excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
  2. Weigh the gel slice in an Eppendorf tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl).
  3. Incubate at 50°C for 10 minutes (or until the gel slice has completely dissolved). To help dissolve the gel, mix by vortexing the tube every 2 to 3 minutes during the incubation.
  4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
  5. Add 1 gel volume of isopropanol to the sample and mix.
  6. To bind DNA, pipet the sample onto the QIAquick column and spin in a microfuge for 1 min.
  7. (Optional): Add 0.5 mL of Buffer QG to QIAquick column and spin again.
  8. To wash the column, add 0.75 mL of Buffer PE to QIAquick column and spin.
  9. Transfer QIAquick column to a clean 1.5 ml microcentrifuge tube or to a provided 2ml collection tube. Centrifuge for 1 min at 13,000 rpm (~17,900 x g).
  10. Place QIAquick column in a clean 1.5 mL microcentrifuge tube.
  11. To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the centre of the QIAquick membrane and centrifuge the column for 1 minute at 13,000 rpm (~17,900 x g). Alternatively, for increased DNA concentration, add 30μl elution buffer, let stand for 1 min, and then centrifuge for 1 minute.



Aim

Perform the ligation of one or more inserts in a plasmid using the In-Fusion cloning kit.


Materials

  • Stellar competent cells (Takara Clontech - Ozyme)
  • Linearized plasmid
  • Purified insert(s)
  • 5X In-Fusion HD Enzyme Premix (Takara Clontech - Ozyme)
  • Control plasmid pUC19
  • Control insert
  • Deionized water
  • Water bath at 50°C
  • 1.5 mL Eppendorf tubes
  • Heating block at 80°C
  • Dry ice

Procedure


  1. Set the mix between insert and linearized vector in molar ratio 2:1 and complete with distilled water to reach a reaction volume of 16 μ L. The optimal quantity of vector is 100-150 ng.
  2. Pre-heat vector and insert for 5 minutes at 80°C.
  3. Put on ice for 3 minutes.
  4. Add 4 μL 5X In-Fusion HD Enzyme Premix and let the cloning occur in a water bath at 50°C.
  5. Set on ice and proceed to transformation in Stellar competent cells.



Aim

Start a liquid culture of transformed bacteria to amplify a plasmid.


Materials

  • Petri Dish with LB agar media + antibiotics carbenicillin 50 μ g/mL or chloramphenicol 25 μ g/mL
  • Sterile LB culture medium
  • Antibiotics: Carbenicillin 50 mg/mL or Chloramphenicol 25 mg/mL
  • Shaking incubator at 37°C
  • Sterile 250 mL Erlenmeyer or 50 mL Falcon tube
  • Inoculation loop
  • Plastic graduated pipette (10 mL or 20 mL)
  • Electric Pipetman (propipet)

Procedure


  1. Add 5 mL of sterile LB culture medium + appropriate antibiotics to a 15 mL falcon tube (or Erlenmeyer).
  2. Using an inoculation loop, touch a colony of transformed bacteria from the petri dish. Immerse the inoculation loop in the liquid medium and stir energetically.
  3. On a new petri dish + antibiotic, spread the rest of the bacterial colony in a zig-zag movement to have a copy plate.
  4. Place the liquid culture in the incubator at 37°C for 14 hours at 180 rpm. Maintain the lids on top using tape but do not close the tubes. The liquid cultures are then good to use for further experiments.
  5. Place the petri dish in the incubator at 37°C for 14 hours and then store it at 4°C.



Aim

Retrieve and purify the amplified plasmids from a culture of transformed bacteria.


Materials

  • 25 mL of overnight liquid bacterial culture of a transformed colony
  • QIAfilter™ Plasmid Midi Kit (Qiagen, 12243)
  • Centrifuges (Eppendorf 5804R, Sigma 1-14 microfuge)
  • Falcon tube 50 mL
  • Electric pipetman (propipet)

Procedure


According to the QIAfilter™ Plasmid Midi Kit's manual


  1. Harvest 25 mL of overnight bacterial culture by centrifugation in a 5804R at 6000 x g for 15 minutes at 4°C.
  2. Re-suspend the pelleted bacterial cells in 4 mL Buffer P1.
  3. Add 4 mL Buffer P2 and mix thoroughly by inverting the tube 4-6 times and incubate at room temperature for 5 minutes.
  4. Add 4 mL Buffer P3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  5. Pour the lysate into the barrel of the QIAfilter cartridge. Incubate at room temperature for 10 minutes, without inserting the plunger.
  6. Equilibrate a QIAgen-tip 100 by applying 4 mL Buffer QBT and allow the column to empty by gravity flow into a collector tube.
  7. Insert the plunger into the Cartridge and filter the cell lysate into the previously equilibrated QIAgen-tip. Let the cleared lysate enter the resin by gravity flow.
  8. Wash the QIAgen tip with 2 x 10 mL Buffer QC.
  9. Elute DNA with 5mL Buffer QF.
  10. Precipitate DNA by adding 3.5 mL of isopropanol at room temperature (RT) to the eluted DNA. Mix and aliquot in 1.5 ml Eppendorf tubes, and centrifuge immediately at 15,000 x g in Sigma 1-14 microfuge for 30 minutes at 4 °C. Carefully decant the supernatant.
  11. Wash DNA with 2mL of 70 % ethanol at RT, and centrifuge at 15,000 x g for 10 minutes. Carefully decant the supernatant.
  12. Air-dry the pellet for 5-10 minutes, and re-dissolve DNA in buffer TE.



Aim

Retrieve and purify the amplified plasmids from a 5 mL culture of transformed bacteria.


Materials

  • 25 mL of overnight liquid bacterial culture of a transformed colony
  • QIAprep™ Spin Miniprep Kit (Qiagen, 27106)
  • Centrifuge
  • 1.5 mL Eppendorf tubes
  • Electric pipetman (propipet)

Procedure


According to the QIAprep™ Spin Miniprep Kit's manual


  1. Pellet 5 mL of overnight bacterial culture by centrifugation in a 5804R at 6000 x g for 3 minutes at room temperature.
  2. Re-suspend the pelleted bacterial cells in 250 μL Buffer P1 and transfer to a 1.5 mL Eppendorf tubes.
  3. Add 250 μ L Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
  4. Add 350 μ L Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  5. Centrifuge for 10 minutes at 13,000 rpm in a Sigma 1-14 micro-centrifuge.
  6. Apply 800 μ L supernatant from step 5 to the QIAprep2.0 spin column by pipetting. Centrifuge in the microfuge for 30-60 s and discard the flow-through.
  7. Wash the QIAprep2.0 spin column by adding 0.75 mL Buffer PE. Centrifuge for 30-60 s and discard the flow-through. Transfer the QIAprep2.0 spin column to the collection tube.
  8. Centrifuge for 1 minute to remove residual wash buffer.
  9. Place the QIAprep2.0 column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 μL Buffer EB. Let stand for 1 minute, and centrifuge in the microfuge for 1 minute at 13,000 rpm.



Aim

Transform a plasmid in E. coli BL21 competent cells for protein expression.


Materials

  • LB agar plates covered with appropriate antibiotics
  • SOC Outgrowth Medium (NEB, B9020S)
  • BL21(DE3)pLysS Singles™ Competent Cells – Novagen (Merck, 70236)
  • Plasmids of interest
  • Shaking incubator at 37 °C (inFORS minitron HT)
  • Stationary incubator at 37 °C
  • Water bath at 42 °C
  • Ice bucket filled with ice
  • Micro centrifuge tubes
  • Sterile spreading rake

Procedure


  1. Remove agar plates from storage at 4°C and let them warm up to room temperature.
  2. Take competent cells out of -80°C and thaw on ice for approximately 5 minutes.
  3. Add 1 μ L of purified plasmid DNA to the tube of competent cells. Tap gently to mix and return to ice.
  4. Incubate the competent cell/DNA mixture on ice for 5 minutes.
  5. Heat shock each transformation tube into a 42°C water bath for 40 seconds.
  6. Put the tubes back on ice for 2 minutes.
  7. Add 650 μ L of SOC medium (without antibiotic) to the bacteria and grow at 37°C and 180 rpm for 60 minutes.
  8. Plate 250 μ L of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.
  9. Incubate plates at 37°C overnight.



Aim

Transform a plasmid in E. coli DH5-α competent cells for amplification and storage.


Materials

  • LB agar plates covered with appropriate antibiotics
  • SOC Outgrowth Medium (NEB, B9020S)
  • DH5-α Competent E. coli (High efficiency) (NEB, C2987 I)
  • Plasmids of interest
  • Shaking incubator at 37 °C
  • Stationary incubator at 37 °C
  • Water bath at 42 °C
  • Ice bucket filled with ice
  • Micro centrifuge tubes
  • Sterile spreading rake

Procedure


  1. Remove agar plates from storage at 4°C and let them warm up to room temperature.
  2. Take competent cells out of -80°C and thaw on ice for approximately 5 minutes.
  3. Add 1 μL (diluted to approx.. 10 ng/uL) of purified plasmid DNA to 100 μL of competent cells. Mix by flicking the bottom of the tube and return to ice.
  4. Incubate the competent cell/DNA mixture on ice for 30 minutes.
  5. Heat shock each transformation tube into a 42°C water bath for 40 seconds.
  6. Put the tubes back on ice for 3 minutes.
  7. Add 650 μ L of SOC medium (without antibiotic) to the bacteria and grow at 37°C and 180 rpm for 40 minutes.
  8. Plate 250 μ L of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic.
  9. Incubate plates at 37°C overnight.


Protein Characterization

Fast Protein Liquid Chromatography

Gel Transfer for Western Blotting

Immunodetection on membrane

IPTG Induction of protein expression

Purification of his-tagged protein from culture med.

SDS-PAGE


Aim

Purify the proteins from a bacterial sample containing our protein of interest with a His-tag (in our case: NGF)


Materials

  • Buffer A (5% glycerol, 50 mM Tris, 200 mM NaCl)
  • Buffer B (5% glycerol, 50 mM Tris, 200 mM NaCl, 250 mM imidazole)
  • Phenylmethylsulfonyl fluoride (PMSF)
  • Sonicator Branson sonifier 450
  • High load centrifuge (Avanti J-265 XPI, rotor JLA-14.50, Beckman Coulter)
  • Fast Protein Liquid Chromatography (FPLC) ÄKTA pure (General Electric) with software Unicorn 6.3
  • Ni-NTA Superflow Cartridge 5mL, (QIAGEN, 1034558)

Procedure


  1. Resuspend the bacterial pellet of the 800 mL culture in 10 mL buffer A and add 10 μM PMSF to prevent protein degradation.
  2. Sonicate the bacteria during 5 cycles of 45 seconds with 1 min intervals at power 6 and 50% duty control.
  3. Centrifuge at 15,000xG for 25 min at 4°C.
  4. Realize the FPLC with the following sequence:
    1. Inject the lysis supernatant at 0.5 mL/min,
    2. Wash with buffer A for 90 min,
    3. Wash with 5% buffer B in buffer A for 30 min,
    4. Increase the proportion of buffer B gradually until reaching 100% during 70 min.



Aim

Transfer the proteins from an SDS-PAGE gel to a nitrocellulose membrane for western blotting.


Materials

  • Gel from an SDS-PAGE (Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis)
  • iBlot® 2 NC regular stacks (Thermofisher-Invitrogen, IB23001)
  • iBlot® 2 gel transfer device (Thermofisher-Invitrogen)

Procedure


According to the iBlot™ 2 Dry Blotting System USER GUIDE .

  1. Unseal the Transfer Stack. Set the Top Stack to one side and discard the white separator. Keep the Bottom Stack in the plastic tray.
  2. Place the Bottom Stack (in the tray) on the blotting surface. Align electrical contacts on the tray with the corresponding electrical contacts on the blotting surface of the iBlot® 2 Gel Transfer Device.
  3. Wet the pre-run gel(s) and place it on the transfer membrane of the Bottom Stack.
  4. Place a pre-soaked (in deionized water) iBlot® Filter Paper on the gel and remove air bubbles using the Blotting Roller (segment of a 10 ml plastic pipette in our case).
  5. Place the Top Stack, over the pre-soaked filter paper. Remove air bubbles using the roller.
  6. Place the Absorbent Pad on top of the Top Stack such that the electrical contacts on the blotting surface of the iBlot® 2 Gel Transfer Device.
  7. Close the lid of the device.
  8. Select the method P0 and touch Start Run. The transfer lasts for 7 minutes.



Aim

Mark the nitrocellulose membrane from the transfer with an anti-his-tag antibody to detect 6his-tagged NGF.


Materials

  • Phosphate-buffered saline 1X (PBS) (Sigma, P4417)
  • Tween20 (VWR, 437082Q)
  • Bovine serum albumine (BSA) (Sigma, A3608)
  • 6x-His tag antibody, Alexa Fluor® 647 conjugate (HIS.H8) (Invitrogen, MA1-21315-A647)
  • Odyssey Gel Imaging Scanner (Li-COR, Nevada)
  • Western blotted nitrocellulose membrane
  • Appropriate recipient for the membrane and low agitation horizontal orbital shaker

Procedure


  1. Wash the transfer membrane in PBST (1L phosphate buffer saline 1X + 1mL Tween20) for 10 min.
  2. Incubate the membrane with 10 mL saturation buffer (95 mL PBS 1X, 3% BSA, 100 μL Tween20) for 1 hour at room temperature.
  3. Wash the membrane with PBST for 1h.
  4. Incubate the membrane with the antibody diluted at 1:500 in 10 mL antibody incubation buffer (10 mL PBS, 3% BSA) for 1 hour at room temperature or overnight at 4°C.
  5. Wash 3 to 5 times for 5 minutes in 10 mL PBST at a higher agitation to decrease background noise.
  6. Reveal the Western Blot using the Odyssey X-ray scanner.



Aim

Derepress the T7 promoter using IPTG to activate NGF or RIP expression.


Materials

  • Desired transformed bacterial cultures on petri dish
  • Sterile LB medium
  • Appropriate antibiotics: Carbenicillin (50 mg/mL) or Chloramphenicol (25 mg/mL)
  • 100 mM Isopropyl β -D-1-thiogalactopyranosid (IPTG) (Sigma-Aldrich, I6758)
  • Erlenmeyer (V = 125 ml)
  • InFORS minitron HT shaking incubator
  • Inoculation loop
  • Pipette p200 + p20 and associated cones
  • Plastic graduated pipette (25 ml)
  • Electric Pipetman (propipet)

Procedure


  1. Resuspend a single transformed colony from a petri dish in 10 mL of LB medium containing the desired antibiotic.
  2. Incubate at 37°C and 180 rpm until the OD600 reaches 0.4 to 0.8 in an inFORS minitron.
  3. Induce the protein expression with IPTG to a final concentration of 0.1 to 0.5 mM depending on the characteristics of the expressed protein.
  4. Incubate at 37°C and 180 rpm for 3 to 5 hours.
  5. Check for protein expression either by staining an SDS-PAGE gel, performing a Western Blot or checking for protein activity, or by mass spectrometry. Compare protein expression in both the total cell extract (soluble + insoluble) and the soluble fraction only.

For large scale protein expression, inoculate 800 mL of liquid LB medium containing the desired antibiotic with a freshly grown colony or 10 mL of fresh liquid preculture. The overnight preculture is first pelleted, washed with LB, twice, then resuspended in 10 ml LB. Then perform steps 2 to 5 using the optimal time/temperature determined in a small-scale trial.




Aim

Purify the proteins from a bacterial sample containing our protein of interest with a His-tag (in our case: NGF)


Materials

  • Buffer A (5% glycerol, 50 mM Tris, 200 mM NaCl)
  • Buffer B (5% glycerol, 50 mM Tris, 200 mM NaCl, 250 mM imidazole)
  • High speed centrifuge (Avanti J-26 XP, rotor JLA-8.100, Beckman Coulter)
  • Peristaltic pump (P1 Pharmacia – GE Healthcare)
  • Ni-NTA Superflow Cartridge 5 mL, (QIAGEN, 1034558

Procedure


  1. Centrifuge 800 mL of induced bacterial culture at 6,238xG during 25min in a JLA-8.100 rotor and Avanti-J26 XP centrifuge.
  2. Carefully take aside 500 mL of supernatant without taking any bacteria from the pellet.
  3. Using the peristaltic pump at 2.5 mL/min, flow 500 mL of supernatant through a 5 mL Ni-NTA superlfow cartridge in a closed loop overnight.
  4. Wash the column with 30 mL Buffer A.
  5. Elute the fixed proteins with 10mL Buffer B in 10 different fractions of 1 mL.



Aim

Separate proteins from a sample according to their molecular mass by electrophoresis (Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis)


Materials

  • Spatula
  • Measuring cylinder
  • NuPAGE™ 4-12% Bis-Tris Gel (Invitrogen, NP0322BOX)
  • Power Pac 300 (BIO-RAD)
  • Mini Gel Tank (Invitrogen, A25977)
  • NuPAGE® MOPS SDS Running Buffer 20X (Novex, NP0001)
  • Protein sample
  • Dry heater block at 90°C
  • 2X SDS-PAGE Sample Buffer (4% SDS, 20% Glycerol, 0,01% bromophenol blue)

Procedure


  1. Prepare protein samples by mixing them with 2X SDS-PAGE Buffer (50% v/v).
  2. Prepare 500 mL of 1X Running Buffer by diluting 25 mL of 20X Buffer in 475 mL of distilled water.
  3. Install the precast gel on the mini gel tank.
  4. Heat the sample at 90 °C for 5 minutes, place on ice.
  5. Deposit up to 20 μL of protein in each well.
  6. Run the gel using the Power Pac 300 for 10 min at 85V and then for 40 min at 125 V.


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Biofilm Assays

Biofilm assay and crystal violet staining

Biofilm PFA fixation


Aim

Test biofilm formation capacity in microtiter plates.


Materials

  • 25 mL of overnight liquid bacterial culture of S. aureus expressing GFP
  • 96-well sterile microtiter plate
  • Crystal violet
  • Distilled water
  • Ethanol 99.8% (Sigma-Aldrich)
  • Acetone (Sigma-Aldrich)

Procedure



Biofilm formation


  1. Dilute to the hundredth an O/N bacterial culture (in LB medium, with appropriate antibiotics).
  2. Distribute 100 μL of diluted culture in each well (in general 4 wells per sample). Don’t’ forget to leave an empty well and a well with LB only as controls.
  3. Incubate plates at 37°C for 24 hours.

Plate washing and reading fluorescence


  1. Discard the supernatant in microbiological waste bin. Do not pipet.
  2. Immerse plate in a container with distilled water (let the water softly enter the wells).
  3. Take the plate out of water and discard water sharply over the waste container.
  4. Repeat this operation two times.
  5. Bang on blotter paper to eliminate residual water.
  6. Read fluorescence (excitation 485nm, emission 510 nm) and export data.

Crystal violet staining


  1. Add 125 μL Crystal violet (1% in water) in each emptied well.
  2. Wait 15 minutes for staining.
  3. Wash 3 times with distilled water as described before.
  4. Bang on blotter paper to eliminate residual water.
  5. Suspend colored biofilm by adding 150 μL ethanol/acetone solution (80:20).
  6. Read OD of 50 μL at 570 nm and export data.


Aim


Materials


Procedure




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