Team:Pasteur Paris/Experiments

""

Text written on this website is not intended for use as protocols, but to give an idea of the main steps and complexity of each experiment. Please follow the links to the full protocols at the bottom of each sliding panel.

Molecular Biology: general protocols

Here we present the basic molecular biology methods we used throughout the project to amplify our plasmids, linearized them and insert our sequences, retrieve them rom bacteria and express proteins.

Bacteria transformation

Liquid culture

Bacterial stocks

DNA extraction from bacterial culture

Enzymatic digestion

Electrophoresis on agar gel

DNA Gel extraction

Ligation of plasmid with DNA insert


Aim

Insert a plasmid of interest into competent bacterial cells, in order to replicate them.



Aim

Grow a colony that have successfully been transformed with one or several plasmids in order to replicate plasmid or to express a protein.



Aim

Stock bacterial culture at -80 °C.


Materials

  • Desired bacterial cultures on petri dish
  • Sterile LB media
  • chloramphenicol (25mg/mL) or carbenicilline(50mg/mL)
  • glycerol 50%

Procedure

In advance:

  • Prepare a stock solution of LB + desired antibiotic in 50 mL falcon tube depending on how many culture you would like to stock in glycerol.
  • Prepare a sterile stock solution of glycerol 50 %.
  1. In 15 ml sterile falcon, add 5 mL of LB media
  2. Vortex the stock solution of antibiotic and add 5 µL to the LB
  3. Using an inoculation loop, touch gently 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 180 rpm for 16h.
  6. After 16 hours, centrifuge the tubes 5 minutes at 3000 rpm.
  7. Discard supernatant.
  8. Resuspend the pellet in 5mL of LB.
  9. Discard supernatant.
  10. Resuspend the pellet in 1 mL of LB + antibiotic.
  11. In a 125 ml erlenmeyer, add 1 mL of bacterial culture in 24 mL of LB + antibiotic.
  12. Incubate the culture at 37˚C 180 rpm.
  13. Measure the OD every hour for the first 3 hours and then every 20 minutes.
  14. When 0,6 < OD < 0,7, withdraw 5 mL of the bacterial liquid culture and add 5 mL of glycerol 50 %.
  15. Vortex.
  16. Aliquot the 10 mL into sterile cryotubes.
  17. Place into dry ice and freeze at -80˚C.

Get full protocol here



Aim

Retrieve amplified plasmids from a liquid culture of transformed bacteria. According to the liquid culture volume, we used the QIAfilter Plasmid Purification kit (for 25 mL culture) or the QIAprep Spin Miniprep kit (for 5 mL culture) from Qiagen.



Aim

Perform restriction enzyme digestion in order to recover linear backbones of the plasmids, extract our inserts from commercial plasmid, or check the success of a ligation.



Aim

Separate DNA fragments according to their molecular weight after an enzymatic digestion, in order to purify inserts or to analyse a plasmid.



Aim

: Extract DNA from an agar gel after an electrophoresis. We used the QIAquick Gel Extraction Kit provided by Qiagen.



Aim

Insert a DNA fragment with appropriate overlaps into a linearized plasmid. We used the In-Fusion HD Cloning Plus provided by Ozyme.


Microfluidics: general protocols

PDMS (Polydimethylsiloxane) is a widely used polymer in microfluidics, for its biocompatibility and transparence, among other qualities. Here we show how to prepare PDMS for microfluidic chips, as well as how to demold them, bond them to other surfaces and treat them for neuron growth. Also, we explain how our molds and chips were fabricated.

PDMS Chip Fabrication

PDMS Chip Demolding

PDMS Chip Bonding

PDMS Chip Treatment for Nerve Growth


Materials

  • Sylgard 184 Elastomer Kit
  • Vacuum pump unit
  • Stove

Protocol

According to manufacturer's instruction.

  1. Mix monomer and curing agent (10:1 proportion) for 30 seconds.
  2. Use a vacuum pump unit and a vacuum bell jar to extract air bubbles until the mixture is clear.
  3. Pour mixture onto mold.
  4. Put mixture+mold in stove at 70 degrees Celsius for 3 hours.

Get full protocol here



Materials

  • Razor blade
  • Biopsy puncher 4mm

Protocol


  1. Cut the borders of the chip with the razor blade.
  2. Extract the chip from its mold.
  3. Drill input and output holes with the biopsy puncher.

Get full protocol here



Materials

  • Plasma cleaner
  • Distilled water
  • Isopropanol
  • Office duct tape
  • Fume hood

Protocol


  1. Take chip and the surface it needs to be bonded to into the fume hood.
  2. Clean chip with duct tape and isopropanol.
  3. Put the chip and the surface into the plasma cleaner.
  4. Expose chip and surface 30 seconds to plasma.
  5. Take the chip and the surface back in the fume hood.
  6. Press the microfluidic chip against the surface.

Get full protocol here



Materials

  • Poly-D-Lysine solution 1.0 mg/mL
  • Laminin

Protocol


  1. Pour poly-D-lysine with concentration 10 &mu g/mL into the chip.
  2. Incubate over night.
  3. Pour laminine with concentration 4 &mu g/mL.
  4. Incubate for a few hours.

Get full protocol here


Microfluidics: membrane filters

Soon enough we realized that we would need something to confine the bacteria, so that it doesn't attack the neurons during our experiments. The solution came as a nanoporous membrane, that would also be used as the conductive element in our system to transmit to the neuron's impulse.

Membrane PEDOT:PSS coating

Membrane PEDOT:Ts and PEDOT:Cl coating



An aqueous solution of PEDOT :PSS can be prepared [1]. We decided to dip the membranes in this solution during the polymerization.


Materials

  • 3,4-Ethylenedioxythiophene
  • Sodium 4-vinylbenzenesulfonate
  • Deionised water
  • Sodium persulfate
  • Iron(III) sulfate hydrate
  • Alumina Oxide Membrane Filters

Protocol


  1. Pour 0.8 g EDOT, 2g PSS and 208 mL water in the glass beaker.
  2. Put the membranes in the solution.
  3. Stir for 10 minutes.
  4. Add 2 g of sodium persulfate and 0.015 g of iron(III) sulfate hydrate.
  5. Stir for 24 hours.
  6. Wash membranes with water and let them dry at room temperature in a Petri dish.

Get full protocol here




PEDOT :Ts and PEDOT :Cl polymers can be obtained by vapor phase polymerization on alumina oxide membranes [1]


Materials

  • 3,4-Ethylenedioxythiophene
  • Iron(III) p-toluenesulfonate hexahydrate for PEDOT :Ts or Iron(III) chloride for PEDOT :Cl
  • 1-butanol
  • Sodium persulfate
  • Iron(III) sulfate hydrate
  • Paper masks

Protocol


  1. Prepare homogenous oxidant solution (1.58 g Iron(III) p-toluenesulfonate hexahydrate and 10 mL butanol for PEDOT:Ts or 1.35 g Iron(III) chloride and 10 mL butanol for PEDOT:Cl)
  2. Dip membranes in oxydant solution.
  3. Let membranes dry at 40◦C.
  4. Place membranes in paper masks on Petri dish lids.
  5. Pour 200 µL EDOT in 50 mL beakers.
  6. Place Petri dish lids on top of the 50 mL beakers, membranes facing the inside of the beakers.
  7. Heat the beakers at 40◦C and stop when membranes darken (takes about 6 minutes).
  8. Wash membranes with butanol and water.
  9. Let membranes dry at room temperature.

Get full protocol here


Microfluidics: well chip

The well chip was designed and assembled by our team. It was used to test the biocompatibility of our membranes, as well as the culture of bacteria in the presence of current. Here we show how the molds were made, how the chip itself was assembled, how well's conductivity was measured and how biofilm culture was performed on it.

PDMS Well Chip Mold Fabrication

PDMS Well Chip Fabrication

PDMS Well Chip Conductivity Measurement




Molds were made of aluminium according to the following plans. Part 1 Mold's center cylinder part is detachable from the bottom to make the demolding ot PDMS easier.


PDMS Well Chip Mold Plans



Materials

  • Molds
  • Syringe without needle
  • Platinum 24mm x 2 mm strip
  • Circular 13mm diameter membrane

Protocol


  1. Prepare 15g of PDMS monomer using our protocol, section 1. Replace step 5 by : Fill the syringe with PDMS. Fill part 1 mold until it's full and part 2 mold until the PDMS layer is more or less 1 cm thick.
  2. Demold the chip following our protocol, section 2. Ignore step 2.
  3. Put membrane and platinum strip on PDMS part 1.
  4. Refer to our protocol, section 3 to bond PDMS part 2 to the PDMS part prepared in the previous step.
  5. Apply a small layer of PDMS with the syringe on the contact zone of the PDMS part 2 and the platinum strip.
  6. Put the chip in the stove for 3 hours.

Get full protocol here



Materials

  • Oscilloscope
  • Function generator
  • Solderless breadboard assembly
  • Electric wires with banana connectors
  • Coaxial cable
  • Male BCN to 2 female banana connectors converter
  • BNC Splitter
  • 1 kOhm resistor

Protocol


  1. Reproduce the following electric circuit.

    2. Electric circuit 1

  2. Set function generator on sine, no offset, 4.5 V amplitude.
  3. Measure Y2 peak-to-peak amplitude and Y2's phase relative to Y1.

Get full protocol here


Microfluidics: microchannel chip

We used the microchannel chip to test the effect of NGF on the neuron's growth.

PDMS Microchannel Chip Mold Fabrication




We were allowed to use the molds made by Institut Curie. We were not involved in the process of their fabrication. Here is a short video we made about how these molds were created.