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.
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.
- Mix monomer and curing agent (10:1 proportion) for 30 seconds.
- Use a vacuum pump unit and a vacuum bell jar to extract air bubbles until the mixture is clear.
- Pour mixture onto mold.
- Put mixture+mold in stove at 70 degrees Celsius for 3 hours.
Materials
- Razor blade
- Biopsy puncher 4mm
Protocol
- Cut the borders of the chip with the razor blade.
- Extract the chip from its mold.
- Drill input and output holes with the biopsy puncher.
Materials
- Plasma cleaner
- Distilled water
- Isopropanol
- Office duct tape
- Fume hood
Protocol
- Take chip and the surface it needs to be bonded to into the fume hood.
- Clean chip with duct tape and isopropanol.
- Put the chip and the surface into the plasma cleaner.
- Expose chip and surface 30 seconds to plasma.
- Take the chip and the surface back in the fume hood.
- Press the microfluidic chip against the surface.
Materials
- Poly-D-Lysine solution 1.0 mg/mL
- Laminin
Protocol
- Pour poly-D-lysine with concentration 10 &mu g/mL into the chip.
- Incubate over night.
- Pour laminine with concentration 4 &mu g/mL.
- Incubate for a few hours.
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
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.
Materials
- Molds
- Syringe without needle
- Platinum 24mm x 2 mm strip
- Circular 13mm diameter membrane
Protocol
- 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.
- Demold the chip following our protocol, section 2. Ignore step 2.
- Put membrane and platinum strip on PDMS part 1.
- Refer to our protocol, section 3 to bond PDMS part 2 to the PDMS part prepared in the previous step.
- Apply a small layer of PDMS with the syringe on the contact zone of the PDMS part 2 and the platinum strip.
- 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
- Reproduce the following electric circuit.
- Set function generator on sine, no offset, 4.5 V amplitude.
- 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.