Team:NYU Shanghai/protocol
Home
Team Members
Attributions
Description
Design
Notebook
Protocol
Results
Piezoelectric Notebook
Parts Overview
Human Practice
Collaboration
Medal Criteria
Competition Results
Description
The proper treatment is very important to extract the best out of the fish scale’s piezoelectric properties. Since the primary paper did not mention the exact time of treatment, we vary the length of demineralization. The moderate size of fish scale (approximately 20mm by 20mm by width) is chosen.
The proper treatment is very important to extract the best out of the fish scale’s piezoelectric properties. Since the primary paper did not mention the exact time of treatment, we vary the length of demineralization. The moderate size of fish scale (approximately 20mm by 20mm by width) is chosen.
Materials/Equipment
Fig: Freshwater Fish from the Local Market (Ctenopharyngodon idellus)
- Fish scale (Grass fish from the local supermarket)
- 20 mM EDTA
- NaOH solution
- deionized water
- NaCl solution
- 0.05M Tris HCl
- Beaker
- Glass dish
Procedures
- Scrape scales and wash by tap water to remove the sticky liquid.
- Wash FS thoroughly with deionized water
- Wash FS with 0.1M NaOH
- Wash FS with a mixture of 1.0 M NaCl, 0.05 M Tris HCl.
- Demineralize FS by immersing under 0.5 M EDTA with stirring.
- We will have 6 sets of immersion durations:
- 0 min (no treatment), 2 hours, 4 hours, 8 hours, 16 hours, 24 hours
Description
For the lamination, we will use demineralized FS, Polypropylene (PP) sheet, and silver paste. For the experimental setting, we used either 1 or 4 fish scales to fabricate one nanogenerator.
For the lamination, we will use demineralized FS, Polypropylene (PP) sheet, and silver paste. For the experimental setting, we used either 1 or 4 fish scales to fabricate one nanogenerator.
Materials
- Demineralized Fish Scales
- Silver paste
- PP film
- Tweezers
Procedures
Fig: Fabrication of Fish Scales with Silver and PP film
- The electrodes are made by painting the silver paste on either side of FS.
- *Silver paste do not over the whole FS to make leading two electrodes.
- Encapsulate 1 or 4 FS with an electrode between two sheets of PP film (80mm by 40mm).
- *The silver paste is elongated to the other side of the nanogenerator to attach leads.
- *Since this FSC nanogenerator (FSCNG) is reversible to produce electricity, we do not distinguish two sides.
- In total, 6 sets of immersion durations * 1 or 4 fish scales = 12 nanogenerators.
Procedures
The Fish-Scale nanogenerator made in the previous steps is now to be examined. The elongated silver paste is attached to the two different electrically conductive leads and connected to the voltmeter. At this stage, we examine the voltage with the voltmeter. Plug the nanogenerators to the voltmeter and place them on the table. Knock those nanogenerators frequently for 10-20 seconds while recording for each nanogenerator.
The Fish-Scale nanogenerator made in the previous steps is now to be examined. The elongated silver paste is attached to the two different electrically conductive leads and connected to the voltmeter. At this stage, we examine the voltage with the voltmeter. Plug the nanogenerators to the voltmeter and place them on the table. Knock those nanogenerators frequently for 10-20 seconds while recording for each nanogenerator.
Materials
- Fabricated Fish Scale nanogenerator
- Voltmeter
- PASCO Capstone Software for recording
Since this part requires 4 days of experiments, each task indicates when to be done (Day 1, Day 2, Day3, and Day 4). The overview of the experimental tasks is shown in Table 1.
Table 1: Overall Schedule of Part 2
| Day | Tasks |
|---|---|
| Day 1 |
|
| Day 2 |
|
| Day 3 |
|
| Day 4 |
|
Description
We order the genes from the company (GenScript China). The plasmid backbones need to produce a high copy and have antibiotic resistance so pUC57 (pMB1 origin and AmpR) plasmid is adopted. We have ordered three parts: the PixCell construct with deg tag (BBa_K2862021, Imperial College 2018), the Relay part (BBa_K3267000) and the Receiver part (BBa_K3267001). Please look at Parts Overviewfor detailed design.
Fig: Schematic Overview of the Relay part (BBa_K3267000) and the Receiver part (BBa_K3267001)
We order the genes from the company (GenScript China). The plasmid backbones need to produce a high copy and have antibiotic resistance so pUC57 (pMB1 origin and AmpR) plasmid is adopted. We have ordered three parts: the PixCell construct with deg tag (BBa_K2862021, Imperial College 2018), the Relay part (BBa_K3267000) and the Receiver part (BBa_K3267001). Please look at Parts Overviewfor detailed design.
Description
We used the BL21 strain for efficient GFP expressions and conducted a heat-shock method for all the transformation throughout our project.
We used the BL21 strain for efficient GFP expressions and conducted a heat-shock method for all the transformation throughout our project.
Materials
- BL21 strain E. coli.
- Pre-constructed Plasmid (BBa_K2862021)
- LB Nutrient Broth
- Water bath
- 37˚C incubator
Procedures
- Prepare competent BL21 bacteria in 1 vial of 50μL and place it on the ice to let it thaw.
- Using a clean pipe each time, add the constructed plasmid DNA solution 1-5 μL to the corresponding tubes. Gently flicking tubes to mix.
- Place them on ice and incubate the tubes for 30mins. While waiting for the incubation, prepare the LB agar plate at RT.
- Conduct heat shock, using foam rack as a holder and put all the tubes into the water bath at 42℃ for 45 sec.
- When heat shock is done, place both tubes back on ice incubate for 2 mins.
- Add 1ml OD LB nutrient broth to the tube. Incubate 37°C 1h with 250rpm shaking
- Flick the closed tubes to mix and resuspend the bacteria well, using a sterile pipet for each tube, pipet 100 μl of the solution onto the agar plates. (for 60mm plate)
- Stack up the plates and tape them together, put group name on the bottom of the stack and place it upside down in 37℃ incubator for 12~16 hours.
Description
The agar plates have to contain some unique modulators in order to convert the electric signal to gene expression. Pyocyanin (Pyo), which is one of the toxins produced by Pseudomonas aeruginosa, is a redox-active molecule to initiate the intracellular redox cycle of artificial parts. Ferricyanide and Ferrocyanide respectively serve as an oxidated and reduced form of the redox-cycling molecules. ([Fe(CN)6]4− ⇌ [Fe(CN)6]3− + e− ) In addition, Sodium sulfite is added to minimize the effect of aerobic oxygen on redox reactions by creating an anaerobic environment in the plates. The experimental sets are assigned as follows (+ indicates the presence and - indicates the absence):
The agar plates have to contain some unique modulators in order to convert the electric signal to gene expression. Pyocyanin (Pyo), which is one of the toxins produced by Pseudomonas aeruginosa, is a redox-active molecule to initiate the intracellular redox cycle of artificial parts. Ferricyanide and Ferrocyanide respectively serve as an oxidated and reduced form of the redox-cycling molecules. ([Fe(CN)6]4− ⇌ [Fe(CN)6]3− + e− ) In addition, Sodium sulfite is added to minimize the effect of aerobic oxygen on redox reactions by creating an anaerobic environment in the plates. The experimental sets are assigned as follows (+ indicates the presence and - indicates the absence):
| Plate # | Name | Plate Profile |
|---|---|---|
| Plate 1 | Sample | +Electcity, +Cell, +Pyo |
| Plate 2 | Control 1 | -Electcity, +Cell, +Pyo |
| Plate 3 | Control 2 | -Electcity, +Cell, -Pyo |
| Plate 4 | Blank | -Electcity, -Cell, +Pyo |
Description
- Pyocyanin
- Ferricyanide/Ferrocyanide
- Sodium sulfite
- 1% Molten Agar
- Ampicillin
- Carbon Graphite Electrodes
- Glue gun
- 90mm plastic plates
- Autoclaved LB broth
Procedures
Fig: Preparing Agar with Necessary Chemicals
- Two 5mm holes are drilled into 9cm agar plates.
- Two electrodes are inserted into the plate penetrating 2mm and glued to the plate.
- *Make sure which electrode is a positive anode or negative cathode.
- 200ml of molten 1% agar is prepared in 4 flasks. Assigned drugs from 5mM Ferrocyanide(R)/5mM Ferricyanide (O), 2.5 uM Pyocyanin, 0.02% Sodium Sulfite, and Ampicillin (100ug/ml) are pipetted into the corresponding flasks.
- *Na2SO3 is necessary to maintain anaerobic condition (to avoid oxidation from the air but limit oxidation from the electrodes)
- After mixing, 100ml of the solution was pipetted onto the 90mm agar plate.
Materials
- Plates with transformed E. coli.
- 37˚C Shaker
- Incubator
- Inoculation tube
- LB broth
Procedures
- Add the needed amount of LB medium into the 15ml conical tube and add corresponding antibiotics (Amp: 100ug/ml).
- Prepare culture tubes for the samples and negative control (no bacteria will be added) with 2ml of LB broth each.
- Picking up colonies from the plate by autoclaved pipet tips and add in the culture tubes by shaking tips.
- Incubate in the 37˚C with 250rpm shaking for 12 hours. For longer inoculation, decrease the shaking speed (~200rpm)
Description
Finally, transformed cell and agar plates with drugs are put together to examine the cellular response to the electric stimulation. This is a very DIY-level set up so that every connection of wires and the placement of dishes are necessary to be checked before proceeding the experiment. The figure below is the picture of the experiment one day checking the voltage difference between anode and cathode. It needs to be ~0.5V to have the redox reactions on the plate [2]. Once the set up is confirmed, 1 hour of electric potential is applied under RT, and plates are placed to the 37˚C incubator overnight. On the next day, take a picture of plates under UV light (302 nm).
Fig: Making Sure that the Electric Setup has 0.5 V.
Finally, transformed cell and agar plates with drugs are put together to examine the cellular response to the electric stimulation. This is a very DIY-level set up so that every connection of wires and the placement of dishes are necessary to be checked before proceeding the experiment. The figure below is the picture of the experiment one day checking the voltage difference between anode and cathode. It needs to be ~0.5V to have the redox reactions on the plate [2]. Once the set up is confirmed, 1 hour of electric potential is applied under RT, and plates are placed to the 37˚C incubator overnight. On the next day, take a picture of plates under UV light (302 nm).
Materials
- The inoculated E. coli. with the PixCell Construct (BBa_K2862021)
- Agar Plates (Plate 1-4)r
- Power Supply/Resistor
- Electric wires with alligator clips
- Incubator
- Voltmeter
- LB broth
- Spreader
- Alcohol ramp
- Microtube
Procedures
- Dilute Inoculated cell by 8 times: take 50 ul and pipet into 350 ul of LB medium
- Spread 100ul of transformed cells containing the PixCell construct on the corresponding agar plate.
- Clip wires on the carbon electrodes from agar plates and make a parallel circuit via the power supply.
- Adjust the knob of the power supply to have 0.5V differences between two electrodes from the same plate. Make sure all the plates has the same potential by using a voltmeter.
- Apply the electric potential for 1 hour at RT, and put all the plates in the 37˚C incubator for overnight.
- On the next day, take a picture under UV light (Wavelength: 302 nm, Exposure: 50 ms)
- *Take a picture of one plate per photo at the exact same spot for accurate intensity.
- Quantify GFP expression level by the pixel intensities of fluorescent light.
Description
This part is almost the same as Part 2: 4 agar plates with different chemicals and transformed E. coli. In contrast to Part 2, all the plates have pyocyanin, but other steps are quite the same. The set up for plates is:
For the Co-culture plate (Plate 1), you should dilute inoculated cells by 8 times and spread 50 ul of each to the plate.
This part is almost the same as Part 2: 4 agar plates with different chemicals and transformed E. coli. In contrast to Part 2, all the plates have pyocyanin, but other steps are quite the same. The set up for plates is:
| Plate # | Name | Plate Profile |
|---|---|---|
| Plate 1 | Co-culture | +Electricity, +Relay Cell, +Receiver Cell |
| Plate 2 | Relay Cell | +Electricity, +Relay Cell |
| Plate 3 | Receiver Cell | +Electricity, +Receiver Cell |
| Plate 4 | Blank | -Electricity, -Cell |
Description
- Two kinds of the transformed BL21 with the Relay part (BBa_K3267000) and the Receiver part (BBa_K3267001)
- Agar plates with electrochemical modulators (Pyo, Ferri/Ferrocyanide, Sodium sulfite) (Plate 1-4)
- Ampicillin
- Electric wires with alligator clips
- Incubator
- Voltmeter
- LB broth
- Spreader
- Alcohol ramp
- Microtube
References
[1] Hall S, McDermott C, Anoopkumar-Dukie S, McFarland AJ, Forbes A, Perkins AV, Davey AK, Chess-Williams R, Kiefel MJ, Arora D, Grant GD (2016) Cellular effects of pyocyanin, a secreted virulence factor of Pseudomonas aeruginosa . Toxins 8:236. https://doi.org/10.3390/toxins8080236
[2] T. Tschirhart, E. Kim, R. McKay, H. Ueda, H.C. Wu, A.E. Pottash, A. Zargar, A. Negrete, J.Shiloach, G.F. Payne, W.E. Bentley. Electronic control of gene expression and cell behaviour in Escherichia coli through redox signalling. Nat Commun, 8 (2017), p. 14030
[1] Hall S, McDermott C, Anoopkumar-Dukie S, McFarland AJ, Forbes A, Perkins AV, Davey AK, Chess-Williams R, Kiefel MJ, Arora D, Grant GD (2016) Cellular effects of pyocyanin, a secreted virulence factor of Pseudomonas aeruginosa . Toxins 8:236. https://doi.org/10.3390/toxins8080236
[2] T. Tschirhart, E. Kim, R. McKay, H. Ueda, H.C. Wu, A.E. Pottash, A. Zargar, A. Negrete, J.Shiloach, G.F. Payne, W.E. Bentley. Electronic control of gene expression and cell behaviour in Escherichia coli through redox signalling. Nat Commun, 8 (2017), p. 14030