iGEM 2019 — Team NYU_Shanghai

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# Team:NYU Shanghai/Notebook

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Team

[Members](https://2019.igem.org/Team%3ANYU_Shanghai/Team)
[Attributions](https://2019.igem.org/Team%3ANYU_Shanghai/Attributions)

Project

[Description](https://2019.igem.org/Team%3ANYU_Shanghai/Description)
[Design](https://2019.igem.org/Team%3ANYU_Shanghai/Design)
[Notebook](https://2019.igem.org/Team%3ANYU_Shanghai/Notebook)
[Protocol](https://2019.igem.org/Team%3ANYU_Shanghai/protocol)
[Results](https://2019.igem.org/Team%3ANYU_Shanghai/Results)
[Parts Overview](https://2019.igem.org/Team%3ANYU_Shanghai/Parts)

Model

[Piezoelectric Notebook](https://2019.igem.org/Team%3ANYU_Shanghai/Model)

Human Practice

[Integrated HP](https://2019.igem.org/Team%3ANYU_Shanghai/Human_Practices)
[Collaboration](https://2019.igem.org/Team%3ANYU_Shanghai/Collaborations)

Awards

[Medal Criteria](https://2019.igem.org/Team%3ANYU_Shanghai/medals)
[Competition Results](https://2019.igem.org/Team%3ANYU_Shanghai/publication)

[Home](https://2019.igem.org/Team%3ANYU_Shanghai)

[Team Members](https://2019.igem.org/Team%3ANYU_Shanghai/Team)
[Attributions](https://2019.igem.org/Team%3ANYU_Shanghai/Attributions)
[Description](https://2019.igem.org/Team%3ANYU_Shanghai/Description)
[Design](https://2019.igem.org/Team%3ANYU_Shanghai/Design)
[Notebook](https://2019.igem.org/Team%3ANYU_Shanghai/Notebook)
[Protocol](https://2019.igem.org/Team%3ANYU_Shanghai/protocol)
[Results](https://2019.igem.org/Team%3ANYU_Shanghai/Results)
[Piezoelectric Notebook](https://2019.igem.org/Team%3ANYU_Shanghai/Model)
[Parts Overview](https://2019.igem.org/Team%3ANYU_Shanghai/Parts)
[Human Practice](https://2019.igem.org/Team%3ANYU_Shanghai/Human_Practices)
[Collaboration](https://2019.igem.org/Team%3ANYU_Shanghai/Collaborations)
[Medal Criteria](https://2019.igem.org/Team%3ANYU_Shanghai/medals)
[Competition Results](https://2019.igem.org/Team%3ANYU_Shanghai/publication)

Responsive Footer

![](https://static.igem.org/mediawiki/2019/c/c9/T--NYU_Shanghai--NotebookTitle.jpg)
![](https://static.igem.org/mediawiki/2019/7/7f/T--NYU_Shanghai--Notebook--CoverPic.jpg)

Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Week 7
Week 8
Week 9
Week 10
Week 11
Week 12
Week 13
Week 14
Week 15
Week 16
Week 17
Week 18&19

![](https://static.igem.org/mediawiki/2019/3/3f/T--NYU_Shanghai--Notebook-Week1.jpg)

**In the lab this week: Yuma, Jialu, Siyi, Tiger, Sally**

##### Tasks: * Make Detailed Proposals/Protocols * Order Genes (pSoxR/pSoxS) * Meet Lab instructors to discuss the schedule

### [05.29] Wednesday

* Wednesday: 9 AM Meet with Jeffery (Lab Technician)

### [05.30] Thursday

* Thursday: Meet with Xu (Lab director)

![](https://static.igem.org/mediawiki/2019/d/d4/T--NYU_Shanghai--Notebook-Week2.jpg)
**In the lab this week: Yuma, Jialu, Siyi, Tiger**

##### Tasks: * Assembly first

### [06.06] Thursday

![](https://static.igem.org/mediawiki/2019/a/aa/T--NYU_Shanghai--Protocol-Fig1.jpg)

* Weekly Meeting (9am-)
* Washing scales (10am-12pm)
* Made stock solutions: 1L of 1.0M NaCl, 1L of 50mM Tris HCl, 0.5L of 0.5M EDTA, 1L of 0.5M NaOH)

![](https://static.igem.org/mediawiki/2019/e/e6/T--NYU_Shanghai--Notebook-Week3.jpg)
**In the lab this week: Yuma, Tiger, Yifan, Siyi, Sally, Sichen, Jialu**

##### Tasks: * Fabricate Fish scale nanogenerator * Examination

### [06.10] Monday

- FSC treatment (Scale, 50mM HCl Tris, 0.5M EDTA, 0.5M NaOH, 1.0M NaCl, Electric leads, silver paste, PP film): One set (step 4 for 15 min) and another sample (~step 3)
- FSCNG Fabrication with PP film (Treated fish scale, silver paste, PP film: Only one fabrication
- PDMS Fabrication (PDMS elastomer, curing agent, plastic container, hotplate, box knife, cutting mat)
- Make more 0.5M EDTA solution (in pH 8 sol) -> 500 mL

### [06.11] Tuesday

- FSCNG Fabrication with PP film (Treated fish scale, silver paste, PP film, adhesive tape (transparent one)) -> silver on one side
- FSCNG Examination (Fabricated Scale, voltage/current sensor)
- PDMS Fabrication (PDMS elastomer, curing agent, plastic container, hotplate, box knife, cutting mat) -> an oven/dessicator is in Rm 703

### [06.12] Wednesday

- FSCNG Fabrication with PP film (Treated fish scale, silver paste, PP film, adhesive tape (transparent one))
- PDMS Fabrication (PDMS elastomer, curing agent, plastic container, hotplate, box knife, cutting mat)

### [06.13] Thursday

- Weekly meeting @ 9:30 AM
- FSCNG Fabrication with PP film (Treated fish scale, silver paste, PP film, adhesive tape (transparent one)) -> PP film did not work, PDMS is more stable
- FSCNG Examination (Fabricated Scale, voltage/current sensor)
- PDMS Fabrication (PDMS elastomer, curing agent, plastic container, hotplate, box knife, cutting mat)
- FSC treatment with EDTA -> start from 11:30AM for 24 hours sample

### [06.14] Friday

- Pick up Treated fish scales
- PDMS Fabrication (PDMS elastomer, curing agent, plastic container, hotplate, box knife, cutting mat)
- FSCNG Examination (Fabricated Scale, voltage/current sensor)

![](https://static.igem.org/mediawiki/2019/4/43/T--NYU_Shanghai--Notebook-Week4.jpg)
**In the lab this week: Yuma, Sally, Yifan, Sichen, Jialu, Siyi**

##### Task: * FSCNG examination with different EDTA treatment timing * Examination on Fish scale piezoelectricity in the solution

### [06.17] Monday

- FSCNG lamination (FSCNG, PDMS uncured agents)

### [06.18] Tuesday

![](https://static.igem.org/mediawiki/2019/f/f5/T--NYU_Shanghai--06.18.19.jpg)

Voltage exam on FSC with PDMS film -> deltaV = 100mV but skeptical

- FSCNG Examination with Sounds (FSCNG, voltage/current sensor, capstone software, sound source)
- Agar plate preparation (agar, Petri dishes, antibiotics)

### [06.19] Wednesday

![](https://static.igem.org/mediawiki/2019/3/34/T--NYU_Shanghai--06.19Pic.jpg)
 Voltage exam on FSC with PDMS film (fish is treated by EDTA for 24 hrs)
Bending: deltaV = 100mV
Tapping: deltaV = 100mV
Sound: deltaV = 20mV- E.coli. transformation (LB broth, agar plate, plasmid, competent bacterial cell, shaker, water bath, 37C incubator) - Finish at 4pm
- FSCNG fabrication with thin PDMS film (silver paste, FSC, thin PDMS)

### [06.20] Thursday

![](https://static.igem.org/mediawiki/2019/4/41/T--NYU_Shanghai--06.20Pic.jpg)
Voltage exam on FSC with PDMS film -> it was not very clear if the gold ring touches to the silver paste -> maybe Polypropylene film is better?
Tapping: deltaV = 50mV- Weekly meeting @ 9:30 AM
- FSCNG Examination (FSCNG, voltage/current sensor, capstone software, sound source): it was not clear if the gold ring touches the silver paste
- Picking up colonies and inoculation (Agar plates with colonies, LB broth, Culture tubes, shaker)

### [06.21] Friday

![](https://static.igem.org/mediawiki/2019/b/ba/T--NYU_Shanghai--06.21Pic.jpg)
Voltage exam on FSC with PP film (deltaV = 20mV)- FSCNG examination
- DNA purification (Shaked culture tube, miniprep kit, nanodrop)
- (GFP purification)
- (Gel Electrophoresis/SDS-PAGE)

![](https://static.igem.org/mediawiki/2019/c/c4/T--NYU_Shanghai--Notebook-Week5.jpg)
**In the lab this week: Yuma, Jialu, Sichen**

### [06.24] Monday

Voltage exam on FSC with PP film

* FSC with pp lamination:

1. One FSC: test tapping (using weight)/ soundwave (speaker in the physics lab), compare which one has better impact on the piezoelectricity
2. Four FSC (simply piled up): tapping and soundwave, compare with one FSC

### [06.25] Tuesday

* FSC with pp lamination:

1. One FSC: test tapping (using weight)/ soundwave (speaker in the physics lab), compare which one has better impact on the piezoelectricity
2. Four FSC (simply piled up): tapping and soundwave, compare with one FSC

### [06.26] Wednesday

- 5 pm ~: Colonies picking up & inoculation (transformed cell plate, culture tube, LB media, 37C shaker)

### [06.27] Thursday

**Morning:**

Agar Plates Preparation - Amp (100ng/mL), Pyo (2.5uM), Fcn (10uM) with 1% agar plate in 90mm dish.

**5PM:**

Spread 400ul of E. coli. sample to each plate, agar plates voltage application (0.5V) begins for DH5a and BL21 with [BBa\_K2862021](http://parts.igem.org/Part%3ABBa_K2862021%3ADesign) (for 16 hrs)

- Weekly Meeting @ 9:30 AM
- 10:30 am~: Agar plate preparation with electrodes/FSC (agar, autoclave, LB powder, antibiotics (pyocyanin, ferricyanide, ferrocyanide)), power supply, long wires
- 5 pm ~: Agar plate voltage application (potentiostat, transformed cells, positive/negative control agar plate with redox modulators and antibiotics, incubator) -- apply 13 hrs
  \*added 400ul

### [06.28] Friday

![](https://static.igem.org/mediawiki/2019/4/4b/T--NYU_Shanghai--06.28Pic.jpg)
9 AM Results
DH5alpha: both with/without electrodes did not glow so much
BL21: both with/without electrodes did glow very well except around electrodes.- Plate fluorescence level examination (voltage applied plate, fluorescence analyzer)

![](https://static.igem.org/mediawiki/2019/c/cb/T--NYU_Shanghai--Notebook-Week6.jpg)
**In the lab this week: Yuma, Sally, Frank, Jialu, Sichen, Jessica**

##### Tasks: * Further examination on pSox Promoter with electrodes and redox modulators * Design new parts

### [07.01] Monday

![](https://static.igem.org/mediawiki/2019/8/83/T--NYU_Shanghai--07.01Pic.jpg)

**Discussed what has gone right and what has gone wrong**

...- NaSO3 was not added -> add it
- Agar was not covering the graphite electrodes wholly -> dig shallower
- Introduction of voltage might have been too long -> compare 3hrs, 6hrs, and overnight
- Ferrocyanide was too much? -> adding Ferricyanide and compare

- Agar plates preparation
- E. coli. Inoculation for BL21 (5 PM)
Read more

### [07.02] Tuesday

![](https://static.igem.org/mediawiki/2019/e/ee/T--NYU_Shanghai--07.02Pic.jpg)

**Morning:**

Agar plates preparation, two 90mm plates for each set (with/without voltage):- Set1: Ferro/Ferri 2.5mM, Amp (100ng/mL), Pyo (2.5uM), sodium sulfite (0.03%)
- Set2: Ferri 5mM, Same others
- Set3: Ferro 5mM, Same others
\*Plate with + sign is the one with voltage, and + sign indicates the positive potential
**5 PM:**- Inoculate transformed BL21 colonies

- Agar plates preparation (Na2SO3, agar, autoclave, LB)
- E. coli. inoculation (5 PM)

### [07.03] Wednesday

![](https://static.igem.org/mediawiki/2019/e/e3/T--NYU_Shanghai--07.03Pic1.jpg)

- **10 AM:** spread 100ul of BL21 in each dish
- **1 PM:** check samples (3hrs): set1, set2, set3

![](https://static.igem.org/mediawiki/2019/9/9a/T--NYU_Shanghai--07.03Pic2.jpg)

**6 PM:** check samples (6hrs): set1, set2, set3

- Electrodes examination with the different drug (Ferro and ferricyanide) - 3hrs, 6hrs, 20hrs (Thursday morning), 100ul of E.coli cell

1. Set1: Ferricyanide 5mM, pyocyanin 2.5uM, sodium sulfite 0.03%, ampicillin 100ug/ml
2. Set2: Ferrocyanide 5mM, same others
3. Set3: Ferricyanide 2.5mM, Ferrocyanide 2.5mM, same others

### [07.04] Thursday

![](https://static.igem.org/mediawiki/2019/5/5e/T--NYU_Shanghai--Notebook--July4.jpg)

**9 AM: check samples (23hrs, overnight): set1, set2, set3**

Conclusion: They are all glowing at some point, but the dish with Ferri and both are glowing more than the dish with Ferro.

- Weekly meeting @ 9:30 AM
- Inoculation (11 AM)
- Agar plates preparation

1. Set1 & 2: Ferricyanide 10mM, pyocyanin 2.5uM or 0uM, sodium sulfite 0.03%, ampicillin 100ug/ml
2. Set3: Ferrocyanide 10mM pyocyanin 2.5uM, sodium sulfite 0.03%, ampicillin 100ug/ml

- Culture tube preparation

1. Set1 & 2: Set1 & 2: Ferricyanide 10mM, pyocyanin 2.5uM or 0uM, sodium sulfite 0.03%, ampicillin 100ug/ml
2. Set3 & 4: Ferrocyanide 10mM pyocyanin 2.5uM or 0uM, sodium sulfite 0.03%, ampicillin 100ug/ml

- Spread cells to agar plates and culture tube and incubate (agar: 1.0V electrode, culture tube: shaking without electrodes. NOTE: 200ul of a cell to the plates, 100ul to the tubes

### [07.05] Friday

- Checking results (16hrs)
- Team member Photoshoot

![](https://static.igem.org/mediawiki/2019/c/c1/T--NYU_Shanghai--Notebook-Week7.jpg)
**In the lab this week: Jialu, Sichen, Sally, Siyi**

##### Tasks: * Finish fish scale experiment/modeling/data analysis * Schedule

### [07.09] Monday

- Make samples of 1 fish scale / 4 fish scales (pile & spread)
- Determine the magnitude of the force we should apply to fish scale

### [07.10] Tuesday

- Knock the fish scale

### [07.11] Wednesday

- Try the soundwave

### [07.12] Thursday

- Weekly meeting @ 9:30 AM

### [07.13] Friday

- Modeling & data analysis

![](https://static.igem.org/mediawiki/2019/5/57/T--NYU_Shanghai--Notebook-Week8.jpg)
**In the lab this week: Tiger**

### [07.19] Friday

Make agar plates for control (empty pUC57 vector):

* Set1: 5mM Ferri, 2.5uM Pyo, 0.02% Sodium Sulfite, Amp x2
* Set2: 5mM Ferro, 2.5uM Pyo, 0.02% Sodium Sulfite, Amp x2
* Set3: 5mM Ferri, 0.02% Sodium Sulfite, Amp x2
* Set4: 5mM Ferro, 0.02% Sodium Sulfite, Amp x2
* \*2 plates for each for with/without electrodes

* make control plates for comparison to make sure target gene is functioning well (Tiger)
* LB Broth autoclave, pour the plates
* 4 plates: 5mM Ferri+ 5mM Ferro+ Pyo +0.02% Na2SO3+ ampicillin (2 with electrodes, two without )
* 4 plates: same but without Pyo

![](https://static.igem.org/mediawiki/2019/6/6f/T--NYU_Shanghai--Notebook-Week9.jpg)
**In the lab this week: Shaoting, Tiger**

##### Tasks: * PUC57 empty vector arrived. * Transformation of the empty vector into BL21 E.coli * Culture with/without electrodes, compare

### [07.22] Monday

* Morning: Transformation: BL21 + empty pUC57 vector
* 5PM: Culture transformed cells (not the plates with drugs, but the plate with just Amp)

* PUC57 empty vector arrived.
* Transformation of the empty vector into BL21 E.coli
* Culture with/without electrodes, compare

### [07.23] Tuesday

**4:28pm: 4 sets of E.coli**

* Set1: 1ml LB + colony from LB21+ plate + 10μl amp
* Set2: 1ml LB + colony from LB21+ plate + 10μl amp
* Set3: 1ml LB + colony from LB21- plate + 10μl amp
* Set4: 1ml LB + colony from LB21- plate + 10μl amp

* pick up colonies and culture, pour the plates, culture overnight

### [07.24] Wednesday

* compare results

![](https://static.igem.org/mediawiki/2019/b/b8/T--NYU_Shanghai--Notebook-Week10.jpg)
**In the lab this week: Shaoting, Tiger**

##### Tasks: More control groups to compare with week 9 experiments

### [07.29] Monday

* Discussion about the Control experiment

### [07.30] Tuesday

* Prepare for the plate and agar

### [08.01] Thursday

* Weekly meeting @ 9:30 AM

![](https://static.igem.org/mediawiki/2019/7/75/T--NYU_Shanghai--Notebook-Week11.jpg)
**In the lab this week: Shaoting, Tiger**

##### Tasks: electric shock for 1 hr followed by overnight culturing -> plates with parts worked properly

### [08.05] Monday

- Making plate for electric culturing: 100ml in total:(100ml dd water + 2.5g agar + 5mM FeCN(O)+ 5mM FeCN(R) + 100μl amp + 0.02g Na2SO3 + 1.25μl pyo)

### [08.06] Tuesday

![](https://static.igem.org/mediawiki/2019/6/6a/T--NYU_Shanghai--08.06Pic.jpg)

50μl cells + 350μl LB- Culture the cells with electric shock for 1 hr, then culture without electric overnight

### [08.08] Thursday

Talked about human practice: we should do NEW APPLICATION TRACK1. Because our project is focusing on the new application of FSC.
2. Waste management is related to our project because the new application is one of the waste reuse.
3. Additionally, fish scale optimization can be the integrated HP because we will ask supermarkets, local markets, and vendors which fish species is the most wasted and we can determine which one is the most cost-effective one to use in our experiment.

![](https://static.igem.org/mediawiki/2019/0/04/T--NYU_Shanghai--Notebook-Week12.jpg)
**In the lab this week: Tiger**

**Task: Ordering the relay cell and receiver cell from the company: this week is a waiting period for the genes to arrive**

![](https://static.igem.org/mediawiki/2019/5/50/T--NYU_Shanghai--Notebook-Week13.jpg)
**In the lab this week: Tiger**

##### Tasks: * Verify New parts + GFP phenotype after the electric shock (1hr) + Protein purification after the electric shock (1hr) 1. Coomassie-stained SDS-PAGE gel

### [08.29] Thursday

* Meeting
  1. Deadlines: track selection, abstract due
  2. Poster: print in NYC
  3. HP
     + research part… ask experts for modeling of collagen
     + (optional) decoration… ask local markets which fish scale is most cost-effective in terms of our result of our experiment.

![](https://static.igem.org/mediawiki/2019/5/5f/T--NYU_Shanghai--Notebook-Week14.jpg)
**In the lab this week: Tiger**

##### * Transform the relay cell and receiver cell gene from the company * Preparation for co-culture task to see the quorum sensing effect

### [09.03] Tuesday

* Check the result

### [09.05] Thursday

* Prepare the agar plate and the gel
* Doing transformation, apply on the plate, overnight culturing

### [09.06] Friday

![](https://static.igem.org/mediawiki/2019/7/77/T--NYU_Shanghai--Notebook--Sep7.jpg)

Transformation for two parts (relay cell and receiver cell), we successfully get a single colony which means the transformation is a success.

* Check the results, stored at 4 ℃

![](https://static.igem.org/mediawiki/2019/5/5d/T--NYU_Shanghai--Notebook-Week15.jpg)
**In the lab this week: Tiger**

### [09.09] Monday

Conducted liquid culture incubation (inoculation), amplified the transformed cell for future experiment, we have: co-culture tube A, co-culture tube B, relay cell, receiver cell, puc57 empty vector,and empty control, the empty control didn’t have bacteria, which means the incubation is success

### [09.10] Tuesday

Making plate for electric culturing: 200ml in total (200ml dd water + 8g LB-agar + 5mM FeCN(O)+ 5mM FeCN(R) + 100μl amp + 0.02g Na2SO3 + 2.5um pyo)

**Sets we have:**

* co-culture in liquid (+elec+pyo+Fe)
* co-culture in liquid (-elec+pyo+Fe)
* co-culture on plate: (add relay cell and receiver cell separately on one plate)(+elec+pyo+Fe)
* only receiver (cell+elec+pyo+Fe)
* only receiver cell (-elec+pyo+Fe)
* only relay cell (-elec+pyo+Fe)
* Puc57 empty vector (+elec+pyo+Fe)
* Puc57 empty vector (-elec+pyo+Fe)

### [09.12] Thursday

![](https://static.igem.org/mediawiki/2019/c/ca/T--NYU_Shanghai--Notebook--Sep12.jpg)

Results: no single colony - Maybe applied too much liquid bacteria on the plate?

![](https://static.igem.org/mediawiki/2019/c/c3/T--NYU_Shanghai--Notebook--Sep12Pic2.jpg)

Result: no glowing for co-culture: (LEFT is the one with two parts; RIGHT is only imperial college part)

![](https://static.igem.org/mediawiki/2019/f/fa/T--NYU_Shanghai--Notebook--Week16.jpg)
**In the lab this week: Tiger**

##### Tasks: 1. Confirm the transformation is okay * Check whether (only) receiver cell colonies glow * Plasmid purification -> Nanodrop * using microscope to check the GFP 2. Prepare for the protein purification and SDS-PAGE Redo the co-culture part Do the culture without electrode, find the best density of the plasmid solution

### [09.16] Monday

![](https://static.igem.org/mediawiki/2019/d/d0/T--NYU_Shanghai--09.16Pic.jpg)

Culturing the cell under RT for over 72 hours- See the **receiver cell glow(+elec) and see the co-culture one glow(-elec)**, but apply too much on the plate, it’s hard to scrape a single colony.

* Check the result, talk with Wenshu and Lina
* Scrap single colony, do liquid incubation

### [09.17] Tuesday

* Prepare SDS-PAGE agar, Electrophoresis gel
* Using GFP microscope to check the GFP
* Prepare more plate, liquid culture the transformed cell for further experiment
* Using GFP microscope to check the GFP

### [09.18] Wednesday

Set up a lot of plate to see the best concentration for growing single colony
Sets we have: 20X,10X, 5X, 2X receiver cell(dilute to 100ul,-elec + pyo and Fe)
20X,10X, 5X, 2X co-culture cell(co-culture on plate)(dilute to 100ul,-elec + pyo and Fe)

### [09.19] Thursday

![](https://static.igem.org/mediawiki/2019/5/56/T--NYU_Shanghai--09.19Pic.jpg)

**Results from last night:** 20X dilute set is still blurry, can see the GFP only on receiver cell- Set up more diluted concentration gradient sets: 50X 100X 500X 1000X receiver cell (+pyo +Fe -elec) and 50X 100X 200X 300X 500X 1000X co-culture on plate cell(same condition)

* Redo the co-culture part incubate overnight
* Do the Electrophoresis

### [09.20] Friday

![](https://static.igem.org/mediawiki/2019/a/a1/T--NYU_Shanghai--09.20Pic.jpg)

As we can see, 1000X dilute can see some small single colony, but the colony is too small, I have no idea how to scrape one.

* Check the results, to see the best culture time
* Check the Electrophore

![](https://static.igem.org/mediawiki/2019/6/65/T--NYU_Shanghai--Notebook--Week17.jpg)
**In the lab this week: Jenny, Tiger**

##### Tasks: * redo the co-culture part (more diluted, 1000X) + Do the culture without electrode, find the best density of the plasmid solution * Do DNA purification and gel electrophoresis

### [09.23] Monday

![](https://static.igem.org/mediawiki/2019/d/d1/T--NYU_Shanghai--Notebook--09.22.jpg)

* 1 day RT culture and 3 hours of 37C culture
* 500X and 1000X receiver kind of glow, (hard to see from the pic but we saw that using our eyes)
* All the co-culture plate don’t glow(except the single colony on the 500X one )

* Scrap single colony, do liquid incubation
**Question:**

1. Why can the only receiver cell plate glow？ Why the expression changed as the concentration changed?
   * It could be because there was no electricity.
2. Why at the same condition, co-culture plate express worse than the receiver cell? (compare 50X two plates and 100X two plates)
   * without electricity, somehow cells cannot express anything.
3. The outside electric stimuli seem doesn’t work that good, why?
   * It is the complicated process to express GFP from electricity because there are two cells involved and multiple modulators associated.
4. At the very beginning of our experiment, we transformed the pixel gene into BL21 why the transformed cell can glow directly? We didn’t add any outside stimuli.
   * what is the pixel gene?

![](https://static.igem.org/mediawiki/2019/c/c5/T--NYU_Shanghai--Notebook--09.22Pic2.jpg)

We think the expression of the GFP has **some correlation with culturing time and concentration,** we're still working on how to find the best condition.

**What we need to know:**

1. Electric potential enhances the GFP expression of co-culture plate
2. The enhanced expression level of LuxR protein in the receiver cell after the electric potential application
3. Fluorescence level measurement (if possible)

### [09.24] Tuesday

* Prepare Agar, DNA electrophoresis gel
* Prepare more plate, liquid culture the transformed cell four further experiment

### [09.25] Wednesday

* Do the gel electrophoresis

### [09.26] Thursday

* Left: receiver cell (150 ul in total (1000x dilution, 2ul receiver cell in 1998 ul LB))
* Right top: Co-culture, (150 ul in total (75 ul each of 1000x dilution receiver cell and relay cell))
* Right-bottom: relay cell (150 ul in total (1000x dilution, 2ul relay cell in 1998 ul LB))
* Left: positive potential, Right: negative potential

![](https://static.igem.org/mediawiki/2019/0/01/T--NYU_Shanghai--Notebook--09.26.jpg)

**Takeaway:**

* there are two types of colonies in the co-culture plate.
* the receiver cell has huge colonies around the negative side.

* Redo the co-culture part incubate overnight

### [09.27] Friday

* Check the results, to see the best culture time
* Check the Electrophore

![](https://static.igem.org/mediawiki/2019/7/70/T--NYU_Shanghai--Notebook--Week19.jpg)
**In the lab this week: Jenny, Tiger**

##### Tasks 1: * Repeat experiment on Imperial College Part (SoxR)

### Day 1

1. Making four 9-cm plates for electric culturing: 100ml in total in each plate

* 100ml dd water total volume + 3.5g LB agar(35g/L) + 5mM FeCN(O)+ 5mM FeCN(R) + 100μl amp + 0.02g Na2SO3 +/- 1.25μl pyo
* Plate 1: Sample (+electcity, +cell, +pyo)
* Plate 2: Control (-electricity, +cell, +pyo)
* Plate 3: Control2 (-electricity, +cell, -pyo)
* Plate 4: Blank (-electricity, -cell,+pyo)
* \*Make a hole and attach electrode only for Plate 1
* \*Do not add Pyocyanin to plate 3

2. Inoculate transformed E.coli. Overnight (~12 hrs)

1. Scrape single colony and dilute in 2ml LB
2. Put in a shaker (37C, 250rpm)

### Day 2

1. Dilute inoculated cell by 8x (50ul cell + 350 ul LB)
2. Spread 100 ul of the diluted cell to each plate
3. Apply electricity for Plate 1 for one hour (in RT for convenience)
4. Culture overnight without electricity (in 37C culture)

### Day 3

1. Take a picture in the UV system (Picture instruction below)
2. Name files as Date\_Imperial\_Plate#.format (eg 191001\_soxR\_Plate3.jpeg)
3. Upload in the result folder in Google Drive

##### Tasks 1: * Repeat experiment on our part (a combination of relay cell and receiver cell) (NYU)

### Day 1

1. Making four 9-cm plates for electric culturing: 100ml in total in each plate

**Materials**

* 100ml dd water + 2.5g agar + 5mM FeCN(O)+ 5mM FeCN(R) + 100μl amp + 0.02g Na2SO3 + 1.25μl pyo
* Plate 1: A combination (+electcity, +cell)
* Plate 2: Relay cell (+electricity, +cell)
* Plate 3: Receiver cell (+electricity, +cell)
* Plate 4: Blank (-electricity, -cell)
* (Plate 5: Receiver cell (+electricity, 1uM +AHL))
* \*Make a hole and attach electrode for Plate 1, 2, 3
* \*Make Plate 5 if Lab has AHL in stock.

2. Inoculate transformed E.coli. (receiver and relay) Overnight (~12 hrs)
   1. Scrape single colony and dilute in 2ml LB
   2. Put in a shaker (37C, 250rpm)

### Day 2

1. Dilute inoculated cell by 8x (50ul cell + 350 ul LB)
   1. \*For plate 2&3, dilute 50 ul relay/receiver cell + 350 LB and add 100 ul of the cell to each plate
   2. For plate 1:
      * dilute 50 ul relay/receiver cell + 350 LB in the separate tube
      * Spread 50 ul of relay cell in the positive electrode half
      * Spread 50 ul of receiver cell in the negative electrode half
2. Spread 100 ul of the diluted cell to each plate
3. Apply electricity for Plate 1 for one hour (in RT for convenience)
4. Culture overnight without electricity (in 37C culture)

### Day 3

1. Take a picture in the UV system
2. Name files as Date\_NYU\_Plate#.format (eg 191001\_NYU\_Plate3.jpeg)
3. Upload in the result folder in Google Drive

\*Repeat at least 4 times for each trial for the statistical test.

\*Form up two teams for each task

\*Double the number of Plates (8 plates in total) if there are enough electrodes

\*https://www.ncbi.nlm.nih.gov/pmc/articles/PMC92623/ (AHL is 1uM)


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# Team:NYU Shanghai/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:NYU Shanghai/Results

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[Piezoelectric Notebook](https://2019.igem.org/Team%3ANYU_Shanghai/Model)

Human Practice

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[Competition Results](https://2019.igem.org/Team%3ANYU_Shanghai/publication)

Responsive Footer

![](https://static.igem.org/mediawiki/2019/b/b2/T--NYU_Shanghai--ResearchTitle.jpg)

[Fish Scale Piezoelectricity](#FishScalePiez)
[Electric Modulation Setup](#ElectricModulation)
[Cell-to-Cell Communication](#CellToCell)

[References](#References)

![](https://static.igem.org/mediawiki/2019/3/3f/T--NYU_Shanghai--FishScaleCover.jpg)

**Description**

Our team came up with the idea of a decentralized source of electric stimulation instead of plugging or using batteries for tissue engineering. The preliminary research showed that fish-scale is rich in type I collagen which is a biocompatible and biodegradable polymer suitable for building the flexible bio-piezoelectric nanogenerator [1]. This can be taken as temporal electric signals which are to be used for the tissue engineering scaffold. Hence we examine different kinds of movements to see if its induced electric potential can be used for the scaffold.

In this section, we tested induced electric potential with a varying treatment duration of demineralization by ethylenediaminetetraacetic acid (EDTA). The freshwater fish scales are soaked in EDTA for 0h, 2h, 4h, 8h, 16h, and 24h. Either 1 or 4 fish scales are fabricated with polypropylene (PP) film with a voltmeter to record the voltage during tapping rigorously. The average voltage generated by the piezoelectrical effect of fish-scale, Uavg, is calculated by Equation 1.

*U avg = 1⁄2 (| U max - U imax| + | U min - U imin|)* (1)

where Uimax and Uimin is the intrinsic potential without tapping.

**Results**

The results are shown in Table 1 and Figure 1, and the sample response in Fig 2 (8 hours & 4 FSs).

| EDTA [h] | #FSC | Uimax[mV] | Uimin[mV] | Umax[mV] | Umin[mV] | Uavg[mV] |
| --- | --- | --- | --- | --- | --- | --- |
| 0 | 1 | -3.29 | -4.7 | -2.35 | -6.11 | 1.175 |
| 0 | 4 | 1.88 | 0 | 3.29 | -1.41 | 1.41 |
| 2 | 1 | -1.41 | -3.29 | 0 | -5.64 | 1.88 |
| 2 | 4 | 0.94 | -0.94 | 2.82 | -5.71 | 3.325 |
| 4 | 1 | 1.41 | 0 | 3.29 | -1.88 | 1.88 |
| 4 | 4 | 1.41 | -0.47 | 8.46 | -5.64 | 6.11 |
| 8 | 1 | 1.41 | -0.94 | 2.82 | -2.35 | 1.41 |
| 8 | 4 | 0.94 | -0.94 | 6.11 | -3.76 | 3.995 |
| 16 | 1 | 1.88 | -0.47 | 3.29 | -3.29 | 2.115 |
| 16 | 4 | 1.41 | 0 | 7.52 | -6.58 | 6.345 |
| 24 | 1 | 0.94 | -0.94 | 3.29 | -3.76 | 2.585 |
| 24 | 4 | 2.82 | 0 | 62.51 | -44.18 | 51.935 |

Table 1: Recorded voltages of each group of fish-scale

![](https://static.igem.org/mediawiki/2019/4/49/T--NYU_Shanghai--ResultsFig1.jpg)

Fig 1: Voltage-Generated by FSCs with different EDTA treatment duration

![](https://static.igem.org/mediawiki/2019/3/3c/T--NYU_Shanghai--ResultsFig2A.jpg)

Fig 2: 4 Fish Scales with 4 hours EDTA responding to tapping

![](https://static.igem.org/mediawiki/2019/4/40/T--NYU_Shanghai--ResultsFig2.jpg)

Fig 3: Setup for Fish Scale Voltage Measurement

From our experiment, it can be concluded that the piezoelectrical effect of fish-scales
increases as the EDTA treatment time increases and larger voltages can be generated by
putting more fish-scales together. Especially, the batch of fish scales (4 FSC) significantly increased its potential after 24 hours of demineralization.

**Summary**

None of the sets was producing enough electric potential to control the electrochemical modulators used later in our projects (~0.5 V [2]). However, there are several improvements that could be made. First, since we tapped the fish scales by hand, the force of each tapping was varying. Second, sometimes we accidentally tapped on the wires instead of the fish scales, which would generate extremely large voltages. Third, more different types of fish-scales can be used such as seawater fish. For future experiments, we can further investigate more experimental groups with the EDTA treatment time ranging from 16hours to 48 hours. Also, not only tapping but also bending and other possible mechanical stress that could be made for the implanted scaffolds.

![](https://static.igem.org/mediawiki/2019/c/c0/T--NYU_Shanghai--ElectricModCover.jpg)

**Description**

The next step is to establish our own method of electrochemical modulation to prove our tissue engineering model is possible. we use two redox biomolecules: pyocyanin (Pyo) for the initiation of gene induction and ferricyanide (Fcn) for electronic control of the expressions [2]. The existing part, [BBa\_K2862021](http://parts.igem.org/Part%3ABBa_K2862021), is used for our initial step. Agar plates are dug by the carbon electrode that gives +0.5V. (Fig 4). The GFP fluorescence level is determined by pixel intensities after a 1-hour electric shock followed by 24-hour post-culturing.

![](https://static.igem.org/mediawiki/2019/d/da/T--NYU_Shanghai--Results--Fig4.jpg)

Fig 4: Electric Modulation on Agar Plate Setup

**Results**

The sample with electricity and pyocyanin (+E/+Pyo) showed the biggest intensity, and the sample without electricity but still with pyocyanin (-E/+Pyo) showed a modest intensity. This clearly proved that our electric signals enhanced the expression level on the existing part [(BBa\_K2862021)](http://parts.igem.org/Part%3ABBa_K2862021).

![](https://static.igem.org/mediawiki/2019/a/ad/T--NYU_Shanghai--Results--Fig5.jpg)

Fig 5: Results of PixCell Construct

**Summary**

As the result is shown in Fig 5, the electric stimulation properly worked on the existing parts in a much simpler method with a normal power supply than the previous method with an expensive potentiostat. This result characterizes the existing part [(BBa\_K2862021)](http://parts.igem.org/Part%3ABBa_K2862021) and improves it since we introduced a much simpler method of electric activation and created the reporter gene of more complicated pathways regulated by electric stimulation, which serves as one of our gold medal criteria.

![](https://static.igem.org/mediawiki/2019/2/26/T--NYU_Shanghai--CellToCellCover.jpg)

**Description**

Finally, we exhibited something novel by using the electrochemical control of genes and proved that complex signaling is possible by our piezoelectric scaffolding for future tissue engineering. We used Quorum-Sensing genes as our model (fig 6) and tested whether electric stimulation can flow down to the GFP expression. Two parts (Relay cell and Receiver cell) are separately transformed in *E. coli.* and mixed on the agar plate to cultured under electric stimulation. Similar to Part 2, the GFP fluorescence level is determined by pixel intensities after a 1-hour electric shock followed by 24-hour post-culturing. There are three sets to show our model in Fig 5: Co-culture of both cells, only relay cell, and only receiver cell.

![](https://static.igem.org/mediawiki/2019/0/0b/T--NYU_Shanghai--Results--Fig6GREEN.jpg)

Fig 6: Quorum Sensing by Electric Stimulation

**Results**

The quantitative result shows the straightforward outcome (Fig 7): only when both Relay and Receiver cell exists (Co-culture), GFP expression is enhanced. Only the Receiver cell has lightly more expression of GFP than only Relay cell. This should be due to the intrinsic LuxR proteins that facilitate the GFP expression only by its own plasmid. Qualitatively, we also observed the spatial expressing patterns of quorum sensing as most of the GFP expressing cells are located as if the glowing colonies are surrounding the positive electrode on the left side (Fig 8). This is because AHL molecules diffused from the Relay cells around the positive electrode and Receiver cells responded to the gradient of Quorum-sensing inducers.

![](https://static.igem.org/mediawiki/2019/8/8b/T--NYU_Shanghai--Results--Fig7.jpg)

Fig 7: Results of Quorum Sensing Cells

![](https://static.igem.org/mediawiki/2019/9/92/T--NYU_Shanghai--ResultsFig8.jpg)

Co-culture Plate under GFP (the left hole is positive anode; the right hole is the negative cathode)

**Summary**

Overall, our parts are proved to be working under the electric stimuli as it is showing the GFP expression with the spatial properties of quorum sensing. The quantitative analysis by pixel intensities is conducted very carefully (using the exact same spot for every picture) to minimize the other factors. Therefore, we combined with qualitative analysis of a single plate to reinforce our results.

**References**

[1] Ghosh, S. K. & Mandal, D. High-performance bio-piezoelectric nanogenerator made with fish scale. Appl. Phys. Lett. 109, 103701 (2016).

[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.](https://www.nature.com/articles/ncomms14030)Nat Commun, 8 (2017), p. 14030