iGEM 2017 — Team NYU_Shanghai

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# Team:NYU Shanghai/Notebook

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iGEM NYU Shanghai 2017 Lab Schedule Notebook

|  |  |  |
| --- | --- | --- |
| Date/Time | What we plan | Summary of what we did |
| Week 1: 4/24-26/2017 | | |
| Monday 4/24/2017  4-6pm | * Inoculate E.coli | * Gathered items needed to be autoclaved |
| Tuesday 4/25/2017  12:30-1:00  3:00-5:30 | * Prepare LB broth * Autoclaved LB broth, pipet tips, and microtubes. * E.coli Inoculation: Prepare liquid culture with LB nutrient broth and E.coli (previously stored in -80C). Inoculate overnight for 12 hours at 37 degrees with shaking. * Spectrophotometer testing with dead E.coli | * Prepared 400mL LB broth * Autoclaved LB broth, pipet tips, and microtubes. * E.coli Inoculation: Prepare liquid culture with LB nutrient broth and E.coli (previously stored in -80C). Inoculate overnight for 12 hours at 37 degrees with shaking. * Spectrophotometer testing with dead E.coli to try to find a correlation between cell concentration and OD600 value (could not find a correlation) |
| Wednesday 4/26/2017  10am-5pm | * Prepare different concentration samples of methanol/ethanol. 10% intervals starting from 10% to 60% * Place E.coli in different samples and perform Spectrophotometer testing to detect the initial and final concentration of E.coli every half an hour | * Prepared 800mL LB broth * Prepared LB agar plates and LB/ampicillin agar plates * Inoculate E.coli * 20% ethanol survival test for 2 hours (15 min intervals for the first hour and 30 min intervals for the second hour) * Correlation test for Bacterial Concentration and OD600- used alive E.coli (found no correlation again) |
| Week 2: 5/31/2017-6/2/2017 | | |
| Wednesday  5/31/2017  2:00-4:00pm | Plan:   * Inoculate E.coli * Spread glycerol stock E.coli on agar plates   Equipment: Sterile hood, spreader, shaker  Reagents: LB broth, agar plates | * Inoculated 6 tubes of E.coli. Started shaking incubator at 3:30pm. * Revived glycerol stock by plating it onto 4 agar plates. Incubate overnight at 37C. |
| Thursday  6/1/2017  1:00-5:00pm | Plan:   * Spread inoculated E.coli on agar plates. * If there is enough E.coli, conduct survival test in ethanol concentration at 10%, 20%, 30%, and 40%.   Equipment: Sterile Hood, spreader, spectrophotometer  Reagents: Ethanol, agar plates, LB broth | * Turn off shaking incubator and placed the tubes in 4C at 10:30am. * 5 E.coli survival trials at 30% and 40% ethanol concentration (15 min intervals for the first hour and 30 min interval for the remaining two hours). * Inoculated 4 tubes of E.coli. Start shaking incubator at 5pm |
| Friday  6/2/2017  9am-1pm | Plan:   * E.coli Survival test at 10% and 20% ethanol concentration. * Try to find the maximum time at the optimal concentration.   Equipment: Sterile Hood, spreader, spectrophotometer  Reagents: Ethanol, LB broth, agar plates | * (2) 5 E.coli survival trials at 10% ethanol concentration. * First method: using ethanol as blank, get rid of LB broth * Second method: made alcohol solution using cell culture LB broth rather than water * Tested for 2 hours. 15 min intervals for the first hour and 30 min intervals for the second hour. * Learned the procedures for trypan blue staining * Competent DH5a E.coli cells arrived today |
| Week 5: 6/5-9/2017 | | |
| Tuesday  6/6/2017  1:00-5:00pm | Plan:   * Inoculate E.coli * Dead E.coli trypan blue staining under microscope   Equipment: Sterile Hood, spreader, microscope, counting grid  Reagents: LB broth, agar plates, trypan blue dye | * Begin development of ethanol/methanol resistant bacteria: Inoculate E. coli in 1% ethanol or methanol |
| Wednesday  6/7/17  9am-5pm | Plan:   * E.coli Survival test at 10% and 5% ethanol concentration. * Inoculate 4 tubes of E.coli   Equipment: Sterile Hood, spreader, shaking incubator, microscope, counting grid  Reagents: Ethanol, agar plates, LB broth, trypan blue dye | * Trypan blue staining  * Could not distinguish E.coli cells from contaminants on the grid  * Ethanol and methanol resistant bacteria:  * Spread plates for 1% resistance; inoculate 2% resistance from the previous 1% inoculation * Spread plates for 2% resistance; inoculate 3% resistance from the previous 2%  * Ordered fluorescence dye |
| Thursday  6/8/17  9am-5pm | Plan:   * Inoculate 1 colony each from the 1% ethanol/methanol plates * Spread plates for 3% resistance; inoculate 4% resistance from the previous 3% * If dye arrives, practice fluorescence microscope   Equipment: Sterile Hood, spreader, shaking incubator, microscope, counting grid  Reagents: Ethanol, agar plates, LB broth, trypan blue dye | * Tried trypan blue staining using new microscope from genetics lab. Was not able to see the E.coli or the counting grids. Abandon using this technique. * Inoculated E.coli in 4% methanol/ethanol solution for 6 hours. Spread onto agar plates to incubate. * Spread 3% E.coli on agar plates |
| Week 6: 6/19-23/2017 | | |
| Monday  6/19/17  4-5pm | Plan:   * Inoculate E.coli | * Inoculated 2 tubes of regular LB broth |
| Tuesday  6/20/17  9-5pm    Amy and Agnes | Plan:   * Prepare E.coli solution * Prepare working solution for the dyes * Learn to use the fluorescent microscope from Professor Wenshu. Perform initial testing together. * Survival test 5% and 10% using the fluorescent microscope * Inoculate E.coli | * Test different concentration of E.coli and dye under fluorescent microscope using the hoechst and propidium iodide dyes. * Gene arrived. Stored in -20 * Inoculated 3 tubes of E.coli |
| Wednesday  6/21/17  9-5pm    Amy, Agnes, Julie, Simba | Plan:   * Survival test 5% and 10% using the fluorescent microscope * Inoculate E.coli   Gene arrives:   * Prepare plasmid * Transformation * Grow overnight on agar plates | * Tested out fluorescence microscope, varying the variables of dye amount and inoculation dilution * Cancelled plans for Gas Chromatography, initiated further research for methanol concentration testing |
| Thursday  6/22/17  9-5pm | * Survival test 5% and 10% using the fluorescent microscope * Calibrating the OD600 measurement for DH5a.  * Inoculate one colony of transformed E.coli | * Tested out fluorescence microscope, varying the variables of dye amount and inoculation dilution * Postponed OD600 calibration, continued Methanol Concentration testing research |
| Week 7: 6/26-30/2017 | | |
| Monday  6/26/17  11am-5pm  Agnes and Eric | Plan:   * Autoclave and pour agar plates * Inoculate E.coli   Materials and Instruments:  Sterile hood, autoclave | * Poured 40 10 mm agar plates and inoculated 3 tubes of E. coli |
| Tuesday  6/27/17  9-5pm  Agnes, Amy, Eric | * Fluorescence microscopy: E. coli ethanol survival testing   Materials and Instruments:  Sterile hood, fluorescence microscope | * Final Day of E.coli viability testing, increased concentration of dyes and found improved results * Inoculated 2 tubes of E. coli |
| Wednesday  6/28/17  9-5pm  Agnes, Amy | * Fluorescence microscopy: E. coli ethanol survival testing   Materials and Instruments:  Sterile hood, fluorescence microscope | * Methanol survival testing: at 20 minutes of varying levels of methanol concentration (10%, 20%, 30%, 40%), we looked at the effect on fluorescence in our E. coli samples * Began methanol clonogenic assay experiment, plated E. coli exposed to varying levels of methanol for 20 minutes (0%, 5%, 10%, 15%, 20%), at 10x dilution |
| Thursday  6/29/17  9-5pm | * Fluorescence microscopy: E. coli ethanol survival testing * Methanol clonogenic assay   Materials and Instruments:  Sterile hood, fluorescence microscope | * Began OD600 Calibration, plated dilutions of 200x, 400x, 500x, 800x, and 1000x * Methanol survival testing continued, varied concentration of dye and exposure to 20% methanol compared to the control |
| Friday  6/30/17  9-5pm | * Methanol clonogenic assay * Fluorescence microscopy: E. coli ethanol survival testing   Materials and Instruments:  fluorescence microscope | * Visualized the results of previous day’s plating and updated lab reports |
| Week 8: 7/3-7/2017 | | |
| Monday  7/3/2017  4-5pm  Agnes and Eric | Plan:   * Inoculate E.coli   Materials and Instruments:   * Sterile room | * Inoculated  3 tubes |
| Tuesday  7/4/17  10-5pm    Julie, Agnes | Plan:   * Calibrating OD 600 value * Inoculate E.coli   Materials and Instruments:   * Sterile room | * Calibration: Plated x200 * Inoculate 2 tubes |
| Wednesday  7/5/17  10-5pm    Julie, Simba, Agnes | Plan:   * Fluorescence Microscope testing * Inoculate E.coli   Materials and Instruments:   * Sterile room * Fluorescence Microscope | * Calibration: Plated x200 and x500 * Inoculate 2 tubes * Fluorescence microscope: varied time, switched water for PBS |
| Thursday  7/6/17  10-5pm    Julie, Agnes | Plan:   * Methanol clonogenic assay * Inoculate E.coli   Materials and Instruments:   * Sterile room * Fluorescence Microscope | * Calibration: Plated x2000 |
| Friday  7/7/17  10-11am | Plan:   * Check on agar plates | * Checked on agar plates |
| Week 9: 7/10-14/2017 Note: Methanol concentration testing will proceed if all chemicals arrive by Tuesday 7/11;  Update: Chemicals have not arrived by Tuesday 7/11 | | |
| Monday  7/10/2017  (10)4-5pm    Agnes, Eric | Plan:   * Inoculate E.coli * (Prepare Magenta Sulfite Colorimetric solutions)   Materials and Instruments:   * Sterile room | * Inoculate 2 tubes E. coli |
| Tuesday  7/11/17  10-5pm | Plan:   * Fluorescence testing * OD600 Calibration * Inoculate E.coli   Materials and Instruments:   * Sterile room * Fluorescence Microscope | * Fluorescence: Varied exposure time (1 min, 5 min, 10 min) to methanol at a concentration of 15% methanol; controls: no exposure, 50% methanol at 10 minutes * OD600 Calibration |
| Wednesday  7/12/17  10-5pm | Plan:   * Pour ampicillin plates * Inoculate E.coli   Materials and Instruments:   * Sterile room | * Methanol Clonogenic assay using 5% and 15% methanol at different incubation times (5, 10, 15, 20) |
| Thursday  7/13/17  10-5pm | Plan:   * Methanol clonogenic assay * Inoculate E.coli   Materials and Instruments:   * Sterile room | * Methanol clonogenic assay with 15% and 10% methanol at different time intervals (5, 10, 15, 20) |
| Friday  7/14/17  10-5pm | Plan:   * Check on agar plates | * Checked on agar plates and took picture |
| Week 10: 7/17-21/2017 Note: Magenta Sulfite Colorimetric testing will proceed if all chemicals arrive by Tue 7/18 | | |
| Monday  7/17/2017  3-5pm    Agnes, Eric | Plan:   * Inoculate E.coli * Spread new E.coli on LB and LB/amp  * 50+500, 10/25/50/75/100   Materials and Instruments:   * Sterile room | * Inoculated E.coli * Plated competent E.coli on agar plates to find suitable plating amount. * Plated normal E.coli on LB/amp plates to check if the antibiotic works |
| Tuesday  7/18/17  10-5pm | Plan:   * Transformation * Methanol clonogenic assay * Inoculate E.coli   Materials and Instruments:   * Sterile room | * Methanol clonogenic assay with different concentrations of methanol 20%-50% |
| Wednesday  7/19/17  10-5pm | Plan:   * Prepare Magenta Sulfite Colorimetric solutions (must be autoclaved) * Methanol clonogenic assay * Inoculate E.coli   Materials and Instruments:   * Sterile room, Autoclave | * Clonogenic assay with different concentrations of ethanol. 10%-40% * Filming |
| Thursday  7/20/17  10-5pm | Plan:   * Magenta Sulfite Colorimetric testing * Inoculate E.coli   Materials and Instruments:   * Sterile room | * Filming * Fluorescent Microscopy with 30% ethanol and 50% methanol |
| Friday  7/21/17  10-115pm | Note: Due to the fact that the chemicals for the Magenta Sulfite Colorimetric testing will be arriving today, we will only go to lab in the morning | * Took the plates out of the incubator. Took pictures * Filmed scenes for video * Chemicals arrived! |
| Week 11: 7/24-28/2017 | | |
| Monday  7/24/2017  1:30-5pm    Agnes, Eric | Plan:   * Inoculate E.coli * Transformation   Materials and Instruments:   * Sterile room | * Inoculated 5 tubes of transformed E.coli * Prepared chemical solutions |
| Tuesday  7/25/17  1-5pm | Plan:   * Magenta Sulfite Colorimetric testing * Inoculate E.coli   Materials and Instruments:   * Sterile room, Microplate reader | * Prepared chemical solutions * Inoculated 2 tubes of transformed E.coli * Reviewed Interlab protocols. Just found out that our school’s plate reader cannot measure fluorescence |
| Wednesday  7/26/17  10-5pm | Plan:   * Magenta Sulfite Colorimetric testing * Methanol clonogenic assay * Inoculate E.coli   Materials and Instruments:   * Sterile room | * Magenta Sulfite Colorimetric |
| Thursday  7/27/17  10-5pm | Plan:   * Magenta Sulfite Colorimetric testing * Inoculate E.coli   Materials and Instruments:   * Sterile room | * Magenta Sulfite Colorimetric |
| Week 11: 7/31-8/4/2017 We will NOT be using the lab this week. Lab work will be moved to Fudan University in order to use their microplate reader for the Interlab. Lab work will continue the following week. | | |
| Week 11: 8/7-8/11/2017 We will not be using the lab this week. Lab work will be moved to Fudan University in order to use their microplate reader for the Interlab. Lab work will continue the following week. | | |
| Week 11: 8/14-18/2017 | | |
| Monday 8/14/20171:30-5pm | Plan:   * Inoculate E.coli * Transformation   Materials and Instruments:   * Sterile room |  |
| Tuesday 8/15/1710-5pm | Plan:   * Magenta Sulfite Colorimetric testing * Inoculate E.coli   Materials and Instruments:   * Sterile room | Same as planed |
| Wednesday8/16/1710-5pm | Plan:   * Magenta Sulfite Colorimetric testing * Inoculate E.coli   Materials and Instruments:   * Sterile room | Same as planned |
| Thursday 8/17/1710-5pm | Plan:   * Magenta Sulfite Colorimetric testing   Materials and Instruments: | Same as planned |

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# Team:NYU Shanghai/Experiments

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* [Judging Form](https://igem.org/2017_Judging_Form?team=NYU_Shanghai)

# Protocols

## Double Staining Fluorescence Microscopy:

Procedure:
Remove 1mL of overnight bacterial suspension into a microcentrifuge tube
Centrifuge at 12,000rpm for 3 minutes
Remove supernatant
Ethanol/Methanol treatment for 30 mins
Centrifuge at 12,000rpm for 3 minutes
Remove ethanol/methanol solution
Resuspense E. coli with PBS (Dilute as much as necessary)
Use 200uL of the diluted E. coli suspension
Add 100uL of Hoechst dye (working concentration: 5ug/mL)
Add 100uL of PI dye (working concentration: 5ug/mL)
Incubate at room temperature under dark conditions for 15-20 minutes.
Clean microscope slides and coverslip and label
Pipett 10uL of bacterial/staining dye solution onto a microscope slide and cover
Observe under brightfield, DAPI, and Texas Red channel

## Clonogenic Assay:

Procedure:
Take the OD600 value of the overnight culture
Take 1mL of the overnight culture and place it into a microcentrifuge tube
Centrifuge at 12,000rpm for 5 mins
Remove the supernatant
Add different concentrations of methanol/ethanol for a certain period of time
Centrifuge at 12,000rpm for 3 mins
Remove the methanol/ethanol supernatant
Dilute 5000x -10000x with LB
Plate the diluted, treated sample onto agar plates
Incubate the plates at 37°C at 220 rpm for 16-18 hours

## Magenta Sulfite Colorimetric:

Procedure:

Preparation of solutions
1. potassium permanganate-phosphoric acid solution
Weigh 15g of potassium permanganate and add into 75 ml 85% phosphoric acid and 350ml of water. After the potassium permanganate has dissolved, control the volume to 500ml. Store the solution in brown bottle.
2.oxalic acid-sulfuric acid
weigh 5g of oxalic acid and dissolve in 1:1 cold sulfuric acid and add water until the 100 ml mark, mix well and store in brown bottle.
3. aniline red- sulfinic acid
weigh 0.1g of aniline red and dissolve with 60ml 80 degree C water. After cooling add 1g sodium sulfite, 1ml of concentrated hydrochloric acid and add water until the 100 ml mark, mix well, filter and store in brown bottle. Let solution stand overnight. if the solution has color, use small amounts of activated charcoal to filter. if the solution turns red it should be discarded.
4. methanol standard solution 1(10mg/ml)
Measure 1.27ml of methanol and place in a 100ml volume controller with a small amount of distilled water, add water up to then 100ml mark, mix well. Store in low temperature
5.methanol standard solution 2 (1mg/ml)
Take 10 ml of the methanol standard solution1 and place in 100 ml volume controller and add water to the 100 ml mark, mix well.
6. 10% sodium sulfite solution

Experimental Procedure
drawing the standard curve (absorbance at 590nm vs. mass of methanol)
Take 0, 0.06, 0.12, 0.18,0.24 and 0.30 ml of methanol (containing 0, 0.06mg, 0.12mg, 0.18mg, 0.24mg and 0.30 mg of methanol) standard solution 2 and place in 2ml microcentrifuge tube. Add water to each microcentrifuge tube up to 0.5ml, and then 0.2 ml of potassium permanganate-phosphoric acid solution, mix well and let the solution sit for 10 min. Add 0.2ml of oxalic acid-sulfinic acid solution, mix well and let the solution sit until the color fades. Then add 0.5ml of aniline red-sulfinic acid solution, mix well and let the solution sit for 0.5 hours (at temperature over 20 degrees centigrade). Transfer 1ml of the solution in each microcentrifuge tube into a cuvette and test the absorbance at 590nm and plot the standard curve of absorbance at 590nm Vs. mass of methanol

Testing
a) methanol concentration 5g/100ml (needs to be diluted 100 times)
Take 0.631ml (0.5 g of methanol) place into test tube and add water up to 10ml. Add 9

transformed E. coli. After 15, 30, 45, and 60 minutes, take 1 ml of solution and centrifuge. Then take 0.005ml/ 0.0025ml of the upper clear solution place in another centrifuge tube, add water until 0.5 ml (diluted 100 times) and then 0.2 ml of potassium permanganate-phosphoric acid solution, mix well and let the solution sit for 10 min. Add 0.2ml of oxalic acid-sulfuric acid solution, mix well and let the solution sit until the color fades. Then add 0.5ml of aniline red-sulfinic acid solution, mix well and let the solution sit for 0.5 hours (at temperature over 20 degrees centigrade). Transfer 1ml of the solution in each microcentrifuge tube into a cuvette and test the absorbance at 590nm. Then plot the values onto the standard curve to find the concentration (needs to be times 200).

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# Team:NYU Shanghai/Results

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* Project
  + [Description](https://2017.igem.org/Team%3ANYU_Shanghai/Description)
  + [Design](https://2017.igem.org/Team%3ANYU_Shanghai/Design)
  + [Protocols](https://2017.igem.org/Team%3ANYU_Shanghai/Experiments)
  + [E.coli Survival Test](https://2017.igem.org/Team%3ANYU_Shanghai/Results)
  + [Magenta Sulfite Colorimetric](https://2017.igem.org/Team%3ANYU_Shanghai/Demonstrate)
  + [Interlab](https://2017.igem.org/Team%3ANYU_Shanghai/InterLab)
  + [Notebook](https://2017.igem.org/Team%3ANYU_Shanghai/Notebook)
* [Part](https://2017.igem.org/Team%3ANYU_Shanghai/Basic_Part)
  + [Basic Part](https://2017.igem.org/Team%3ANYU_Shanghai/Basic_Part)
* [Safety](https://2017.igem.org/Team%3ANYU_Shanghai/Safety)
* [Human Practice](https://2017.igem.org/Team%3ANYU_Shanghai/HP/Silver)
  + [Community Outreach](https://2017.igem.org/Team%3ANYU_Shanghai/HP/Silver)
* [Awards](https://2017.igem.org/Team%3ANYU_Shanghai/Applied_Design)
  + [Medal](https://2017.igem.org/Team%3ANYU_Shanghai/Applied_Design)
* [Judging Form](https://igem.org/2017_Judging_Form?team=NYU_Shanghai)

# E.coli Survival Test

Since we did not know if E.coli can survive in methanol solution, we decided to test the E.coli survival rate in different concentrations of methanol and ethanol using three different methods: Spectrophotometry, double staining fluorescence microscopy, and clonogenic assay.

## Spectrophotometry Our first method, spectrophotometry, was designed based on the fact that when a bacterium dies, its membrane breaks and all the proteins are released. The spectrophotometer is a machine that can be used to detect the concentration of these proteins by measuring the light absorbance of a certain wavelength. By measuring the change in protein concentration after adding methanol, we sought to find the cell death rate according to the formula found in the literature, but when actually put into practice, the reading fluctuated considerably and so was unable to give us an accurate result, probably because of the protein settling and degradation in methanol. Double Staining Fluorescence Microscopy The second method we tried is double staining fluorescence microscopy. Two fluorescent dyes, propidium iodide and hoechst 33342, both stain bacterial DNA, but they differ in their permeability and in their fluorescence. Hoechst 33342 is a nucleic acid stain that emits blue fluorescence when bound to double stranded DNA. This dye is highly permeable and can enter the cell membranes of both dead and live E.coli. The other important fluorescent dye we used is propidium iodide, which emits red light when bound to DNA. Unlike hoechst, propidium iodide can only penetrate the cell membrane when E.coli is undergoing lysis, indicating that they are dead or starting to die, making it a very useful tool to test the survival rate. We had to play around with different conditions to see which ones will give us the best visual result. For example, we tested different dilutions for the bacterial suspension, different concentrations of the dyes, incubation time, dye ratio and amount. We also had difficulties at first finding our E.coli. There were a lot of residues on both side of the coverslips that we accidentally mistaken as E.coli. After deep cleaning our microscope slides and coverslips with the ultrasonic cleaner, we finally identified our E.coli under 400x magnification. After numerous testings, we found that 50% methanol is deadly to the E.coli, as indicated by all the red fluorescent signals. We overlapped the blue and red images to visualize how much E.coli has died. The purple denote bacteria which are dead, while the blue show those which are still alive. Additionally, we determined that ethanol had a more detrimental effect on the E.coli, with 30% being the fatal concentration. ![](https://static.igem.org/mediawiki/2017/9/99/Ecolinyush1.jpg) ![](https://static.igem.org/mediawiki/2017/4/4d/Ecolinyush2.jpg)Clonogenic Assay In order to determine whether or not our fluorescence microscope results were reliable and accurate, we utilized another method called clonogenic assay to test the E.coli survival rate in ethanol/methanol solutions. This method involves applying a treatment to a sample of cells and then spreading these cells on agar plates to allow them to grow overnight. The next day, you can observe distinct colonies of bacteria on the agar plates. The number of colonies able to grow on the plate signifies the number of live bacteria originally deposited onto the plate. Comparing the number of colonies on the treated plate to an untreated plate gives a rough idea of the percentage of cells that survived the treatment. For our experiment, we first determined the ideal dilution of our bacterial suspension for a visible result. Without a high enough dilution, it will result in a bacterial lawn rather than in distinct colonies. After diluting our overnight bacterial suspension by 10,000 times, we were able to obtain fairly decent results. As for the treatment, we tested various concentrations of ethanol and methanol, ranging from 10% to 50%, with varying incubation times. After incubating the E.coli with the treatment for a certain period of time, we removed the methanol/ethanol solution, diluted the sample using LB nutrient broth, and spread 10uL of that sample on the agar plates. As we increase the concentration of ethanol/methanol, we observed a slight decrease in the number of colonies and the colony size. In the figure shown here of our methanol treated E. coli, the number of colonies significantly decrease in size at 40% and do not even grow at 50%. Similarly, in the ethanol treatment test, there appears to be very little change between the control and the 20% ethanol treatment, but no growth at all under 30% treatment. These results -- that no colonies form at 30% ethanol nor 50% methanol, are consistent with the results we found using the fluorescence microscope technique. ![](https://static.igem.org/mediawiki/2017/b/b2/Ecolinyush3.png) ![](https://static.igem.org/mediawiki/2017/3/31/Ecolinyush4.png)

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