iGEM 2021 — Team NEYCFLS_China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NEYCFLS China/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NEYCFLS China/Engineering

* [ANTI-Virus](https://2021.igem.org/Team%3ANEYCFLS_China)
* Project

  :   [Project Description](https://2021.igem.org/Team%3ANEYCFLS_China/Description)
  :   [Experiment](https://2021.igem.org/Team%3ANEYCFLS_China/Experiments)
  :   [Implementation](https://2021.igem.org/Team%3ANEYCFLS_China/Implementation)
  :   [Proof Of Concept](https://2021.igem.org/Team%3ANEYCFLS_China/Proof_Of_Concept)
* [Modeling](https://2021.igem.org/Team%3ANEYCFLS_China/Model)
* Engineering

  :   [Engineering](https://2021.igem.org/Team%3ANEYCFLS_China/Engineering)
  :   [Parts](https://2021.igem.org/Team%3ANEYCFLS_China/Parts)
  :   [Contribution](https://2021.igem.org/Team%3ANEYCFLS_China/Contribution)
* Human Practices

  :   [Human Practices](https://2021.igem.org/Team%3ANEYCFLS_China/Human_Practices)
  :   [Tie Dye](https://2021.igem.org/Team%3ANEYCFLS_China/Tie_Dye)
  :   超链接

      [Arts](https://2021.igem.org/Team%3ANEYCFLS_China/Art)
      :   超链接

          [Education](https://2021.igem.org/Team%3ANEYCFLS_China/Communication)
* Team

  :   [About Us](https://2021.igem.org/Team%3ANEYCFLS_China/Team)
  :   [Attributions](https://2021.igem.org/Team%3ANEYCFLS_China/Attributions)
  :   [Collaboration](https://2021.igem.org/Team%3ANEYCFLS_China/Collaborations)
  :   [Partnership](https://2021.igem.org/Team%3ANEYCFLS_China/Partnership)

![](https://static.igem.org/mediawiki/2021/3/36/T--NEYCFLS_China--engineCov.png)

![](https://static.igem.org/mediawiki/2021/c/c1/T--NEYCFLS_China--Experiment1.png)
![](https://static.igem.org/mediawiki/2021/f/f1/T--NEYCFLS_China--Experiment2.png)
* To test the powder we had acquired, we first ran a thin-layer chromatography test on the powder using 20% methanol and 80% DCM. The results showed that the powder we had acquired was mostly indigo, but also had a little bit of impurities in it. Then, we ran an IR test and Proton NMR test on the powder. From the results, we are sure that we have indigo, but it contained some impurities. Then, we created our indigo with biosynthetic methods

![](https://static.igem.org/mediawiki/2021/7/7f/T--NEYCFLS_China--Experiment333.png)
* Biosynthesis of tryptophan using E.coli involves three major steps. First, glucose in the bacteria is condensed to phosphoenolpyruvate (PEP) in the central metabolism pathway. Then, PEP undergoes the common aromatic pathway, where it is converted to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) then to chorismate (CHA). At this point, the genes prephenate dehydrogenase (tyrA) and prephenate dehydrase (pheA) were blocked to prevent the CHA from being converted to tyrosine, and, with the help of tryptophanase BA (trpBA), we were able to convert the CHA into our desired tryptophan. Finally, after another enzyme called tryptophanase-A (tnaA) will convert all of our synthesized tryptophan to indole.

![](https://static.igem.org/mediawiki/2021/4/43/T--NEYCFLS_China--Genetcs1.png)
* Meanwhile, pTacFd911 will produce an enzyme called Flavin-containing Monooxygenase, abbreviated as FMO, that spontaneously converted indole to indoxyl, or 3-hydroxyindole, over the course of 14 hours.
* Finally, we got our indigo by mixing appropriate dilutions of whole fermentation broth (i.e., cells and medium ) with dimethylsulfoxide (DMSO) to solubilize the indigo, which has been converted from indoxyl by oxidation when in contact with air. Then, we proportionally mixed the indigo and silver nanoparticles. If we were to create a dye solution that contained 100 ppm of silver nanoparticles and, we would add 0.1g of AgNP to 9.9 g of indigo and add the entire mixture to 100 mL of water.
* After we had dyed our cloth, we used computer models to estimate and help visualize the effectiveness of the mask against viruses and bacteria. We tested the effectiveness using some concentrations of indigo and silver, then gave the data to our modeling and CS team for processing. Here is the model they have made.
* Another comes in when we have the results from our survey on the suitable price for our masks, and the relationship between concentrations and effectiveness. Using this model, we can find a balance between the price and performance, and also the amount of the two ingredients we would have to use for one mask.

![](https://static.igem.org/mediawiki/2021/2/2e/T--NEYCFLS_China--ender.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NEYCFLS China/Experiments

* [ANTI-Virus](https://2021.igem.org/Team%3ANEYCFLS_China)
* Project

  :   [Project Description](https://2021.igem.org/Team%3ANEYCFLS_China/Description)
  :   [Experiment](https://2021.igem.org/Team%3ANEYCFLS_China/Experiments)
  :   [Implementation](https://2021.igem.org/Team%3ANEYCFLS_China/Implementation)
  :   [Proof Of Concept](https://2021.igem.org/Team%3ANEYCFLS_China/Proof_Of_Concept)
* [Modeling](https://2021.igem.org/Team%3ANEYCFLS_China/Model)
* Engineering

  :   [Engineering](https://2021.igem.org/Team%3ANEYCFLS_China/Engineering)
  :   [Parts](https://2021.igem.org/Team%3ANEYCFLS_China/Parts)
  :   [Contribution](https://2021.igem.org/Team%3ANEYCFLS_China/Contribution)
* Human Practices

  :   [Human Practices](https://2021.igem.org/Team%3ANEYCFLS_China/Human_Practices)
  :   [Tie Dye](https://2021.igem.org/Team%3ANEYCFLS_China/Tie_Dye)
  :   超链接

      [Arts](https://2021.igem.org/Team%3ANEYCFLS_China/Art)
      :   超链接

          [Education](https://2021.igem.org/Team%3ANEYCFLS_China/Communication)
* Team

  :   [About Us](https://2021.igem.org/Team%3ANEYCFLS_China/Team)
  :   [Attributions](https://2021.igem.org/Team%3ANEYCFLS_China/Attributions)
  :   [Collaboration](https://2021.igem.org/Team%3ANEYCFLS_China/Collaborations)
  :   [Partnership](https://2021.igem.org/Team%3ANEYCFLS_China/Partnership)

![](https://static.igem.org/mediawiki/2021/2/26/T--NEYCFLS_China--experimentCover.png)

* **Experimental Summary**

* Our experiment will have four very simple steps, we will first purify and test the major component of dye, the compound that we think is responsible for the unique blue color in dyed cloth. The next step would be manufacturing large amounts of this compound using biosynthetic methods. Step three, is to put together our biosynthetic indigo, with silver nanoparticles to create our ANTI-Virus dye. Finally, using this dye we will color our cloths,
  and send it to the biology group to test it with ATCC 25922.
* **Step One: Purifying and Testing the Major Component of Dye**

* According to folktale, a substance called indigo was responsible for both the properties of the dye. Skeptically, we bought some of the dye sold by artisans and started to experiment with it.
* ![](https://static.igem.org/mediawiki/2021/c/c1/T--NEYCFLS_China--Experiment1.png)

* However, for our experiment, using traditional methods of , we were not able to get an amount of indigo adequate enough for IR and NMR confirmation. We found a way to first remove a large amount of the impurities, and then try to take out the indigo.
* We started by adding a 50% aqueous ethanol solution to the powder, and started stirring it for about an hour. Then, we vacuum filtered the system, dried the filtrate, and repeated the process again for a few times with other organic solvents: petroleum ether, ethyl acetate, n-butanol, and chloroform. Then, we evaporated off the chloroform, leaving us with a mixture that mainly contained indigo, along with other impurities.
* ![](https://static.igem.org/mediawiki/2021/f/f1/T--NEYCFLS_China--Experiment2.png)

* The last part, would be to test out the substance that we had acquired, we first ran a thin-layer chromatography test on the powder using 20% methanol and 80% DCM. The results showed that the powder we had acquired was mostly indigo, but also had a little bit of impurities in it. Then, we ran an IR test and Proton NMR test on the powder. From the results, we are sure that we have indigo, but it contained some impurities.* ![](https://static.igem.org/mediawiki/2021/7/7f/T--NEYCFLS_China--Experiment333.png)
* **Step Two: Producing Indigo Using E.Coli**

* (Note: Due to Chinese university policies, our team wasn't able to get into any university laboratory. The information that we have provided for step two of our experiment it's mainly theoretical, and most of the strains and types of bacteria that we have included in this section and the genetics section, came from articles that we have bought from Google Scholar. During our research, we received help from one of our seniors at University of North Carolina, Chapel Hill. We also talked to another professor who had a Ph.D. in biotechnology, and was familiar with work using CRISPR. Both of them helped us make sure that our experimental methods could work, and could be implemented on a non-theoretical scale.)
* Now we know that indigo is the most important substance in terms of dyeing cloth, our next step is to produce indigo using synthetic biological methods with the help of our friend—E. Coli, starting by producing an amino acid called Tryptophan.
* ![](https://static.igem.org/mediawiki/2021/9/98/T--NEYCFLS_China--Experiment5.png)

* Biosynthesis of tryptophan using E.coli involves three major steps. First, glucose in the bacteria is condensed to phosphoenolpyruvate (PEP) in the central metabolism pathway. Then, PEP undergoes the common aromatic pathway, where it is converted to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) then to chorismate (CHA). At this point, the genes prephenate dehydrogenase (tyrA) and prephenate dehydrase (pheA) were blocked to prevent the CHA from being converted to tyrosine, and, with the help of tryptophanase BA (trpBA), we were able to convert the CHA into our desired tryptophan. Finally, after another enzyme called tryptophanase-A (tnaA) will convert all of our synthesized tryptophan to indole.
* Tyrosine is a nonessential amino acid the body makes from other amino acids called phenylalanine and tryptophan. It is an essential component for the production of several important brain chemicals called neurotransmitters, including epinephrine, norepinephrine, and dopamine. However for us, who are using E. Coli to produce Tryptophan, we have to get rid of the enzymes that convert Trp to Tyr (Y). Thus we are using FB-01 to knock out the gene that boosts this conversion.
* ![](https://static.igem.org/mediawiki/2021/e/ed/T--NEYCFLS_China--Experiment6.png)

* ![](https://static.igem.org/mediawiki/2021/5/57/T--NEYCFLS_China--Experiment7.png)

* Meanwhile, pTacFd911 will produce an enzyme called Flavin-containing Monooxygenase, abbreviated as FMO, that spontaneously converted indole to indoxyl, or 3-hydroxyindole, over the course of 14 hours.
* Finally, we assayed indigo by mixing appropriate dilutions of whole fermentation broth (i.e., cells and medium ) with dimethylsulfoxide (DMSO) to solubilize the indigo, which has been converted from indoxyl by oxidation when in contact with air. Then centrifuging the mixture to remove the biomass, we read the absorbance of the supernatant at 620 nm and calculated our biosynthetic indigo concentrations by comparison to a standard curve constructed using synthetic indigo.
* To sum it up, we will produce tryptophan in our E. Coli while knocking out the jeans that convert tryptophan into tyrosine, have the bacteria's own mechanisms convert tryptophan into indole, and give it another gene that will produce an enzyme that converts indole into indoxyl.
* **Step Three: Producing the ANTI-Virus Dye**

* Producing the dye was probably the easiest step of our experiment. There were two different ways we could do this.
* First, we could proportionally mix the indigo and silver nanoparticles. If we were to create a dye solution that contained 100 ppm of silver nanoparticles and, we would add 0.1g of AgNP to 9.9 g of indigo and add the entire mixture to 100 mL of water. However, this would make it very hard to change the concentration of AgNP and indigo at the same time. This was not a problem for us, for we had already calculated the best combination of indigo added and AgNP in the indigo.
* Thus our second way is simply preparing a solution of indigo, then adding a prepared aqueous solution of AgNP into the indigo before dyeing. Because of its flexibility, this was the method we used in dyeing our cloths for efficiency test and the method we prepare to use for implementing ANTI-Virus in the real world.

![](https://static.igem.org/mediawiki/2021/f/f7/T--NEYCFLS_China--Experiment8.png)

* **Step Four: Dyeing Fabric Using ANTI-Virus Dye**

![](https://static.igem.org/mediawiki/2021/4/41/T--NEYCFLS_China--Experiment9.png)

* To dye our fabric, we would have to first turn indigo into a solution of leucoindigo. Our dyeing solution uses 2 grams of lye (sodium hydroxide) per 100 mL of water, and 2 grams of sodium dithionite per gram of ANTI-Virus dye. All of these main ingredients were put in a large beaker and brought to a boil on a heating mantle with stirring. Then, after the solution turned clear, we turn off the heat and transferred all of the solution into another beaker, where we added the cloth that was to be dyed. The cloth was put in the dye solution for 10 minutes and taken out. After most of the leucoindigo had reacted with the oxygen in the air, which was characterized by the dark blue color, we put the cloth back into the solution and repeated the process for another time.

![](https://static.igem.org/mediawiki/2021/2/2c/T--NEYCFLS_China--Experiment10.png)
* **References**
* [[1] https://2021.igem.org/Team:NEYCFLS\_China/JFF](https://2021.igem.org/Team%3ANEYCFLS_China/JFF)
* [[2] The Synthesis and Application of an Organic Dye Indigo: Introduction and Discussion of an Oversea Undergraduate Experiment, Zhipeng WANG, April 2016](http://www.dxhx.pku.edu.cn/CN/10.3866/PKU.DXHX20160483)
* [[3] Indigo and Tyrian Purple – From Ancient Natural Dyes to Modern Organic Semiconductors, Eric Daniel Głowacki et al., June 2012](https://onlinelibrary.wiley.com/doi/abs/10.1002/ijch.201100130)
* [[4] Tryptophan, Wikipedia, (n.d.)](https://en.wikipedia.org/wiki/Tryptophan)
* [[5] Phosphoenolpyruvic acid, Wikipedia, (n.d.)](https://en.wikipedia.org/wiki/Phosphoenolpyruvic_acid)
* [[6] 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate, Wikipedia, (n.d.)](https://en.wikipedia.org/wiki/3-Deoxy-D-arabino-heptulosonic_acid_7-phosphate)
* [[7] Chorismic acid, Wikipedia, (n.d.)](https://en.wikipedia.org/wiki/Chorismic_acid)
* [[8] Tyrosine, Wikipedia, (n.d.)](https://en.wikipedia.org/wiki/Tyrosine)
* [[9] An overview of microbial indigo-forming enzymes, Andrea N. Fabara et al., December 2019](https://link.springer.com/article/10.1007/s00253-019-10292-5)
* [[10] Application of metabolic engineering to improve both the production and use of biotech indigo, A Berry et al., 2002](https://academic.oup.com/jimb/article/28/3/127/5989506?login=true)
* [[11] Development of L-tryptophan production strains by defined genetic modification in Escherichia coli, Chun Zou et al., December 2011](https://doi.org/10.1007/s10295-011-0978-8)
* [[12] Effectiveness of Silver Nanoparticles against SARS-CoV-2, ZhiHu, Marvin Zhou,](https://zhuanlan.zhihu.com/p/267306439)

![](https://static.igem.org/mediawiki/2021/2/2e/T--NEYCFLS_China--ender.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NEYCFLS China/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)