iGEM 2021 — Team HK_CPU-WFN-WYY

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# Team:HK CPU-WFN-WYY/Notebook

HK\_CPU-WFN-WYY: Home page

[![](https://static.igem.org/mediawiki/2021/2/21/T--HK_CPU-WFN-WYY--IMG_3206.PNG)](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY)

**TEAM**

[Team](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Team)
 [Attributions](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Attributions)
 [Collaborations](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Collaborations)

**PROJECT**

[Communication](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Communication)
 [Contribution](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Contribution)
 [Description](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Description)
 [Experiment](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Experiment)
 [Engineering](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Engineering)
 [Notebook](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Notebook)
 [Proposed Implementation](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Implementation)
 [Results](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Results)

**PARTS**

[Parts Collection](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Part_Collection)
 [Parts](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Parts)

 [**SAFETY**](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Safety)
 [**HUMAN PRACTICES**](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Human_Practices)

 [**MODELING**](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Model)

# Notebook

| DNA no. | Abbreviation | Parts registry ID |
| --- | --- | --- |
| DNA 1 | Piri6 (PiSp\_Trichonephila clavipes\_6) | BBa\_K3816009 |
| DNA 2 | Masp2\_2 | BBa\_K3816015 |
| DNA 3 | Piri2 (PiSp\_Trichonephila clavipes\_2) | BBa\_K3816005 |
| DNA 4 | Masp2\_1 | BBa\_K3816014 |
| DNA 5 | Piri5 (PiSp\_Trichonephila clavipes\_5) | BBa\_K3816008 |
| DNA 6 | Masp1\_102 | BBa\_K3816011 |
| DNA 7 | Piri4 (PiSp\_Trichonephila clavipes\_4) | BBa\_K3816007 |
| DNA 8 | Tusp (Tubuliform silk\_Nephila Antipodiana) | BBa\_K3816002 |
| DNA 9 | Masp1\_103 | BBa\_K3816001 |

## June

1. Literature review
2. programmatically finding target sequences from NCBI
3. manually screening sequences, creating gene constructs and primer pairs (for screening and PCR) in Benchling
4. order reagents

## 1/8-7/8

1. Test amplification of target genes by low-fidelity DNA polymerase of DNA 1-9

## 8/8-14/8

1. Test amplification of target genes by low-fidelity DNA polymerase of DNA 1-9
2. DNA amplification by high-fidelity DNA polymerase of DNA 1-9

## 15/8-21/8

1. 1st Digestion of DNA1,2,3,4,6,7,8,9 with Sac I and Sal l
2. Ligation of DNA 1,2,3,4,6,7,8,9
3. Transformation of DNA 1,2,3,4,6,7,8,9
4. Incubation overnight
5. colony PCR
6. DNA amplification by high-fidelity DNA polymerase of DNA 1-9

## 22/8-28/8

1. 2nd Digestion of DNA 1,2,3,4,6,7,8,9 with Sal1 and Sac1, plasmid
2. Ligation
3. Transformation
4. Miniprep for plasmid (transformation and streak plate)
5. Incubation
6. Run gel
7. Gene clean
8. Colony PCR
9. cell culture
10. Miniprep to extract plasmid (TR -ve)

## 29/8-4/9

1. Colony PCR
2. run gel
3. LB culture
4. miniprep

## 5-11/9

1. Colony PCR and gel electrophoresis
2. 1st digestion of DNA 1,2,3,4 with Sac1
3. Gene clean of DNA 2,3,4 without gel
4. 2nd digestion of DNA 1,2,3,4 with Sal1
5. Run gel (agarose) of DNA 1,2, 3, 4, plasmid and ladder to check digestion product
6. gene clean with PE and PW wash
7. Ligation of DNA 1,2,3,4 (DNA2,3,4 overnight)
8. Transformation DNA1
9. PCR DNA 8,9

## 12/9-18/9

1. Double digest (DNA 6-9+Plasmid)
2. Run gel (DNA 6-9+Plasmid)
3. Gene clean with PE and PW wash of plasmid
4. Ligation DNA 2-4,6-9(DNA 2-4 with mighty mix)
5. Transformation of DNA 2, 3, 4, 6, 7, 8, 9 and plasmid
6. Spread plate with DNA 2,3,4, 6, 7 or plasmid for each plate
7. Prepare competent cells of E.coli DH5a

## 19/9-25/9

1. Check colonies 6,7(DNA 6:failed, DNA 7:positive but colony too small)
2. Mini. prep
3. Run gel
4. Ligation, transformation and spread plate using old stock
5. Colony PCR of DNA 3, 7with F primer and R primer
6. Incubation
7. Checking the competent cells by transformation
8. Prepare competent cells of E.coli DH5a and E.coli LMG194

## 26/9-2/10

1. Digestion DNA1,7,8,9 and plasmid
2. Gel electrophoresis
3. Ligation
4. Testing PCR
5. Cell culture
6. Miniprep 1,2 (DNA 7)

## 4/10-10/10

1. Double digestion with gene clean without gel(DNA 2,3,4)
2. Gene clean of without gel eluted with new elution kit with smaller product volume(DNA 2,3,4)
3. Ligation using new ligase and buffer(DNA 2,3,4)
4. Transformation (DNA1,2,3,4,8,9) and spread plate
5. Colony PCR(DNA 1,8,9)
6. PCR screening of 15 colony, 5 in each plate, with FWD primer and REV primer(DNA2,3,4)
7. Gel electrophoresis of DNA 1,8,9,
8. Liquid culture (recombinant plasmids transformants)
9. Re-digestion of DNA 1,8,9(Sac1 and Sal1)2,3,4(using XbaI, HindIII of miniprep product from some colonies to check if there is insert)
10. Miniprep
11. Liquid culture and miniprep (to increase concentration of plasmid)
12. Run gel (DNA 7 miniprep product)

## 12/10

1. Sending miniprep products to sequencing company

### Sponsors:

[![](https://static.igem.org/mediawiki/2021/8/88/T--HK_CPU-WFN-WYY--Home_Sponsor_idt.svg)](https://sg.idtdna.com/pages)[![](https://static.igem.org/mediawiki/2021/2/2f/T--HK_CPU-WFN-WYY--Home_Sponsor_benchling.svg)](https://www.benchling.com/)

[![](https://static.igem.org/mediawiki/2021/5/51/T--HK_CPU-WFN-WYY--Mathworks.png)](https://www.mathworks.com/)[![](https://static.igem.org/mediawiki/2021/3/30/T--HK_CPU-WFN-WYY--Home_Sponsor_twist.svg)](https://www.twistbioscience.com/)

### Contact us!

[![](https://static.igem.org/mediawiki/2021/6/64/T--HK_CPU-WFN-WYY--Home_IG_icon.svg)](https://www.instagram.com/igem_cpuwfnwyy/) @hk.cpuwfnwyy

![](https://static.igem.org/mediawiki/2021/1/1f/T--HK_CPU-WFN-WYY--Home_Gmail_icon.svg) hk.cpuwfnwyy@gmail.com

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# Team:HK CPU-WFN-WYY/Engineering

HK\_CPU-WFN-WYY: Home page

[![](https://static.igem.org/mediawiki/2021/2/21/T--HK_CPU-WFN-WYY--IMG_3206.PNG)](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY)

**TEAM**

[Team](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Team)
 [Attributions](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Attributions)
 [Collaborations](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Collaborations)

**PROJECT**

[Communication](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Communication)
 [Contribution](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Contribution)
 [Description](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Description)
 [Experiment](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Experiment)
 [Engineering](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Engineering)
 [Notebook](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Notebook)
 [Proposed Implementation](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Implementation)
 [Results](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Results)

**PARTS**

[Parts Collection](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Part_Collection)
 [Parts](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Parts)

 [**SAFETY**](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Safety)
 [**HUMAN PRACTICES**](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Human_Practices)

 [**MODELING**](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Model)

# Engineering

### Design

**Choice of chassis**
To test whether spider silk will have variable properties when different spidroins are mixed, our aim is to engineer some recombinant *E. coli* to express the spidroins that we are interested in so that we can test our hypothesis.
We chose *E. coli* as our chassis as it is easy to handle and has a rapid growth rate compared to other chassis, such as yeast or mammalian cell lines. Also, it is easy to transform, which is crucial point of concern in a high school setting.
**Choice of strains**
In the process of creating the recombinant plasmid and expressing spidroin, we will use both the Dh5α strain and the BL21 strain. Due to the nature of digest and ligate assembly, the chance of the recombinant plasmid in concern being formed is very low.
To amplify and separate the required plasmid from other undesired plasmids, we will first transform the plasmids into *E. coli* Dh5α so that they can be plated, selected and screened with colony PCR.
During the process, the recombinant *E. coli* will divide and replicate to produce more copies of itself, as well as making copies of the desired recombinant plasmid.
As *E. coli* Dh5α grows rapidly, using this strain enables the rapid production of large amounts of plasmids in a short period of time. However, it is has reduced protein expression capabilities.
To compensate for this, we will extract the desired recombinant plasmid from *E. coli* Dh5α by miniprep, and transforming *E. coil* BL21 with the miniprepped plasmids.
This way, we can take advantage of the superior expression capabilities of *E. coli* BL21 for expressing the spidroins that we need, as well as using Dh5α for plasmid amplification.
**Assembly of plasmid**
To assemble the recombinant plasmid that we want, which contains the gene of interest and other regulatory sequences required for expression, such as ribosome binding site and indicible promoter, we will use digest and ligate assembly to create the plasmid.
We chose digest and ligate assembly as this is cheaper and easier to implement compared to other methods like Gibson assembly.
The backbone of our choice is the [pET SUMO](https://www.thermofisher.com/order/catalog/product/K30001#/K30001) expression plasmid because we have already obtained this plasmid, and continue using it will reduce the financial burden of our team.
This plasmid also fits our use case as we want the expression to be inducible so that it can be finely controlled. We modified the plasmid by removing the SUMO tag, as it may interfere with the folding of spidroin.
The backbone also included a his-tag, which aids in the purification process.
We ordered the gene of interest from IDT, which removes the hassle of obtaining real spiders and extracting DNA/ RNA from them.
**Testing the design**
To test the design, we will assemble it in the lab and transform it into *E. coli* as mentioned above. After transformation, we will culture the recombinant *E. coli* in LB broth and add IPTG to express the spidroin.
We then extract the spidroin via his-tag chromatography and check if the result is as expected or not by SDS-PAGE. If the result is as expected, we should see a band on the gel at the position corresponding to its molecular weight.

### Build

**Assembly coniderations**
As there is only one component to be inserted into the plasmid backbone, that is, our gene of interest, we decided to use digest and ligate assembly. Due to the nature of digest and ligate assembly, scar sites will be introduced.
However, it is expected that the scar sites will not interfere with the expression of the protein as translation only starts at the start codon and should end at the stop codons. Thus, the ribosome should ignore the scar sites before and after the gene of interest.
**Verification and expression of plasmid**
To verify the assembled plasmid actually contains the insert, we will perform colony PCR on the colonies that have grown on an antibiotic-agar plate. The miniprepped plasmid will then be used for sequencing to confirm sequence integrity.
We will use sanger sequencing as it is the most reliable sequencing method. The primers that will be used binds to the plasmid backbone and should be able to sequence the full insert.
We also double-check by doing a restriction digest on the miniprepped plasmid, and see if the digest profile matches the theoretical digest profile. Since we used two restriction sites in the assembly process, we do not have to worry about inverted inserts.
To express the spidroin in concern, we have to transfer the plasmid to another chassis, namely from *E. coli* Dh5α to *E. coli* BL21. To do this, we will take the miniprepped plasmid and do another transformation into *E. coli* BL21 for expression.
**Validation of expression products**
To ensure the spidroin expressed is what we want, we will perform SDS-PAGE on the extracted protein. It is expected that we should see a band present according to the molecular weight of the spidroin.

### Test

**Testing for expression**
To validate that the spidroins are expressed correctly, we will perform SDS-PAGE on the extracted proteins. A vertical gel electrophoresis tank will be needed. This will provide qualitative measurements of the spidroin in concern.
To quantify the spidroins being expressed, western blotting can be performed. However, due to the financial and instrumental constraints, we are unable to perform this step.
To ensure the proteins detected is not contamination, we will also run negative samples as well as eluates during the protein extraction process to ensure that the protein band we detected is actually spidroin.
To enhance the specificity of the test, western blotting can be used if possible. After SDS-PAGE, sliver stain will be used to visualize the proteins in the acrylamide gel.

### Sponsors:

[![](https://static.igem.org/mediawiki/2021/8/88/T--HK_CPU-WFN-WYY--Home_Sponsor_idt.svg)](https://sg.idtdna.com/pages)[![](https://static.igem.org/mediawiki/2021/2/2f/T--HK_CPU-WFN-WYY--Home_Sponsor_benchling.svg)](https://www.benchling.com/)

[![](https://static.igem.org/mediawiki/2021/5/51/T--HK_CPU-WFN-WYY--Mathworks.png)](https://www.mathworks.com/)[![](https://static.igem.org/mediawiki/2021/3/30/T--HK_CPU-WFN-WYY--Home_Sponsor_twist.svg)](https://www.twistbioscience.com/)

### Contact us!

[![](https://static.igem.org/mediawiki/2021/6/64/T--HK_CPU-WFN-WYY--Home_IG_icon.svg)](https://www.instagram.com/igem_cpuwfnwyy/) @hk.cpuwfnwyy

![](https://static.igem.org/mediawiki/2021/1/1f/T--HK_CPU-WFN-WYY--Home_Gmail_icon.svg) hk.cpuwfnwyy@gmail.com

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

# Team:HK CPU-WFN-WYY/Experiments

# 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.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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

# Team:HK CPU-WFN-WYY/Results

HK\_CPU-WFN-WYY: Home page

[![](https://static.igem.org/mediawiki/2021/2/21/T--HK_CPU-WFN-WYY--IMG_3206.PNG)](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY)

**TEAM**

[Team](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Team)
 [Attributions](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Attributions)
 [Collaborations](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Collaborations)

**PROJECT**

[Communication](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Communication)
 [Contribution](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Contribution)
 [Description](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Description)
 [Experiment](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Experiment)
 [Engineering](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Engineering)
 [Notebook](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Notebook)
 [Proposed Implementation](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Implementation)
 [Results](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Results)

**PARTS**

[Parts Collection](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Part_Collection)
 [Parts](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Parts)

 [**SAFETY**](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Safety)
 [**HUMAN PRACTICES**](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Human_Practices)

 [**MODELING**](https://2021.igem.org/Team%3AHK_CPU-WFN-WYY/Model)

# Results

Below shows the sizes of the DNA fragments:

| Gene | whole gene size (bp) |
| --- | --- |
| Piri6 | 1337 |
| Masp2\_2 | 947 |
| Piri2 | 533 |
| Masp2\_1 | 908 |
| Piri5 | 860 |
| Masp1\_102 | 935 |
| Piri4 | 911 |
| Tusp | 1157 |
| Masp1\_1 | 1253 |

### PCR

PCR was used to replicate DNAs. Below are the gel photos showing the result of PCR.

![](https://static.igem.org/mediawiki/2021/8/8d/T--HK_CPU-WFN-WYY--electrop1.png)

Figure 1a: 1%agarose gel showing the results of PCR (the bands of masp2\_2(947bp), prir2(533bp), masp2\_1(908bp), masp1\_102(935bp), piri4(911bp), tusp(1157bp), masp1\_1(1253bp))

![](https://static.igem.org/mediawiki/2021/2/2d/T--HK_CPU-WFN-WYY--unnamed_%282%29.png)

Figure 1b: 1%agarose gel showing the results of PCR (the band of piri6(1337bp))

![](https://static.igem.org/mediawiki/2021/d/d4/T--HK_CPU-WFN-WYY--unnamed_%283%29.png)

Figure 1c: 1%agarose gel showing the results of PCR (the band of piri5(860bp))

As shown in the figures, all genes were replicated successfully except Piri5 and Masp1\_103.

### Digestion

Restriction enzymes sacI and salI were used to digest the inserts and backbone. The bands of successfully digested DNA fragments should travel slightly faster than that of undigested DNA fragments.

Same as before, the genes were extracted via gene clean and stored at -20°C

![](https://static.igem.org/mediawiki/2021/3/3b/T--HK_CPU-WFN-WYY--Digestion1.png)

Figure 2a: 1%agarose gel showing the results of digestion (the bands of piri6, tusp and masp1\_1)

![](https://static.igem.org/mediawiki/2021/1/18/T--HK_CPU-WFN-WYY--Digestion2.png)

Figure 2b: 1% agarose gel showing the results of digestion using SalI and SacI (masp2\_2, prir2, masp2\_1)

As shown on the figure, piri6 , tusp and masp1\_1, masp2\_2, prir2, masp2\_1 were digested successfully and extracted via gene clean.

### Colony PCR

After incubation, the colonies were picked to perform colony PCR in order to differentiate between undigested plasmid transformants and recombinant plasmids transformants.

The colonies with the correct band size were extracted from the plate and cultured by liquid culture in order to get a large amount of plasmids for further analysis.

![](https://static.igem.org/mediawiki/2021/2/24/T--HK_CPU-WFN-WYY--Colony1.jpg)

Figure 3a: 1%agarose gel showing the results of colony PCR. 6 colonies were picked for piri6. Correct band sizes and location were labelled.

![](https://static.igem.org/mediawiki/2021/7/75/T--HK_CPU-WFN-WYY--Colony2.jpg)

Figure 3b: 1%agarose gel showing the results of colony PCR. 8 colonies were picked for tusp. Correct band sizes and location were labelled.

![](https://static.igem.org/mediawiki/2021/a/a0/T--HK_CPU-WFN-WYY--Colony3.png)

Figure 3c: 1%agarose gel showing the results of colony PCR. 8 colonies were picked for tusp. Correct band sizes and location were labelled.

### Re-digestion

Since the target gene and plasmids were cut and separated, there should have 2 bands in gel electrophoresis, one was the band of plasmid, which is around 5000bp, and one was the band of target gene, the size depended on the type of genes.

![](https://static.igem.org/mediawiki/2021/c/c8/T--HK_CPU-WFN-WYY--ColonyDigest1.png)

Figure 4a: 1%agarose gel showing the results of digestion of plasmid obtained from the colonies having +ve results in colony PCR. Correct band sizes and location were labelled.

![](https://static.igem.org/mediawiki/2021/f/f4/T--HK_CPU-WFN-WYY--ColonyDigest2.png)

Figure 4b: 1%agarose gel showing the results of digestion of plasmid obtained from the colonies having +ve results in colony PCR. Correct band sizes and location were labelled.

As shown in figures,the recombinant plasmids that inserted Piri6 and tusp are matched the expectations. They were sent to sequencing company to check the insert integrity to ensure spidoin produced will have the expected amino acid sequence.

### Sequencing

The sequencing results were returned in the form of .ab1 files. They were then imported into Benchling for alignment with the expected sequence to check if there are any mutations detected. Comparisons have shown that no mutations are found in some of the samples. They will then be used for protein expression.

![](https://static.igem.org/mediawiki/2021/a/a6/T--HK_CPU-WFN-WYY--SeqResults1.png)

Figure 5a: Sequence alignment for TuSp

![](https://static.igem.org/mediawiki/2021/8/8f/T--HK_CPU-WFN-WYY--SeqResults2.png)

Figure 5b: Sequence alignment for Piri6

### Sponsors:

[![](https://static.igem.org/mediawiki/2021/8/88/T--HK_CPU-WFN-WYY--Home_Sponsor_idt.svg)](https://sg.idtdna.com/pages)[![](https://static.igem.org/mediawiki/2021/2/2f/T--HK_CPU-WFN-WYY--Home_Sponsor_benchling.svg)](https://www.benchling.com/)

[![](https://static.igem.org/mediawiki/2021/5/51/T--HK_CPU-WFN-WYY--Mathworks.png)](https://www.mathworks.com/)[![](https://static.igem.org/mediawiki/2021/3/30/T--HK_CPU-WFN-WYY--Home_Sponsor_twist.svg)](https://www.twistbioscience.com/)

### Contact us!

[![](https://static.igem.org/mediawiki/2021/6/64/T--HK_CPU-WFN-WYY--Home_IG_icon.svg)](https://www.instagram.com/igem_cpuwfnwyy/) @hk.cpuwfnwyy

![](https://static.igem.org/mediawiki/2021/1/1f/T--HK_CPU-WFN-WYY--Home_Gmail_icon.svg) hk.cpuwfnwyy@gmail.com