iGEM 2018 — Team Hong_Kong_JSS

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# Team:Hong Kong JSS/Notebook

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PROJECT

[Description](http://2018.igem.org/Team%3AHong_Kong_JSS/Description)
[Design](http://2018.igem.org/Team%3AHong_Kong_JSS/Design)
[Experiments](http://2018.igem.org/Team%3AHong_Kong_JSS/Experiments)
[Notebook](http://2018.igem.org/Team%3AHong_Kong_JSS/Notebook)
[InterLab](http://2018.igem.org/Team%3AHong_Kong_JSS/InterLab)
[Model](http://2018.igem.org/Team%3AHong_Kong_JSS/Model)
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[Demonstrate](http://2018.igem.org/Team%3AHong_Kong_JSS/Demonstrate)
[Improve](http://2018.igem.org/Team%3AHong_Kong_JSS/Improve)
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PARTS

[Parts Overview](http://2018.igem.org/Team%3AHong_Kong_JSS/Parts)
[Basic Parts](http://2018.igem.org/Team%3AHong_Kong_JSS/Basic_Part)
[Composite Parts](http://2018.igem.org/Team%3AHong_Kong_JSS/Composite_Part)
[Part Collection](http://2018.igem.org/Team%3AHong_Kong_JSS/Part_Collection)

[SAFETY](http://2018.igem.org/Team%3AHong_Kong_JSS/Safety)

HUMAN PRACTICES

[Human Practices](http://2018.igem.org/Team%3AHong_Kong_JSS/Human_Practices)
[Education & Engagement](http://2018.igem.org/Team%3AHong_Kong_JSS/Public_Engagement)

AWARDS

[Applied Design](http://2018.igem.org/Team%3AHong_Kong_JSS/Applied_Design)
[Entrepreneurship](http://2018.igem.org/Team%3AHong_Kong_JSS/Entrepreneurship)
[Hardware](http://2018.igem.org/Team%3AHong_Kong_JSS/Hardware)
[Measurement](http://2018.igem.org/Team%3AHong_Kong_JSS/Measurement)
[Model](http://2018.igem.org/Team%3AHong_Kong_JSS/Model)
[Plant](http://2018.igem.org/Team%3AHong_Kong_JSS/Plant)
[Software](http://2018.igem.org/Team%3AHong_Kong_JSS/Software)

[JUDGING FORM ⇗](http://igem.org/2018_Judging_Form?team=Hong_Kong_JSS)

[**Hong Kong JSS**](http://2018.igem.org/Team%3AHong_Kong_JSS)

About

[Our Team](http://2018.igem.org/Team%3AHong_Kong_JSS/Team)
[Collaboration](http://2018.igem.org/Team%3AHong_Kong_JSS/Collaborations)
[Contact](http://2018.igem.org/Team%3AHong_Kong_JSS/Contact)

Project

[Description](http://2018.igem.org/Team%3AHong_Kong_JSS/Description)
[Design](http://2018.igem.org/Team%3AHong_Kong_JSS/Design)
[Experiment](http://2018.igem.org/Team%3AHong_Kong_JSS/Experiments)
[Results](http://2018.igem.org/Team%3AHong_Kong_JSS/Results)
[Attributions](http://2018.igem.org/Team%3AHong_Kong_JSS/Attributions)
[Notebook](http://2018.igem.org/Team%3AHong_Kong_JSS/Notebook)
[InterLab](http://2018.igem.org/Team%3AHong_Kong_JSS/InterLab)

Parts

[Parts Overview](http://2018.igem.org/Team%3AHong_Kong_JSS/Parts)
[Basic Parts](http://2018.igem.org/Team%3AHong_Kong_JSS/Basic_Part)
[Composite Parts](http://2018.igem.org/Team%3AHong_Kong_JSS/Composite_Part)

[Safety](http://2018.igem.org/Team%3AHong_Kong_JSS/Safety)

Human Practices

[Human Practices](http://2018.igem.org/Team%3AHong_Kong_JSS/Human_Practices)
[Education and Engagement](http://2018.igem.org/Team%3AHong_Kong_JSS/Public_Engagement)

[Judging Form](http://igem.org/2018_Judging_Form?team=Hong_Kong_JSS)

# Notebook

### Interview with Goldfish Market shops

Date: 12th April

We have interviewed a number of shop owners in Goldfish Market to know more about whether they are facing the problem of water containing heavy metals. We have also asked about how do they tackle the problem and the frequency of doing it.

### Video Meeting with Team:Hong\_Kong-CUHK

Date: 13th April

We have our first video meeting with Team:Hong\_Kong-CUHK. We have briefly introduced both of our projects and exchanged some idea on it. We have also discussed the details of our collaboration, including borrowing their lab for our interlab experiment and suggesting a meetup for all Hong Kong teams that will attend iGEM this october.

### Ordering of MT1A gene

Date: 14th May

We have ordered our design MT1A gene part from IDT through the provided 20 kb of DNA. Thanks IDT for the generosity so that we can reduce the cost of our projects.

### Ordering of EhMT1 gene

Date: 6th June

We have ordered our design EhMT1 gene part from IDT through the provided 20 kb of DNA. Thanks IDT for the generosity so that we can reduce the cost of our projects.

### Collect Water Sample from Goldfish Market

Date: 25th June

We have collected 11 sample from Goldfish Market to study the seriousness of water containing copper content in a business aspect. It is pleasure that a number of shops had cooperated with us by providing numerous water sample.

### Learning of Wiki Building

Date: 4th-5th July

We have learned how to build our Wiki page from our senior members. We hope that our Wiki page can help judges and other iGEM teams to know more about our project.

### Team Wiki

Date: 4th-7th July

We have started creating the wiki web page. We started creating the content for wiki. The first draft of all contents are done.

### Interlab Study

Date: 17th-23th July

We have completed our interlab study in Team:Hong\_Kong-CUHK’s lab. We hope that the interlab study can increase the accuracy of measuring GFP in different labs.

### Assemble of DIY Colorimeter

Date: 20th-21st July

We have designed and assembled a DIY colorimeter to record the blue level out of RGB values of water samples so that we can know the absorbance of heavy metal without using a professional colorimeter. We hoped that this could help the public to measure absorbance of heavy metal in a easier way.

### Wiki page list

Date: 28th July

A brief page list is created for wiki. The list listed the pages we need to create so that we can have a clear direction while creating the wiki content.

### Meeting with Team:Hong\_Kong\_HKUST

Date: 2nd August

We have our first meeting with Team:Hong\_Kong-HKUST in our school. We have exchanged our project and raised a few questions for both projects. We have also discussed some ideas in terms of biosafety.

### iGEM Training Camp

Date: 3rd-5th August

We held the Joint School Biotech Training Camp 2018 from 3rd to 5th of August with the aim of training high school students to participate iGEM competition in the future. A total of eight high schools have joined the camp and students are taught with different laboratory skills such as PCR and transformation. There is also a talk by professor Chau,King Lau. We hoped the camp can introduce iGEM to the public.

### Experiment

Date:15th - 20th August

We designed protocols for our wet lab. We immediately started our experiment after that.

First of all, we calibrated the colorimeter, which was the machine we used to do measurement of absorbance of copper solution and cell culture in SOB medium in our experiment, by undergoing assay 2, which will be detailedly explained in the experiment page, in order to found out the optimal wavelength of filter in colorimeter and concentration range of copper(II) sulphate aqueous solution after copper testing kit treatment.

Secondly, we underwent assay 3, which will also be detailedly explained in the experiment page, to generate a standard curve of absorbance against copper concentration, allowing us to estimate the copper concentration from the absorbance with colorimeter after adding copper test solution copper.

After then, we did assay 4 on the two successfully transformed parts BBa\_K2578610(EhMT1) and BBa\_K2578810(MT1A), which the process took two days. On the first day of assay 4, we grew up cell culture that will be used in the assay in SOB medium with ampicillin for 18 ours. On the second day, we put our genetically engineered bacterias and control factor empty vector bacteria into copper(II) sulphate solution, and compare their copper absorption ability my measuring the concentration of the copper(II) sulphate solutions inoculated with different experimental bacterias, with the aid of colorimeter and standard curve.

On the same day we finished doing assay 4, we did assay 5, in order to confirm that bacteria can’t pass through the kind of dialysis tubing we used to make the bacterial copper filtration device but copper ions can, as we have the responsibility to prove our product can help the bacteria inside absorb copper ions in water and won’t cause leakage of bacteria to the environment, causing pollution.

### Data collection

Date: 20th August

We collected the result of the wet lab. The experimental result of assay 4 disproves our hypothesis about the enhancing ability on copper absorption of bacteria by the metallothionein in vivo expression, which it reveals that the engineered bacteria give a similar ability on copper absorption with bacteria not engineered (control experiment empty vector bacteria). Therefore, we gathered and discussed the experimental result, trying to find out the reason for the disprovement of our hypothesis, which several reasons were discussed like the fluctuation of cross-species gene expression.

Besides, the result of assay 5 confirms that the kind of dialysis tubing we used is impermeable to our engineered bacteria but permeable to copper ions, proving our device won’t cause biopollution on the environment normally and allow bacterias inside the device absorb copper ion from the aqueous environment.

### Interview Experts

Date: 20th August

We have interviewed a number of experts in the aspect of aquaponics through phone to learn about the impacts of high copper concentration brings to the environment. We will like to thanks Mr. Gary Wong for helping us.

### Ordering of MymT gene

Date: 27th August

We have ordered our designed MymT gene part from IDT through the provided 20 kb of DNA. Thanks IDT for the generosity so that we can reduce the cost of our projects.

### Title and abstract

Date: 5th September

We have created the title and abstract for our team. The draft is corrected and uploaded.

### iGEM sharing session

Date: 22th September

We have hosted a sharing session between Team Hong\_Kong\_JS\_S,Team Hong\_Kong\_UST and Team HK\_HCY\_LFC. Each team presented their project to each other, and different teams asked questions based on their project. We hoped that this can improve the presentation at iGEM for each team.

[〈

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 Attributions](http://2018.igem.org/Team%3AHong_Kong_JSS/Attributions)

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 InterLab

〉](http://2018.igem.org/Team%3AHong_Kong_JSS/InterLab)

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## Hong Kong JSS

### Contact

hkjsigem@gmail.com


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# Team:Hong Kong JSS/Experiments

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[HOME](https://2018.igem.org/Team%3AHong_Kong_JSS)

TEAM

[Team Members](https://2018.igem.org/Team%3AHong_Kong_JSS/Team)
[Collaborations](https://2018.igem.org/Team%3AHong_Kong_JSS/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AHong_Kong_JSS/Description)
[Design](https://2018.igem.org/Team%3AHong_Kong_JSS/Design)
[Experiments](https://2018.igem.org/Team%3AHong_Kong_JSS/Experiments)
[Notebook](https://2018.igem.org/Team%3AHong_Kong_JSS/Notebook)
[InterLab](https://2018.igem.org/Team%3AHong_Kong_JSS/InterLab)
[Model](https://2018.igem.org/Team%3AHong_Kong_JSS/Model)
[Results](https://2018.igem.org/Team%3AHong_Kong_JSS/Results)
[Demonstrate](https://2018.igem.org/Team%3AHong_Kong_JSS/Demonstrate)
[Improve](https://2018.igem.org/Team%3AHong_Kong_JSS/Improve)
[Attributions](https://2018.igem.org/Team%3AHong_Kong_JSS/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AHong_Kong_JSS/Parts)
[Basic Parts](https://2018.igem.org/Team%3AHong_Kong_JSS/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AHong_Kong_JSS/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AHong_Kong_JSS/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AHong_Kong_JSS/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AHong_Kong_JSS/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AHong_Kong_JSS/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AHong_Kong_JSS/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AHong_Kong_JSS/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AHong_Kong_JSS/Hardware)
[Measurement](https://2018.igem.org/Team%3AHong_Kong_JSS/Measurement)
[Model](https://2018.igem.org/Team%3AHong_Kong_JSS/Model)
[Plant](https://2018.igem.org/Team%3AHong_Kong_JSS/Plant)
[Software](https://2018.igem.org/Team%3AHong_Kong_JSS/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Hong_Kong_JSS)

[**Hong Kong JSS**](https://2018.igem.org/Team%3AHong_Kong_JSS)

About

[Our Team](https://2018.igem.org/Team%3AHong_Kong_JSS/Team)
[Collaboration](https://2018.igem.org/Team%3AHong_Kong_JSS/Collaborations)
[Contact](https://2018.igem.org/Team%3AHong_Kong_JSS/Contact)

Project

[Description](https://2018.igem.org/Team%3AHong_Kong_JSS/Description)
[Design](https://2018.igem.org/Team%3AHong_Kong_JSS/Design)
[Experiment](https://2018.igem.org/Team%3AHong_Kong_JSS/Experiments)
[Results](https://2018.igem.org/Team%3AHong_Kong_JSS/Results)
[Attributions](https://2018.igem.org/Team%3AHong_Kong_JSS/Attributions)
[Notebook](https://2018.igem.org/Team%3AHong_Kong_JSS/Notebook)
[InterLab](https://2018.igem.org/Team%3AHong_Kong_JSS/InterLab)

Parts

[Parts Overview](https://2018.igem.org/Team%3AHong_Kong_JSS/Parts)
[Basic Parts](https://2018.igem.org/Team%3AHong_Kong_JSS/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AHong_Kong_JSS/Composite_Part)

[Safety](https://2018.igem.org/Team%3AHong_Kong_JSS/Safety)

Human Practices

[Human Practices](https://2018.igem.org/Team%3AHong_Kong_JSS/Human_Practices)
[Education and Engagement](https://2018.igem.org/Team%3AHong_Kong_JSS/Public_Engagement)

[Judging Form](https://igem.org/2018_Judging_Form?team=Hong_Kong_JSS)

# Experiment

# Assay

In order to design suitable experiments and to test the effectiveness of our bacteria, we carry out five assays. Firstly, as we hope our result is beneficial and useful to the society, we performed Assay 1 to find out the range of concentration of copper ions in the water we use daily. Then, we used assay 2 to determine which copper concentration, wavelength of filter and colorimeter will generate the best results. After that, we made our standard curve by assay 3 so that we can use it to estimate the change in concentration of copper in assay 4. We also conducted assay 5 and assay 6 to test the feasibility of the device that we proposed which the designs of our assays will be discussed detailedly below.

## Assay 1 : Heavy metal test of water sample collected from Goldfish Market and households

### Abstract of assay

Measuring the copper concentration of water sample collected from Goldfish Market and households with the aid of Salifert Copper Test Kit.

### Aim of Experiment

To know the copper concentration of both water samples from households and Goldfish Market, and determine the seriousness of copper pollution problem in Hong Kong.

### Protocols

1. Add 2 mL of water sample into Salifert Profit test kit test vial.
2. Add 5 drops of the Salifert Profit Copper reagent to the test vial and swirl gently for 5 seconds.
3. Allow to stand for 10 minutes .
4. Hold the test vial in front of eyes, compare the colour of solution in the test vial with the colour chart provided by the Salifert copper test kit.

![](https://static.igem.org/mediawiki/2018/8/8d/T--Hong_Kong_JSS--exp01.jpeg)

Colour Chart

### Reference

- About Salifert Copper Test Kit:

[https://www.marinedepot.com/test\_kits\_salifert\_copper\_information-ap.html"](https://www.marinedepot.com/test_kits_salifert_copper_information-ap.html)

## Assay 2 : Absorption spectrum of copper solution after adding API copper test solution

### Abstract of assay

In a high school labobatory, we do not have any specialised equipments for testing copper concentration directly. Moreover, using naked eye and color chart of testing kits to determine the copper concentration is not accurate enough for our purpose. Therefore, we modified the protocol and using 2 colorimeter to measure the absorbance of reaction mixture.

### Aim of Experiment

The aim of these trials is finding the optimal wavelength of filter in colorimeter and concentration range of copper(II) sulphate aqueous solution after copper testing kit treatment. These settings and initial concentration were selected for performing assay 3 and 4.

### Protocols

**Test 1. Absorption spectrum of 10 mg/L copper solution**

1. Prepare 10 mg/L Copper solution from Copper(II) sulphate powder.
2. Transfer 5 ml Copper solution to a test tube.
3. Add 10 drops of API Copper testing solution into the copper solution.
4. Wait 30 seconds.
5. Transfer 2.5 ml of the reaction mixture into a clean cuvette. Insert the cuvette into colorimeter.
6. Measure the absorption spectrum of the solution at different wavelength by colorimeter No. 6 and No. 9.

**Test 2. Absorbance of copper solution at different concentration**

1. Label four test tubes 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, 10 mg/L.
2. Add 4.5, 4, 3.5, 3 ml of distilled water into the test tubes respectively.
3. Add 0.5, 1, 1.5, 2 ml of copper into the test tubes respectively.
   Add 175 ul of API Copper ions testing solution into each tube.
4. Wait 30 seconds.
5. Measure the absorbance of each solution (in cuvette) by using colorimeter.

To further improve the accuracy and consistency of future experiments, the unit of API test solution has been changed from 5 drops to 175 ul which is approximately the same volume. Because using drops has a larger error than using micropipette when dripping.

### Reference

- Method of handling SMART 2 colorimeter :

<https://www.omega.com/manuals/manualpdf/M3780.pdf>

- API copper test kit instructions :

<https://www.apifishcare.com/product.php?p=downloads&id=584>

## Assay 3 : Standard curve of absorbance against copper concentration in SOB medium

### Abstract of assay

Prepare 5 standard copper solution in an ascending order start from 0 mg/L to 10 mg/L. Using diluted water to standardize the colorimeter to scan blank. Use colorimeter to measure the absorbance of the six standard solution ascendingly according to their concentration. Relationship between absorbance and copper concentration can be established.

### Aim of Experiment

To plot a standard curve of absorbance against copper concentration, allowing us to estimate the copper concentration from the absorbance with colorimeter after adding copper test solution copper.

### Protocols

1. Dilute 1000 mg/L copper solution into 100 mg/L.
2. Label five test tubes 0 mg/L, 2 mg/L, 2.5 mg/L, 5 mg/L, 7.5 mg/L, 10 mg/L.
3. Mix the copper solution with SOB medium according to following table.

| Concentration of copper in SOB medium (mg/L) | Volume of SOB medium (mL) | Volume of 100 mg/L copper solution (uL) |
| --- | --- | --- |
| 0 | 2.5 | 0 |
| 2 | 2.45 | 50 |
| 2.5 | 2.4375 | 62.5 |
| 5 | 2.375 | 125 |
| 7.5 | 2.3125 | 187.5 |
| 10 | 2.25 | 250 |

4. Add 175 ul of API Cu2+ test solution into each tube
5. Measure the absorbance of each solution by using SMART 2 colorimeter.
6. Record the result and construct a standard curve.

### Reference

- Method of handling SMART 2 colorimeter :

<https://www.omega.com/manuals/manualpdf/M3780.pdf>

## Assay 4 : Test for copper adsorption ability on engineered *E. coli*

### Abstract of assay

In order to test the copper adsorption ability of *E. coli* transformed with Metallothionein (MT) gene. The engineered *E. coli* were grew in SOB medium overnight. Copper(II) sulphate solution were added to the bacterial culture. The concentration of copper ions were measured after 0 hour, 2 hours and 4 hours respectively. The absorption ability were determined by comparing the copper concentration before and after the incubation.

### Aim of Experiment

The aim of doing these assays was to test whether the three MT genes can enhance the copper absorbing ability of *E. coli*, and which of these MT genes has the most significant effect.

### Protocols

**Growing up cell culture**

1. Label *E. coli* cell culture with EhMT1 gene inserted as A1,MT1A gene inserted as B1, MymT gene inserted as C1, and empty vector as E1.
2. Dissolve 7.02 g SOB medium in 250 ml distilled water.
3. Autoclave six 250ml empty conical flask.
4. Autoclave six flask of 40 ml SOB medium in 250 ml conical flask.
5. Prepare 1000X ampicillin (100mg/ml) by dissolving 1g ampicillin in 10 ml distilled water.
6. **Caution**: Wear face protection (mask, safety goggles etc.), gloves and turn off air conditioner, in order to prevent powder getting into our body or flowing to the environment. (Ampicillin is corrosive and irritant)
7. Wait until the SOB medium to cool down to room temperature.
8. Label the two flask with A1/B1/C1 and E1 respectively.
9. Add 40 uL of 1000X ampicillin into each flask with 40 ml SOB medium.
10. Inoculate respective cell culture into 2 conical flask with SOB medium.
11. Put magnetic stirrer into A1/B1/C1 and E1 then leave it in room temperature for 18 hours.

**Comparing the copper absorption ability of engineered *E. coli***

1. Rinse the test tubes with distilled water then autoclave it.
2. Label 12 test tubes (A1/B1/C1, E1 & SOB/0, 2, 4 & 10 mg/L, 2 mg/L) as the following table.

| Cell culture, copper concentration | 1st incubation period | 2nd incubation period | 3rd incubation period |
| --- | --- | --- | --- |
| A1/B1/C1, 10 mg/L | 0 hours | 2 hours | 4 hours |
| A1/B1/C1, 2 mg/L | 0 hours | 2 hours | 4 hours |
| E1, 10 mg/L | 0 hours | 2 hours | 4 hours |
| E1, 2 mg/L | 0 hours | 2 hours | 4 hours |

3. Transfer 4.5ml of cell culture in 250ml conical flask into respective test tubes.
4. Prepare 50 ml of 100 mg/L copper solution by mixing 5 ml of 1000 mg/L copper solution with 45 ml of distilled water.
5. Transfer 0.5ml of 100 mg/L copper solution into all respective test tubes.
6. Prepare 50 ml of 20 mg/L copper solution by mixing 1 ml of 1000 mg/L copper solution with 49 ml of distilled water.
7. Transfer 0.5ml of 20 mg/L copper solution into all respective test tubes.
8. Incubate all 0hrs, 2hrs, 4hrs test tubes at 37’°Cat its respective time.
9. After incubation, transfer cell culture and copper mixture to two 1.5ml tubes.
10. Centrifuge all tubes at 6000g for 3 mins at 4’C.
11. Extract 2.5ml of supernatant into a cuvette.
12. Transfer 175ul of API copper test solution into each.
13. Wait for 1 minute. Measure the result with a colorimeter using water as blank (440nm).
14. Record the result and run the procedure for three times for all A1, B1 and C1. Using E1 as control experiment.

### Reference

- API copper test kit instructions :

<https://www.apifishcare.com/product.php?p=downloads&id=584>

- Method of handling SMART 2 colorimeter :

<https://www.omega.com/manuals/manualpdf/M3780.pdf>

## Assay 5 : Test for the permeability of dialysis tubing

### Abstract of assay

We designed a bacterial copper filtration device which can circulate *E. coli* culture inside dialysis tubing. We believe that due to the pore size of dialysis tubing, bacteria cannot move across the tubing while copper ion can, thus allowing the bacteria to absorb copper ion in water without letting the transgenic bacteria out to the environment.

### Aim of Experiment

1. Confirm copper ion can pass through the dialysis tubing
2. Confirm *E. coli* cannot pass through the dialysis tubing

### Protocols

**Testing if copper ion can pass through the dialysis tubing**

1. 100mg/L copper (II) solution was prepared using anhydrous copper (II) sulphate.
2. 10mg/L copper (II) solution was prepared by diluting the 100mg/L copper (II) solution.
3. 180mL of 10mg/L copper (II) solution was added to a 500ml beaker.
4. 20mL of distilled water was added into a 20cm long dialysis tubing.
5. Two knots were tied at two ends of the dialysis tubing.
6. The dialysis tubing was immersed into the beaker as shown in the below figure.

   ![](https://static.igem.org/mediawiki/2018/f/f1/T--Hong_Kong_JSS--exp02a.png)
7. After 2 hours of immersion, the dialysis tubing was removed from the beaker and rinsed with distilled water .
8. The dialysis tubing was cut open and the solution inside was isolated.
9. To tested for copper concentration of the solution, 2.5 mL of the solution was added to a cuvette.
10. 175uL of the API copper test solution was added to the cuvette.
11. After 1 minute, the absorbance was measured with a colorimeter, the result was then compared to standard curve to calculate the copper concentration.
12. The experiment was repeated with 8 and 24 hours of immersion.

**Testing if bacteria can pass through the dialysis tubing.**

1. 180mL of SOB solution and 180uL of Ampicillin (100mg/mL) were added to the beaker.
2. 20mL of SOB solution and 20uL of Ampicillin (100mg/mL) were added into a 20cm long dialysis tubing.
3. Two knots were tied at two ends of the dialysis tubing.
4. The dialysis tubing was immersed into the beaker as shown in the below figure.

   ![](https://static.igem.org/mediawiki/2018/6/62/T--Hong_Kong_JSS--exp03a.png)
5. The SOB with ampicillin in the beaker was inoculated with Ampicillin-resistance *E. coli*.
6. After 24 hours, bacteria growth inside and outside of the dialysis tubing were determined by observing the turbidity of solution.

### Reference

- API copper test kit instructions :

<https://www.apifishcare.com/product.php?p=downloads&id=584>

- Method of handling SMART 2 colorimeter :

<https://www.omega.com/manuals/manualpdf/M3780.pdf>

## Assay 6 : Testing the cardboard colorimeter

### Abstract of assay

In this experiment, we used standard copper (II) solutions to test the reliability of the cardboard colorimeter. The measurement was taken using iphoneX with an iOS Apps “ColorMeter”. Other smartphone (iOS or android) with camera and similar color identification Apps can also be used. In this experiment, since we are detecting an orange color change, a contrasting blue background is used and we will be monitoring the change in blue intensity of the RGB color codes. The absorbance of the solution were calculated by the fomular A = Log10 (I0/I) under Beer’s rule.

### Aim of Experiment

Evaluate the accuracy of the cardboard colorimeter by plotting a standard curve

### Protocols

1. 100mg/L copper (II) solution was prepared using anhydrous copper (II) sulphate.
2. 2.5mg/L, 5mg/L, 7.5mg/L and 10mg/L were prepared by diluting the 100mg/L copper (II) solution.
3. 2.5mL of each copper (II) solution were added to a labeled test tubes.
4. 2.5mL of distilled water was added to a labeled test tube as blank.
5. 175uL of API copper test solution was added to each of the labeled test tubes in step 3-4.
6. After 1 minute, the blue intensity of each of the solution was measured by the smartphone using a color identification Apps “ColorMeter”.
7. The absorbance of the solution were calculated by the fomular A = Log10 (I0/I) whereas I0 is the blue intensity of the blank (distilled water) while I is the blue intensity of the tested sample.
8. The standard curve of absorbance against copper (II) concentration was plotted using the results in step 7.

### Reference

- API copper test kit instructions :

<https://www.apifishcare.com/product.php?p=downloads&id=584>

- Principle of the cardboard colorimeter:

- Beer–Lambert law

<https://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law>

- ColorMeter Apps on iOS

<https://itunes.apple.com/hk/app/colormeter-rgb-colorimeter/id713258885?mt=8>

# Plasmid Cloning

## Composite part cloning

### Abstract

As we ordered our plasmid by custom gene synthesis, which the backbone of the plasmid is pUCIDT (Amp), we did miniprep to obtain the plasmid and did cloning after we conducted assays 1-5. To ligate our gene of interest, MymT producing gene (BBa\_K2578410), EhMT1 producing gene (BBa\_K2578610) and MT1A producing gene (BBa\_K2578810), with the pSB1C3 backbone, which is the shipping standard backbone, we first did restriction enzyme digestion on the ordered plasmid and pSB1C3 backbone by using Pst1 and EcoR1, then we did gel extraction followed by gel purification, and ligate the gene of interest with the pSB1C3 backbone. After that, we transformed that to *E.coli* and obtain the DNA by miniprep.

### Aim

To ligate the gene of interest with the standard backbone pSB1C3, in order to meet the requirement of part submission.

### Protocol

**Miniprep using the Invitrogen™ PureLink™ Quick Plasmid Miniprep Kit**

Before Starting

1. Grow transformed *E. coli*(TOP10) ( BBa\_K2578810, BBa\_K2578610, BBa\_K2578410 ) in 1–5 mL LB medium overnight.

Purification Procedure Using Centrifugation

1. Centrifuge 5 mL of the overnight LB-culture. Remove all medium.
2. Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous.
3. Add 250 μL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogeneous. Do not vortex. Incubate the tube at room temperature for 5 minutes.
4. Add 350 μL Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 × g for 10 minutes.
5. Load the supernatant from step 4 onto a spin column in a 2-mL wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the flow-through and place the column back into the wash tube.
6. (OP) Add 500 μL Wash Buffer (W10) with ethanol to the column. Incubate the column for 1 minute at room temperature. Centrifuge the column at 12,000 × g for 1 minute. Discard the flow-through and place column back into the wash tube.
7. Add 700 μL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 × g for 1 minute. Discard the flow-through and place the column into the wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the wash tube with the flow-through.
8. Place the Spin Column in a clean 1.5-mL recovery tube. Add 75 μL of preheated TE Buffer (TE) to the center of the column. Incubate the column for 1 minute at room temperature.
9. Centrifuge the column at 12,000 × g for 2 minutes (the recovery tube contains the purified plasmid DNA). Discard the column. Store plasmid DNA at 4°C (short-term) or store the DNA in aliquots at −20°C (long-term).

**Restriction enzyme digestion**

1. Prepare one 8-tube strip, BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul), dH2O, NEB Buffer 2, BSA and Restriction Enzymes: EcoRI, PstI.
2. Add 250 ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul to every reaction.
3. Add 2.5ul of NEBuffer 2 to every reaction.
4. Add 0.5ul of BSA to every reaction.
5. Add 0.5ul of EcoRI to every reaction.
6. Add 0.5ul of PstI to every reaction.
7. There should be a total volume of 20 ul of solution in every reaction. Mix well and spin down briefly.
8. Incubate the restriction digest at 37°C for 30 minutes, and then 80°C for 20 minutes to heat kill the enzymes. We incubate in a thermal cycler with a heated lid.

**Gel electrophoresis**

Prepare Agarose Gel

1. Measure 0.6 g Agarose powder and add it to a 200 ml conical flask.
2. Add 60 ml TAE Buffer to the flask to make 1% agarose gel.
3. Cover the opening with aluminium foil.
4. Melt the agarose using the hot plate until the solution becomes clear .
5. Let the solution cool to about 50-55°C using water bath, swirl the flask occasionally to cool evenly.
6. Add 6 ul of SYBR Green dye into the solution. (SYBR Green is light-sensitive, keep it dark).
7. Seal the ends of the casting tray with two layers of tap
8. Place the combs in the gel casting tray.
9. Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear milky white).
10. Carefully pull out the combs and remove the tape.
11. Place the gel in the electrophoresis chamber.
12. Add enough TAE Buffer (About 120 ml) so that there is about 2-3 mm of buffer over the gel.

Loading The Gel

1. Record the order each sample will be loaded on the gel, including who prepared the sample, the DNA template - from what organism did the DNA came from, controls and ladder.
2. Add 2 ul loading buffer on parafilm. (Same number as sample)
3. Carefully pipette 10 ul of each sample and mix with loading buffer.
4. Load the mixed sample to the corresponding well.
5. Pipette 10 ul of the DNA ladder marker into at least one well of each row on the gel.

Running The Gel

1. Place the lid on the gel box, connecting the electrodes.
2. Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected. (Remember – “Run to Red”)
3. Turn on the power supply to about 120 volts.
4. Check to make sure the current is running through the buffer by looking for bubbles forming on each electrode.
5. Check to make sure that the current is running in the correct direction by observing the movement of the blue loading dye – this will take a couple of minutes (it will run in the same direction as the DNA).
6. Let the power run until the blue dye approaches the end of the gel.
7. Turn off the power.
8. Disconnect the wires from the power supply.
9. Remove the lid of the electrophoresis chamber.
10. Using gloves, carefully remove the tray and gel.

**DNA purification from gel by using Invitrogen™ PureLink™ Quick Gel Extraction Kit**

Excising and Dissolving the Gel

1. Equilibrate a water bath or heat block to 50°C.
2. Excise a minimal area of gel containing the DNA fragment of interest.
3. Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g.
4. Add Gel Solubilization Buffer (L3) to the excised gel in the tube size indicated in the following table:

| Gel | Tube | Buffer L3 Volume |
| --- | --- | --- |
| ≤2% agarose | 1.7-mL polypropylene | 3:1 (i.e., 1.2 mL Buffer L3: 400 mg gel piece) |
| >2% agarose | 5-mL polypropylene | 6:1 (i.e., 2.4 mL Buffer L3: 400 mg gel piece) |

5. Place the tube with the gel slice and Buffer L3 into a 50°C water bath or heat block. Incubate the tube at 50°C for 10 minutes . Invert the tube every 3 minutes to mix and ensure gel dissolution.

   **Note**: High concentration gels (>2% agarose) or large gel slices may take longer than 10 minutes to dissolve.
6. After the gel slice appears dissolved, incubate the tube for an additional 5 minutes .
7. For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
8. Purify the DNA using a Centrifuge.

Purifying DNA Using a Centrifuge

1. Add ethanol to the Wash Buffer (W1) according to the label on the bottle provided by the testkit set.
   Pipet the dissolved gel piece onto a Quick Gel Extraction Column inside a Wash Tube. Use 1 column per 400 mg of agarose gel.
   **Note**: The column reservoir capacity is 850 µL.
   Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
2. Add 500 µL Wash Buffer (W1) containing ethanol to the column.
3. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
4. Centrifuge the column at maximum speed for 1–2 minutes . Discard the flow-through.
5. Place the column into a Recovery Tube. Add 50 µL Elution Buffer (E5) to the center of the column. Incubate the tube for 1 minute at room temperature.
6. Centrifuge the tube at >12,000 × g for 1 minute.
7. Store the purified DNA in elution tube at 4°C for immediate use or at −20°C for long-term storage.

**Ligation**

1. Add 2 ul of digested plasmid backbone (25 ng).
2. >Add 2 ul of EcoRI-HF PstI digested fragment.
3. Add 1 ul T4 DNA ligase buffer.

   Note: Do not use quick ligase.
4. Add 0.5 ul T4 DNA ligase.
5. Add water to 10 ul.
6. Ligate 16C/30 min, heat kill 80C/20 min.
7. Transform with 1-2 ul of product.

**Single tube transformation**

1. Resuspend DNA with 10µl dH20. Pipet up and down several times, let sit for a few minutes . Resuspension will be red from cresol red dye.
2. Label 1.5ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.
3. Thaw competent cells on ice, this may take 10-15 minutes for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
4. Pipette 50µl of competent cells into 1.5 ml tube, 50µl in a 1.5ml tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5ml tube for your control.
5. Pipette 1µl of resuspended DNA into 1.5 ml tube, pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
6. Pipette 1µl of control DNA into 2 ml tube, pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
7. Close 1.5ml tubes, incubate on ice for 30 minutes, tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
8. Heat shock tubes at 42°C for 45 sec, 1.5ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
9. Incubate on ice for 5 minutes, Return transformation tubes to ice bucket.
10. Pipette 950µl SOC media to each transformation, SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.
11. Incubate at 37°C for 1 hours, shaking at 200-300rpm
12. Pipette 100 µL of each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
13. Spin down cells at 6800g for 3 minutes and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates, spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
14. Incubate transformations overnight (14-18 hours) at 37°C, incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
15. Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.

### Reference

1. Restriction Digestion :
[http://parts.igem.org/Help:Protocols/Restriction\_Digest](http://parts.igem.org/Help%3AProtocols/Restriction_Digest) [https://drive.google.com/file/d/146gJ1lDpryZF4geOJkIYtExIa0tkdQAE/view?usp=sharing](https://drive.google.com/file/d/146gJ1lDpryZF4geOJkIYtExIa0tkdQAE/view?usp=sharing )2. DNA purification from gel Invitrogen™ PureLink™ Quick Gel Extraction Kit :
<https://assets.thermofisher.com/TFS-Assets/LSG/manuals/purelink_quick_gel_extraction_kit_qrc.pdf>3. Ligation :
[http://parts.igem.org/Help:Protocols/Ligation](http://parts.igem.org/Help%3AProtocols/Ligation)4. Single tube transformation :
[http://parts.igem.org/Help:Protocols/Transformation](http://parts.igem.org/Help%3AProtocols/Transformation)5. Miniprep using the Invitrogen™ PureLink™ Quick Plasmid Miniprep Kit :
<https://assets.thermofisher.com/TFS-Assets/LSG/manuals/purelink_quick_plasmid_qrc.pdf>

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# Results

## Assay 1 : Heavy metal test of water sample collected from Goldfish Market and households

### Abstract of assay

Measuring the copper concentration of water sample collected from Goldfish Market and households with the aid of Salifert Copper Test Kit.

### Aim of Experiment

To know the copper concentration of both water sample from households and Goldfish Market, and determine the seriousness of copper pollution problem in Hong Kong.

### Raw data link:

<https://docs.google.com/spreadsheets/d/1Nold56q7CW6Xpp56Njz2uUGoDbThRocBv--VVLLVoos/edit?usp=sharing>

Table 1.1: Result of shops water samples:

| Copper Concentration (ppm) | Number of samples | Percentage (%) |
| --- | --- | --- |
| 0 | 0 | 0 |
| 0.1 | 10 | 91 |
| 0.25 | 1 | 9 |
| 0.5 | 0 | 0 |

Remark:ppm = mg/L

Table 1.2: Result of households water samples:

| Copper Concentration (ppm) | Number of samples | Percentage (%) |
| --- | --- | --- |
| 0 | 3 | 16 |
| 0.1 | 6 | 31 |
| 0.25 | 4 | 9 |
| 0.5 | 6 | 32 |

Remark:ppm = mg/L

### Graphed data:

Graph 1.1: Copper concentration of water samples from shops

![](https://static.igem.org/mediawiki/2018/a/a2/T--Hong_Kong_JSS--hp-24.png)

Graph 1.2: Copper concentration of water samples from households

![](https://static.igem.org/mediawiki/2018/3/3f/T--Hong_Kong_JSS--hp-25.png)

Graph 1.3: Distribution of copper concentration of water samples from shops

![](https://static.igem.org/mediawiki/2018/4/44/T--Hong_Kong_JSS--hp-22.png)

Graph 1.4: Distribution of copper concentration of water samples from households

![](https://static.igem.org/mediawiki/2018/a/ad/T--Hong_Kong_JSS--Graph_1.4.png)

### Discussion

After collecting the water samples from shops and households,our team tried to test the copper concentration in water by using Salifert Copper Profi test kit.The test result showed that the copper level is not very high, it is in the range of 2 mg/L standard.

It is obvious to see that the copper concentration in household is higher than in shops.

We started to wonder why the copper concentration in household is way much higher than in shops. We figured that shops in Goldfish market usually monitor their water quality in order to keep fishes healthy. Moreover, when the heavy metal concentration in water is too high, shopkeepers will use bio-rings to remove the excess heavy metal. They will also use heavy metal precipitating agent after replacing water. But the procedures of replacing water is complicated as they need to relocate large number of fishes.That is why not many shopkeepers prefer to use replacing water as a method to remove excess heavy metal. As shopkeepers monitor the water quality regularly, shops have lower copper concentration than in households.

## Assay 2 : Absorption spectrum of copper solution after adding API copper test solution

### Abstract of assay

In a high school labobatory, we do not have any specialised equipments for testing copper concentration directly. Moreover, using naked eye and color chart of testing kits to determine the copper concentration is not accurate enough for our purpose. Therefore, we modified the protocol and using 2 colorimeters to measure the absorbance of reaction mixture.

### Aim of Experiment

The aim of these trials is to find the optimal wavelength of filter in colorimeter and concentration range of copper(II) sulphate solution after copper testing kit treatment. These settings and initial concentration were selected for performing assay 3 and 4.

### Raw data link:

<https://docs.google.com/spreadsheets/d/1nlB9lhyvsyBQvvC7mKIuv_oJOk9hNN_gjTaATzl9HV8/edit?usp=sharing>

Table 2.1: Absorption spectrum of 10 mg/L copper solution (Test 1)

| Wavelength of filter / nm | Absorbance using colorimeter No.6 | Absorbance using colorimeter No.9 |
| --- | --- | --- |
| 440 | 1.07 | 1.08 |
| 470 | 0.95 | 1.02 |
| 490 | 0.65 | 0.72 |
| 520 | 0.56 | 0.61 |
| 550 | 0.42 | 0.42 |
| 580 | 0.32 | 0.32 |

Table 2.2: Absorbance of copper solution at different concentration (Test 2)

| Concentration of copper solution/mg/L | Absorbance using colorimeter No.6 | Absorbance using colorimeter No.9 |
| --- | --- | --- |
| 1 | 0.00 | 0.00 |
| 2 | 0.06 | 0.05 |
| 3 | 0.01 | 0.01 |
| 4 | 0.02 | 0.03 |
| 10 | 1.07 | 1.08 |
| 100 | 2.00 | 2.00 |
| 1000 | 2.00 | 2.00 |

### Graphed data:

[https://docs.google.com/spreadsheets/d/16T8ravctt9e7uaUzUs\_6XqdsAiWYF2-jd5cRdRg8Qo4/edit?usp=sharing](https://docs.google.com/spreadsheets/d/16T8ravctt9e7uaUzUs_6XqdsAiWYF2-jd5cRdRg8Qo4/edit?usp=sharing )

Graph 2.1: Absorption spectrum of 10 mg/L copper solution

![](https://static.igem.org/mediawiki/2018/6/63/T--Hong_Kong_JSS--re-201.png)

Graph 2.2: Absorbance of copper solution at different concentration (1 to 10 mg/L)

![](https://static.igem.org/mediawiki/2018/e/e5/T--Hong_Kong_JSS--re-202.png)

Graph 2.3: Absorbance of copper solution at different concentration (10 to 100 mg/L)

![](https://static.igem.org/mediawiki/2018/e/e0/T--Hong_Kong_JSS--re-203.png)

### Discussion

In test 1, after testing the absorption spectrum of copper(II) sulphate solution using API copper test kit and colorimeter, the absorbance using the 440 nm filter is the highest which is around 1 absorbance unit, others with nm 440 above have absorbances of 1 and below, this shows that the higher the wavelength the lower the absorbance. We choose the 440 nm filter for the following assays because the wavelength that gives the most sensitivity (lower detection limit) for a test factor is the complementary color of the test sample.

In test 2, concentrations of 4 mg/L and below show a absorbance unit below 0.1, the test results of concentrations of 100mg/L and above all show the same absorbance which is 2 absorbance units, the maximum unit of absorbance this colorimeter can measure, and the concentration of 10mg/L shows an absorbance unit around 1,as it is the closest measurable concentration before reaching 100 mg/L, we choose this concentration for the following assays as the maximum concentration.

To further improve the accuracy and consistency of future experiments, the unit of API test solution has been changed from 5 drops to 175 ul which is approximately the same volume. As adding drops has a higher chance of error without accurate measurements, using 175 ul can increase the accuracy in volume by extracting using micropipette. This can further improve the accuracy for our experiments.

Table 2.3: Accuracy of colorimeter No.6 and No.9

| Raw data for colorimeter No. 9 | | | | | |
| --- | --- | --- | --- | --- | --- |
| copper concentration (mg/L) / absorbance (440nm) | 1 | 2 | 3 | 4 | 5 |
| 0 mg/L (blank) | 0.02 | 0.01 | 0 |  |  |
| 2.5 mg/L | 0.1 | 0.09 | 0.11 |  |  |
| 5 mg/L | 0.27 | 0.2 | 0.2 | 0.19 | 0.4 |
| 7.5 mg/L | 0.21 | 0.25 | 0.23 | 0.25 | 0.34 |
| 10 mg/L | 0.46 | 0.34 | 0.32 | 0.29 | 0.45 |
| Raw data for colorimeter No. 6 | | | | | |
| copper concentration (mg/L) / absorbance (440nm) | 1 | 2 | 3 | 4 |  |
| 0 mg/L (blank) | 0 | 0 | 0 |  |  |
| 2.5 mg/L | 0.08 | 0.08 | 0.08 |  |  |
| 5 mg/L | 0.17 | 0.16 | 0.17 |  |  |
| 7.5 mg/L | 0.23 | 0.23 | 0.23 |  |  |
| 10 mg/L | 0.28 | 0.24 | 0.3 | 0.29 |  |

In the above data, colorimeter no.6 shows a smaller value range than colorimeter no.9 in all concentrations, it can be deduced that the accuracy of colorimeter no.6 is higher than that of no.9, so we decided to use colorimeter no.6 in the following assays to improve accuracy.

## Assay 3 : Standard curve of absorbance against copper concentration in SOB medium

### Abstract of assay

Prepare 5 standard copper solution in an ascending order start from 0 mg/L to 10 mg/L. Using diluted water to standardize the colorimeter to scan blank. Use colorimeter to measure the absorbance of the six standard solution ascendingly according to their concentration. Relationship between absorbance and copper concentration can be established.

### Aim of Experiment

To plot a standard curve of absorbance against copper concentration, allowing us to estimate the copper concentration from the absorbance with colorimeter after adding copper test solution copper. Since SOB medium is yellow in color, the absorbance of SOB medium without copper ions have to be studied.

### Raw data link:

<https://docs.google.com/spreadsheets/d/1jlsSUDPuEC-BaEUjWhntt9rtd7S0Zn5-evJ01uvujyk/edit?usp=drivesdk>

Table 3.1: Absorbance of copper solution at different concentration

| Concentration of copper solution/mg/L | Absorbance at 440nm | | | |
| --- | --- | --- | --- | --- |
| Repeat 1 | Repeat 2 | Repeat 3 | Average |
| 0 | 0.2 | 0.19 | 0.19 | 0.19 |
| 2 | 0.36 | 0.37 | 0.38 | 0.36 |
| 2.5 | 0.42 | 0.44 | 0.43 | 0.43 |
| 5 | 0.66 | 0.65 | 0.67 | 0.66 |
| 7.5 | 0.89 | 0.87 | 0.87 | 0.88 |
| 10 | 1.13 | 1.12 | 1.14 | 1.13 |

### Graphed data:

Graph 3.1: Standard Curve of absorbance at 440 nm against copper concentration in SOB medium

![](https://static.igem.org/mediawiki/2018/9/9b/T--Hong_Kong_JSS--ren--301.png)

Remark: Error bar is range of data

### Discussion

In this standard curve, The absorbance value was recorded against SOB medium with 6 different copper concentration. The test was repeated twice to obtain an average value of the absorbance value. An equation was generated to estimate the concentration of copper ions in SOB medium from absorbance at 440 nm. y = 0.0939x + 0.186. y is absorbance of solution in 440 nm while x is the concentration of copper solution in SOB medium. The result of the standard curve shows a linear relationship between absorbance and copper concentration. When the copper concentration increases, the absorbance increases. Water is used as blank reference. The absorbance of SOB medium without copper ions is 0.19 because SOB medium is yellow in color. In assay 4, SOB medium is used for culture medium. Therefore, it is essential to study the relationship between absorbance and copper conconcentartion in SOB medium background. The result show that SOB medium will not affect the linear relationship between absorbance and copper concentration in 0 to 10 mg/L range.

## Assay 4 : test for copper absorption ability of engineered *E.coli*

### Abstract of assay 4A

In preparation of this assay, transformed *E.coli* with MT plasmids (*Homo sapiens* and *Elsholtzia haichowensis* MT1 gene) and plasmid vector without insert (as control setup), are inoculated as *E.coli* colonies and incubated for another 18 hours in SOB medium. We then transferred the transformed *E.coli* in SOB medium to copper(II) sulphate solution, with concentration 2 mg/L and 10 mg/L, in a ratio of 1:9. *E.coli* was removed by centrifugation after 0 hours, 2 hours and 4 hours and 2.5 ml of supernatant was extracted to test for the absorbance after adding 175 ul of copper test solution. Using the standard curve produced by calibrating absorbance of solution against copper concentration in assay 3, we can estimate the copper concentration after incubation. Copper adsorption ability of the engineered *E.coli* can be studied from the information obtained.

Graph 3.1: Standard Curve of absorbance at 440 nm against copper concentration in SOB medium

![](https://static.igem.org/mediawiki/2018/9/9b/T--Hong_Kong_JSS--ren--301.png)

We first conducted the assay with the 10mg/L copper(II) sulphate solution to determine the range of copper ion absorption by bacteria. *E.coli* transformed with *Homo sapiens* MT1 gene, *Elsholtzia haichowensis* MT1 gene are transferred to 10mg/L copper(II) sulphate solution after different incubation interval. And we used bacteria transformed with empty vector and SOB solution as control setups.

We measured the absorbance of the solution and the copper concentration is determined by using the standard curve.

### Results

Table 4.1 Change of absorbance in 0, 2, 4 hours with engineered bacteria

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|| Sample | Absorbance | | | copper concentration, mg/L | | |
| --- | --- | --- | --- | --- | --- | --- |
| 0 h | 2 h | 4 h | 0 h | 2 h | 4 h |
| *Elsholtzia haichowensis* MT1 gene | 1.14 | 0.95 | 0.95 | 10.2 | 8.1 | 8.1 |
| 1.11 | 1.02 | 0.96 | 9.8 | 8.9 | 8.2 |
| *Homo sapiens* MT1 gene | 1.14 | 1.02 | 1.01 | 10.2 | 8.9 | 8.8 |
| 1.14 | 0.99 | 0.85 | 10.2 | 8.6 | 7.1 |
| Empty Vector | 1.14 | 0.98 | 0.94 | 10.2 | 8.5 | 8 |
| 1.14 | 0.94 | 0.78 | 10.2 | 8 | 6.3 |
| SOB medium | 1.18 | 1.18 | 1.18 | 10.6 | 10.6 | 10.6 |
| 1.18 | 1.18 | 1.18 | 10.6 | 10.6 | 10.6 |

Table 4.2 Copper absorption by engineered bacteria in 0, 2, 4 hours (10 mg/L)

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|| Sample | Inital Concentration of copper, mg/L | Change of concentration, mg/L | | % change of concentration | |
| --- | --- | --- | --- | --- | --- |
| after 2 hour | after 4 hour | after 2 hour | after 4 hour |
| *Elsholtzia haichowensis* MT1 gene | 10.2 | -2 | -2 | -19.90% | -19.90% |
| 9.8 | -1 | -1.6 | -9.70% | -16.20% |
| *Homo sapiens* MT1 gene | 10.2 | -1.3 | -1.4 | -12.60% | -13.60% |
| 10.2 | -1.6 | -3.1 | -15.70% | -30.40% |
| Empty Vector | 10.2 | -1.7 | -2.1 | -16.80% | -21.00% |
| 10.2 | -2.1 | -3.8 | -21.00% | -37.70% |
| SOB medium | 10.6 | 0 | 0 | 0.00% | 0.00% |
| 10.6 | 0 | 0 | 0.00% | 0.00% |

Table 4.3 Average copper absorption by engineered bacteria in 0, 2, 4 hours

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|| Sample | average initial concentration,mg/L | average chage of concentration, mg/L | | average % change of concentration | |
| --- | --- | --- | --- | --- | --- |
| after 2 hour | after 4 hour | after 2 hour | after 4 hour |
| *Elsholtzia haichowensis* MT1 gene | 10 | -1.5 | -1.8 | -15% | -18% |
| *Homo sapiens* MT1 gene | 10.2 | -1.45 | -2.25 | -14% | -22% |
| Empty Vector | 10.2 | -1.9 | -2.95 | -19% | -29% |
| SOB medium | 10.6 | 0 | 0 | 0% | 0% |

Graph 4.1: Effect of engineered bacteria on copper concentration

![](https://static.igem.org/mediawiki/2018/e/e5/T--Hong_Kong_JSS--graph4.1.jpeg)

Graph 4.2: Average decrease in copper concentration after incubation with engineered bacteria

![](https://static.igem.org/mediawiki/2018/e/ee/T--Hong_Kong_JSS--graph4.2.png)

\*Error Bar is standard deviation of estimated copper concentration

Graph 4.3: Percentage decrease in copper concentration after incubation with engineered bacteria

![](https://static.igem.org/mediawiki/2018/5/55/T--Hong_Kong_JSS--Graph4.3.png)

From the result, that is a significant decrease in copper concentration of the solution with *E.coli* with empty vector while comparing it to the SOB solution. While there is no change in the concentration for SOB medium, there is a decrease of 19% of copper concentration for *E.coli* after 2 hours of incubation and a decrease of 29% after 4 hours of incubation. Showing that *E.coli* itself are able to absorb copper ions.

Also, we could see that the copper concentration of the solution with *Homo sapiens* MT1 decrease 0.96 mg/L(14%) after 2 hours and 1.63 mg/L(22%) after 4 hours. Comparing with the control setup, we found the decreases of copper concentration in two setups are similar. Thus, we interpreted that the copper absorbed by *E.coli* transformed with *Homo sapiens* MT1 is similar to that absorbed by untransformed *E.coli*, meaning the present of *Homo sapiens* MT1 gene does not have significant effect in absorbing copper.

The copper concentration of the solution with *Elsholtzia haichowensis* EhMT1 decreased 1.49 mg/L(15%) within 2 hours and 3.02 mg/L(18%) within 4 hours. The concentration change of it and the control setup are similar so it is also interpreted that the copper absorbed by *E.coli* transformed with *Elsholtzia haichowensis* MT1 is similar to that absorbed by untransformed *E.coli*, meaning the present of *Elsholtzia haichowensis* MT1 gene does not have significant effect in absorbing copper.

To conclude, *E.coli* itself can absorb copper ions, *E.coli* with empty vector and transformed bacteria with the *Elsholtzia haichowensis* MT1 gene or *Homo sapiens* MT1 gene show similar copper ion absorbing ability.

### Abstract of assay 4B

According to the World Health Organization’s guidelines for drinking water quality[1], 2 mg/L is the guideline value for copper concentration. We found the copper concentration would decrease around 2 mg/L after incubating the bacteria in 10 mg/L copper solution for 4 hours. We would like to know whether the concentration would decrease to 0 mg/L if the bacteria is incubated in 2 mg/L copper solution, or would the solution reach an equilibrium. Upon the question, we conducted the test of incubating transformed *E.coli* in 2 mg/L copper solution to investigate it.

### Results

Table 4.4 Change of absorbance in 0, 2, 4 hour with engineered bacteria

| Sample | Absorbance | | | copper concentration, mg/L | | |
| --- | --- | --- | --- | --- | --- | --- |
| 0 h | 2 h | 4 h | 0 h | 2 h | 4 h |
| *Elsholtzia haichowensis* MT1 gene | 0.37 | 0.37 | 0.35 | 2.0 | 2.0 | 1.7 |
| *Homo sapiens* MT1 gene | 0.44 | 0.34 | 0.28 | 2.7 | 1.6 | 1.0 |
| Empty Vector | 0.37 | 0.34 | 0.34 | 2.0 | 1.6 | 1.6 |
| Empty Vector | 0.42 | 0.34 | 0.29 | 2.0 | 1.6 | 1.1 |

Table 4.5 Copper absorption by engineered bacteria in 0, 2, 4 hours(2 mg/L)

| Sample | Initial concentration,mg/L | Change of concentration, mg/L | | % change of concentration | |
| --- | --- | --- | --- | --- | --- |
| after 2 hour | after 4 hour | after 2 hour | after 4 hour |
| *Elsholtzia haichowensis* MT1 gene | 2.0 | 0.0 | -0.2 | -0.0% | -10.9% |
| *Homo sapiens* MT1 gene | 2.7 | -1.1 | -1.7 | -39.4% | -63.0% |
| Empty Vector | 2.0 | -0.3 | -0.3 | -16.3% | -16.3% |
| Empty Vector | 2.5 | -0.9 | -1.4 | -34.2% | -55.6% |

Graph 4.4: Effect of engineered bacteria on copper concentration (2 mg/L)

![](https://static.igem.org/mediawiki/2018/c/c0/T--Hong_Kong_JSS--graph4.4.png)

We conducted this assay with two cultures of transformed *E.coli* with empty vector. While there is a decrease of 0.3mg/L (16.3%) in the medium with an initial copper concentration of 2.0mg/L, there is also a decrease of 1.4mg/L(55.6%) in the one with an initial concentration of 2.5mg/L.

For the transformed bacteria with *Elsholtzia haichowensis* MT1 gene, there is a decrease of 0.2 mg/L (10.9%) after 4 hours of incubation in 2mg/L copper solution.

For transformed *E.coli* with *Homo sapiens* MT1 gene, there is a decrease of 1.1mg/L (39.4%) after 2 hours of incubation and 1.7mg/L(63.0%) after 4 hours of incubation in 2mg/L copper solution.

Table 4.6 Comparing copper absorption by engineered bacteria with different initial copper concentration

| Sample | Initial concentration,mg/L | Change of concentration, mg/L | | % change of concentration | |
| --- | --- | --- | --- | --- | --- |
| after 2 hour | after 4 hour | after 2 hour | after 4 hour |
| *Elsholtzia haichowensis* MT1 gene | 9.8 | -1.0 | -1.6 | -9.7% | -16.2% |
| 2.0 | -0.0 | -0.2 | -0.0% | -10.9% |
| *Homo sapiens* MT1 gene | 10.2 | -1.6 | -3.1 | -15.7% | -30.4% |
| 2.7 | -1.1 | -1.7 | -39.4% | -63.0% |
| Empty Vector(Set A) | 10.2 | -1.7 | -2.1 | -16.8% | -21.0% |
| 2.0 | -0.3 | -0.3 | -16.3% | -16.3% |
| Empty Vector(set B) | 10.2 | -2.1 | -3.8 | -21.0% | -37.7% |
| 2.5 | -0.9 | -1.4 | -34.2% | -55.6% |

Graph 4.5: Decrease in copper concentration with different initial copper concentration for EhMT1 transformant

![](https://static.igem.org/mediawiki/2018/3/31/T--Hong_Kong_JSS--graph4.5.png)

For transformed bacteria with *Elsholtzia haichowensis* MT1 gene. It shows similar percentage decrease of copper concentration when they are tested in different initial copper concentration. While there is a decrease of 1.60mg/L (16.2%) when the *E.coli* are incubated in 10mg/L copper solution for 4 hours, there is also a decrease of 0.23 mg/L (10.9%) for solution with *E.coli* incubated in 2mg/L copper solution for the same amount of time. It shows that the net absorption of transformed bacteria with *Elsoltzia haichowensis* MT1 gene is concentration dependent.
.

Graph 4.6: Decrease in copper concentration with different initial copper concentration for MT1A transformant

![](https://static.igem.org/mediawiki/2018/b/b6/T--Hong_Kong_JSS--graph4.6.png)

For transformed bacteria with *Homo Sapiens* MT1 gene. The difference of the percentage decrease of copper concentration when they are tested in different initial copper concentration is large. While there is a decrease of 3.09mg/L (30.4%) when the *E.coli* are incubated in 10mg/L copper solution for 4 hours, there is also a decrease of 1.70 mg/L (63%) for solution with *E.coli* incubated in 2mg/L copper solution for the same amount of time.

However, when we look at the data, the initial absorbance it show is 0.44 that the concentration of copper in the solution is 2.7mg/L according to the standard curve which it supposed to be 2.0mg/L. It shows that this set of data is unreliable. We hoped to conduct the assay again to obtain a more accurate set of data but we failed to do so due to time limit.

Graph 4.7: Decrease in copper concentration with different initial copper concentration for empty vector transformant

![](https://static.igem.org/mediawiki/2018/a/a1/T--Hong_Kong_JSS--Graph4.7.png)

For the transformed bacteria with empty vector (Set A) it shows similar percentage decrease of copper concentration when they are tested in different initial copper concentration. While there is a decrease of 1.70mg/L (16.8%) when the *E.coli* are incubated in 10mg/L copper solution for 2 hours, there is also a decrease of 0.32 mg/L (16.3%) for solution with *E.coli* incubated in 2 mg/L copper solution for the same amount of time. It shows that the net copper ion absorption rate of transformed bacteria with empty vector (Set A) is concentration dependent.

For Set B of *E.coli* transformed with empty vector, the difference of the percentage change between the two different initial copper concentration is large, while there is a decrease of 3.83mg/L (33.7%) when the *E.coli* are incubated in 10mg/L copper solution for 4 hours, there is also a decrease of 1.38mg/L (55.6%) for solution with *E.coli* incubated in 2mg/L copper solution for the same amount of time. However, we notice that this set of data has a similar problem with the one of *Homo Sapiens* MT1 gene that the initial copper concentration deduced from the standard curve is much larger than it supposed to be. The concentration deduced from the standard is 2.5mg/L which the theoretical concentration is 2.0mg/L. Therefore, the data set B is unreliable, but we failed to perform it again due to time limit.

To conclude, the copper concentration does not decrease from 2 mg/L to 0 mg/L after 4 hours as we expected. Instead, we found that the copper concentration decreased by a similar percentage as the solutions did in the 10 mg/L test. Therefore, we can conclude that the net copper ion absorption rate is concentration dependent.

Graph 4.8: Assay summary of decrease in copper concentration with different initial copper concentration

![](https://static.igem.org/mediawiki/2018/f/fb/T--Hong_Kong_JSS--g4.8.png)

### Conclusion

Here are the few points that we would like to draw after the assays

1. Transforming plasmid with metallothioneins might not be able to enhance *E.coli*’s copper absorption ability.

2. By comparing the percentage of change in copper concentration between solutions with different transformed *E.coli* incubated, we cannot observed a significant difference between *E.coli* cloned with empty vectors, cloned with *Elsholtzia haichowensis* MT1 gene or *Homo sapiens* MT1 gene.

3. The reason behind might be the failure of expressing eukaryotic genes in prokaryotic cells such as *E.coli*.

4. By comparing the results from the assays using 2mg/L and 10mg/L, we found that the percentage change of copper concentration is similar between empty vector *E.coli* and transgenic *E.coli*, and both of them can absorb around 20-30% of copper ions after 4 hours of incubation.

### Prospect

In the future, we would like to improve our device by lowering the copper concentration more effectively, causing a greater percentage decrease. Also, we aim to use the findings to design a device to absorb copper ions in water.

When water flows through a dialysis tubing containing *E.coli*, the copper ions will be absorbed by the bacteria, creating a concentration gradient between water inside the dialysis tubing and the water outside the device. Therefore, copper ions diffuse into the tubing and water with low concentration of copper diffuses out from the tubing by osmosis continuously. In order to test if the dialysis tubing is permeable to copper ions and the feasibility of our device, assay 6 is carried out.

Reference: [1]<http://www.who.int/water_sanitation_health/publications/drinking-water-quality-guidelines-4-including-1st-addendum/en/>

## Assay 5 : Test for the permeability of dialysis tubing

### Abstract of assay

We designed a bacterial copper filtration device which can circulate *E.coli* culture inside dialysis tubing. We believe that due to the pore size of dialysis tubing, bacteria cannot move across the tubing while copper ion can, thus allowing the bacteria to absorb copper ion in water without letting the transgenic bacteria out to the environment. The setup of the experiment were shown in the below diagrams.

![](https://static.igem.org/mediawiki/2018/f/f1/T--Hong_Kong_JSS--exp02a.png)
![](https://static.igem.org/mediawiki/2018/2/26/T--Hong_Kong_JSS--exp03.png)

### Aim of Experiment

1. Confirm copper ion can pass through the dialysis tubing

2. Confirm *E.coli* cannot pass through the dialysis tubing

### Raw data:

Concentration of copper (II) ion inside dialysis tubing were measured at 0, 2, 8 and 24 hours after immersion.

| Hour(s) of immersion | Copper (II) ion concentration (mg/L) |
| --- | --- |
| 0 | 0 |
| 2 | 2.42 |
| 8 | 4.69 |
| 24 | 6.05 |

The turbidity of the Ampicillin SOB solution outside and inside of the dialysis tubing were observed after 24 hours.

|  | Outside dialysis tubing | Inside dialysis tubing |
| --- | --- | --- |
| Turbidity (-/+) | + | - |

### Graphed data:

We had confirmed that copper ion can pass through the dialysis tubing, more than 6 mg/L of copper can be detected inside dialysis tubing after 24 hours.

![](https://static.igem.org/mediawiki/2018/6/67/T--Hong_Kong_JSS--assay5_dialysis_copper.jpg)

On the other hand, *E.coli* cannot pass through as after 24 hours, the SOB seeded with Ampicillin-resistance bacteria became turbid while SOB inside dialysis tubing remain clear after 24 hours.

## Assay 6 : Testing the cardboard colorimeter

### Abstract of assay

In this experiment, we used standard copper (II) solutions to test the reliability of the cardboard colorimeter. The measurement was taken using iphoneX with an iOS Apps “ColorMeter”. Other smartphone (iOS or android) with camera and similar color identification Apps can also be used. In this experiment, since we are detecting an orange color change, a contrasting blue background is used and we will be monitoring the change in blue intensity of the RGB color codes. The absorbance of the solution were calculated by the formula A = Log10 (I0/I) under Beer’s rule.

### Aim of Experiment

Evaluate the accuracy of the cardboard colorimeter by plotting a standard curve.

### Raw data:

| Copper Concentration (mg/L) | Blue intensity (RGB) | Absorbance\* |
| --- | --- | --- |
| 0 | 214 | 0 |
| 2.5 | 148 | 0.1602 |
| 5 | 79 | 0.4328 |
| 7.5 | 42 | 0.7072 |
| 10 | 20 | 1.0294 |

\*Absorbance was calculated by log10(Io/I), whereas Io is blue intensity of the blank, which is 214. I is the blue intensity of the measured sample.

### Graphed data:

The standard curve of absorbance against copper (II) concentration was plotted in the graph below.

![](https://static.igem.org/mediawiki/2018/e/e2/T--Hong_Kong_JSS--ren--601.png)

From the graph, we can see that the correlation coefficient (R2) value is >0.98, indicating that there is a strong linear relationship between the absorbance and copper (II) ion concentration from 0 to 10 mg/L. It also implicates that the measurement done with our cardboard colorimeter is reliable.

## Plasmid Cloning result

After ordering the plasmids containing BBa\_K2578610 and BBa\_K2578810, we used the restriction enzymes EcoRI and PstI to digest the plasmids. Then the plasmids were verified and purified by gel electrophoresis. As seen in figure 7.1, the size of EhMT1 producing gene, BBa\_K2578610 (737 bp) and MT1A producing gene, BBa\_K2578810 (1664 bp) are similar to our predicted result. The DNA fragments used to undergo gel electrophoresis include prefix and suffix.

![](https://static.igem.org/mediawiki/2018/e/eb/T--Hong_Kong_JSS--re-602a.png)

| Lane | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
| Gene of interest | BBa\_K2578610 | BBa\_K2578610 | BBa\_K2578810 | BBa\_K2578810 |

Figure 7.1 Restriction enzyme digestion of plasmids by EcoRI and PstI

After verifying the validity of gene ordered from IDT, we did gel purification to extract the gene of interest , BBa\_K2578610 ( 737 bp) and BBa\_K2578810 ( 1664 bp ), were being separated from pUCIDT in the above gel electrophoresis. For the next step, we ligated our gene of interest into plasmid backbone pSB1C3, transformed the plasmid to our competent cell *E.coli* (TOP10), and grew them in separated nutrient agar plate with chloramphenicol. Afterwards, we picked single cell colony and cultured them in liquid nutrient medium. The plasmids with pSB1C3 background were extracted by miniprep protocol. After plasmid extraction, we applied the plasmids to gel electrophoresis to check the existence of the plasmid. It was proven that our plasmid wasn’t lost in either growing up cell and mini prep.

![](https://static.igem.org/mediawiki/2018/a/ac/T--Hong_Kong_JSS--re-603a.png)

Figure 7.2 Plasmids extracted from *E.coli* (TOP10)

To verify the gene of interest, we then did restriction digestion on the plasmid obtained, and did a gel electrophoresis again on the digested plasmid. Figure 7.3 shows the position of pSB1C3 ( 2070 bp), BBa\_K2578610 ( 737 bp) and BBa\_K2578810 (1664 bp), which are similar to our prediction, which means the plasmid cloned is validated to contain the gene of interest. The DNA fragments used to undergo gel electrophoresis include prefix and suffix.

![](https://static.igem.org/mediawiki/2018/9/9e/T--Hong_Kong_JSS--re-604a.png)

| Lane | 1 | 2 | 3 |
| --- | --- | --- | --- |
| Gene of interest | BBa\_K2578610 | BBa\_K2578810 | / |
| Plasmid backbone | pSB1C3 | pSB1C3 | Linearised pSB1C3 |

Figure 7.3 Restriction enzyme digestion of plasmids consisting of gene of interest and pSB1C3 by EcoRI and PstI

Lastly, after doing the above verifying work, we made a conclusion that our parts are correctly cloned and these two composite parts (BBa\_K2578610 and BBa\_K2578810) were submitted to the iGEM headquarters on 3 Oct 2018.

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## Hong Kong JSS

### Contact

hkjsigem@gmail.com