iGEM 2019 — Team Hong_Kong_JSS

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# Team:Hong Kong JSS/Notebook

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**Hong Kong JSS**

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**Notebook**


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# Team:Hong Kong JSS/Experiments

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**Hong Kong JSS**

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**Experiments**

*** [Generating The Standard Curve](#section 1)
* [Quantifying Copper Concentration in Water Samples](#section 2)
* [Bacterial Copper Adsorption Test](#section 3)
* [Testing the Bacterial Copper Adsorption Device (B-CAD)](#section 4)**

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Generating The Standard Curve

**Aim**
A standard curve is a type of graph used as a quantitative research technique. It is used for quantifying the copper level of our samples in different experiments. According to Beer’s Law, A=Ebc, under ideal conditions, a substance’s concentration and its absorbance are directly proportional. Making use of the commercially available API copper test kit, absorbance of multiple samples with known copper concentration is measured and graphed, which then allows the concentration of the unknown samples to be determined by interpolation on the graph.

**Materials and Equipments**

|  |  |
| --- | --- |
| * 500ml volumetric flask * 10ml pipette * 200ul and 1000ul micropipette * Electronic balance | * Colorimeter and cuvettes * API copper testing kit * Anhydrous copper (II) sulphate * Distilled water |

**Procedure**

Preparation of copper standard solutions:

1. 500ml of 1000mg/L copper solution was prepared by adding 1.255g anhydrous copper solution to a 500ml volumetric flask.
2. Distilled water was added to the flask until it reached the graduation marking.
3. The flask was gently inverted several times until all copper (II) sulphate dissolved.
4. A serial dilution was done to prepare standard copper solution of 10, 8, 6, and 2 mg/L

Measuring the absorbance of standard copper solutions:

1. 2.5ml of copper standard copper solution was added to a cuvette.
2. 175ul of API copper testing solution was added to the cuvette.
3. The solution was mixed by pipetting up and down for 10 seconds.
4. Wait 20 seconds for the color to develop.
5. The absorbance (440nm) of the solution was measured by a colorimeter.

Determine the copper level of a sample:

1. Absorbance several copper standard solutions were measured.
2. The graph was plotted by absorbance against concentration.
3. The absorbance of the sample was measured.
4. Copper concentration of the samples was determined by interpolation the absorbance measured on the graph.

![](https://static.igem.org/mediawiki/2019/f/f1/T--Hong_Kong_JSS--stdcurve.jpg)

An example of stand curve used in our experiments.

Quantifying Copper Concentration in Water Samples

**Aim**
Water samples collected from various locations in Hong Kong were tested for the copper level. We aimed to investigate the severity of copper pollution in different types of waters, coming from freshwater habitats, aquarium/aquaculture, seawater and tap water.

![](https://static.igem.org/mediawiki/2019/1/17/T--Hong_Kong_JSS--water_sample_collection.jpg)

Water samples collected in Hong Kong.

**Materials and Equipments**

|  |  |
| --- | --- |
| * 200ul and 1000ul micropipette * Colorimeter and cuvettes | * Water samples * API copper testing kit |

**Procedure**

1. 2.5ml of water samples was added to a cuvette.
2. 175ul of API copper testing solution was added to the cuvette.
3. The solution was mixed by pipetting up and down for 10 seconds.
4. Wait 20 seconds for the color to develop.
5. The absorbance (440nm) of the solution was measured by a colorimeter.
6. The copper concentration of the samples was determined by interpolating the absorbance measured on the standard curve

Bacterial Copper Adsorption Test

Aim
In this experiment, we aimed to test the copper adsorption ability of our genetically modified bacteria. The bacteria will be mixed with copper solution and the concentration of the mixture will be measured at different time intervals.

**Materials and Equipments**

|  |  |
| --- | --- |
| * 200ul and 1000ul micropipette * Colorimeter * Electronic balance * Conical flasks * API copper testing kit | * SOB medium * Ampicillin / Kanamycin * IPTG (optional) * 100mg/L copper solution * Distilled water |

**Procedure**
Preparation of bacterial culture:

1. 7.02 g SOB powder was dissolved in 250 ml distilled water to prepare the SOB medium.
2. 40 ml SOB medium was added to 250 ml conical flask.
3. Each of the conical flask with SOB medium was sterilized by autoclave.
4. Prepare 1000X ampicillin (100mg/ml) by dissolving 1g ampicillin in 10 ml distilled water. *Caution: Wear mask, safety goggles and gloves when handling Ampicillin powder as it is corrosive and irritating.*
5. 40 uL of 1000X ampicillin was added into each flask with 40 ml SOB medium.
6. A single colonie of respective cell culture was inoculated into conical flask with SOB medium.
7. The flasks were incubated in a shaking incubator at 37℃ for 18 hours.

Remarks:

1. According to the selection marker of the plasmid or bacteria strain, Kanamycin or other antibiotics may be used instead of ampicillin.
2. In some experiments, the bacteria needed to be induced by IPTG. To prepare IPTG stock (1000X), 2.38 g of IPTG was dissolved in 10 mL of distilled water. The solution was filter sterilized with a 0.22μm syringe filter.

![](https://static.igem.org/mediawiki/2019/7/78/T--Hong_Kong_JSS--expt_cell_grow.jpeg)

Part 1 of the experiment: Grow up of cell culture and incubation with copper solution.

Testing of the copper absorption ability of genetically engineered *E. coli*:
To take the measurement of the initial copper concentration,

1. 4.5 ml of SOB medium was mixed with 0.5 ml of 100mg/L copper solution inside a test tube.
2. 2.5 ml of the mixture was transferred to a cuvette.
3. 175ul of API copper testing solution was added to the cuvette.
4. The solution was mixed by pipetting up and down for 10 seconds.
5. Wait 20 seconds for the color to develop.
6. The absorbance (440nm) of the solution was measured by a colorimeter.
7. Copper concentration of the samples was determined by interpolation the absorbance measured on the standard curve.

To measure the copper absorption ability of the bacteria,

1. 4.5 ml of bacterial culture was added into 2 test tubes. (labeled as 2 hrs and 4 hrs)
2. 0.5 ml of 100mg/L copper solution was added into each of the test tubes. The final copper concentration of each test tube was 10mg/L.
3. All of the tubes were incubated in a shaking incubator at 37℃.
4. At 2 and 4 hours, the mixture inside each test tube was transferred to two 1.5ml microcentrifuge tubes.
5. The bacteria inside microcentrifuge tubes were spun down at 6000g for 3 minutes.
6. 2.5 ml of supernatant from the two microcentrifuge tubes were extracted to a cuvette.
7. 175ul of API copper testing solution was added to the cuvette.
8. The solution was mixed by pipetting up and down for 10 seconds.
9. Wait 20 seconds for the color to develop.
10. The absorbance (440nm) of the solution was measured by a colorimeter.
11. Copper concentration of the samples was determined by interpolation the absorbance measured on the standard curve.

![](https://static.igem.org/mediawiki/2019/1/1a/T--Hong_Kong_JSS--expt_copper_test.jpeg)

Part 2 of the experiment: Measuring the copper concentration of the mixture.

Testing the Bacterial Copper Adsorption Device (B-CAD)

**Aim**
In this experiment, we aimed to test our B-CAD in a small scale but realistic situation. We obtain aquaponic water from a local aquaponic system and adjust the copper concentration (to 5, 10 or 15 mg/L) by adding copper (II) sulphate, this simulate copper polluted aquaponic water. We load our genetically modified bacteria to the B-CAD and circulate them through the “copper polluted” aquaponic water. The copper level of the system was measured every 2 hours for 2 days to evaluate if our B-CAD can lower copper level in water.

**Materials and Equipments**

|  |  |
| --- | --- |
| * 200ul and 1000ul micropipette * Colorimeter * Electronic balance * Conical flasks * Test tubes * API copper testing kit | * SOB medium * Ampicillin / Kanamycin * IPTG (optional) * 100mg/L copper solution * Aquaponic water * Distilled water |

**Procedure**
Preparation of bacterial culture:

1. 7.02 g SOB powder was dissolved in 250 ml distilled water to prepare the SOB medium.
2. 40 ml SOB medium was added to 250 ml conical flask.
3. Each of the conical flask with SOB medium was sterilized by autoclave.
4. Prepare 1000X ampicillin (100mg/ml) by dissolving 1g ampicillin in 10 ml distilled water. *Caution: Wear mask, safety goggles and gloves when handling Ampicillin powder as it is corrosive and irritating.*
5. 40 uL of 1000X ampicillin was added into each flask with 40 ml SOB medium.
6. A single colonie of respective cell culture was inoculated into conical flask with SOB medium.
7. The flasks were incubated in a shaking incubator at 37℃ for 18 hours.

Remarks:

1. According to the selection marker of the plasmid or bacteria strain, Kanamycin or other antibiotics may be used instead of ampicillin.- In some experiments, the bacteria needed to be induced by IPTG. To prepare IPTG stock (1000X), 2.38 g of IPTG was dissolved in 10 mL of distilled water. The solution was filter sterilized with a 0.22μm syringe filter.

Testing of the B-CAD,

1. 5 L of aquaponic water was added to a water tank.- 1000mg/L copper solution was added to the tank until the final concentration of copper reach 10mg/L.
   - 950 ml of aquaponic water was added to the reservoir of the B-CAD.
   - 50 ml of bacterial culture was added to the reservoir of the B-CAD.
   - The peristaltic pump and magnetic stirrer of the B-CAD was switched on.
   - 3 ml of water sample was collected from the water tank and B-CAD every 12 hours for water testing.
   - The 3 ml of water sample was separated into two 1.5ml microcentrifuge tubes.
   - The bacteria inside microcentrifuge tubes were spun down at 6000g for 3 minutes.
   - 2.5 ml of supernatant from the two microcentrifuge tubes were extracted to a cuvette.
   - 175ul of API copper testing solution was added to the cuvette.
   - The solution was mixed by pipetting up and down for 10 seconds.
   - Wait 20 seconds for the color to develop.
   - The absorbance (440nm) of the solution was measured by a colorimeter.
   - Copper concentration of the samples was determined by interpolating the absorbance measured on the standard curve.

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# Team:Hong Kong JSS/Results

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**Hong Kong JSS**

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[Characterisation](https://2019.igem.org/Team%3AHong_Kong_JSS/Contribution)
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[Judging Form](https://igem.org/2019_Judging_Form?id=3076)

**Results**

*** [Revisit To Our 2018 iGEM Project](#section 1)
* [Characterisation of MymT](#section 2)
* [Improving The Composite Parts Design](#section 3)
* [New Parts: CgMT](#section 4)
* [Gene Knockout experiments](#section 5)**

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**Achievement**

![](https://static.igem.org/mediawiki/2019/0/0b/T--Hong_Kong_JSS--SUPER.png)

1. We characteristised previous part BBa\_K1980002 (iGEM 2016 Oxford) to show it can enhance copper absorption in *E. coli*
2. New parts - Metallothionein from *Corynebacterium glutamicum* (CgMT) (BBa\_K3076100) and showed it can enhance copper absorption in *E. coli*
3. We tested several copper exporter knockout strain of *E. coli* and found *cusF* knockout could enhance copper absorption in *E. coli*

Revisit To Our 2018 iGEM Project

In 2018, we tried to enhance the copper absorption ability of *E. coli* by ectopically express copper binding metallothionein, the plant *Elsholtzia haichowensis* EhMT1 and human  *Homo sapiens*  MT1A. Our expression vector is pSB1C3 and the *E.coli* strain was TOP10. The diagram below shows our 2018 parts design.

![](https://static.igem.org/mediawiki/2019/1/15/T--Hong_Kong_JSS--2018EHMTHSMT.jpg)

Our 2018 parts design.

However, *E. coli* transformed with pSB1C3-EhMT1 ([BBa K2578611](http://parts.igem.org/Part%3ABBa_K2578611))and pSB1C3-MT1A ([BBa K2578811](http://parts.igem.org/Part%3ABBa_K2578811)) did not show any increase in copper absorption ability. In the copper absorption test, the percentage decrease of copper in both EhMT1 and MT1A transformant are similar to the empty vector control.

![](https://static.igem.org/mediawiki/2019/9/99/T--Hong_Kong_JSS--2018EHMTHSMT_results.jpg)

Results of our 2018 project, we failed to enhance copper absorption ability of the *E. coli*.

It could be due to several reasons, and one of them could be experimental error. We evaluated our experiment procedure in 2018 and identified the following potential errors. This year, we improved and repeat our experiments accordingly:

1. Incubation temperature

   |  |  |
   | --- | --- |
   | **Potential error** | Last year, we incubated the bacterial cultures at room temperature with a magnetic stirrer. The growth and health of the bacteria can be easily affected by the changing temperature. |
   | **Improvement** | We improved our experiment by using a shaking incubator at room temperature to 37℃. |

2. Bacteria concentration

   |  |  |
   | --- | --- |
   | **Potential error** | Last year, our experiments are done using bacteria culture grown for 18 hours. However, the growth rate of different bacteria strains can be different and this can affect bacterial concentration. |
   | **Improvement** | We controlled the bacteria concentration in our experiment by fixing it to absorbance at 440nm = 1.6. We diluted the bacterial culture with SOB medium to ensure all bacterial cultures were all started in the same concentration. |

However, after we improved our protocol and repeated the experiments, the results are the same. *E.coli* TOP10 cells transformed with pSB1C3-EhMT1 and pSB1C3-MT1A could not absorb more copper than the empty vector control. We deducted the some reasons for the results:

1. The composite parts we designed are not working.
2. The protein was not expressed properly, it could be due to the fact that E.coli as a prokaryote, cannot fold the eukaryotic protein properly, so there is no functional protein.

![](https://static.igem.org/mediawiki/2019/e/ea/T--Hong_Kong_JSS--ehmt1_mt1a_repeat2019.jpg)

EhMT1 and MT1A expressing *E. coli* cannot absorb more copper than normal bacteria.

Characterisation of MymT

Refer to one of the reasons mentioned above, the eukaryotic protein could be expressing improperly. It may be due to the prokaryotic based system in *E. coli* which is unfavorable for folding eukaryotic protein properly and possibly leads to no functional protein. As a result, a new experiment was carried out for the MymT, a cysteine-rich protein. It has been shown by iGEM16\_Oxford  [Part:BBa\_K1980002](http://parts.igem.org/Part%3ABBa_K1980002) that *E.coli* expressing MymT can absorb more copper.

In our experiment, Mymt was cloned into our pBS1C3 plasmid using the same design as [above](#above). We expected the transformed bacteria can absorb more copper. Without a doubt, it reduced the copper level in water by 11.4% after 4 hours. The reduction is significantly different from the control, however, it didn’t reach our expectations.

![](https://static.igem.org/mediawiki/2019/0/05/T--Hong_Kong_JSS--mymt2019.jpg)

MymT transformants can absorb more copper than normal bacteria, but the reduction is not very significant.

Improving The Composite Parts Design

In order to obtain a better result, we tried to test for CgMT ([BBa K3076100](http://parts.igem.org/Part%3ABBa_K3076100)), a new part never been used by any other iGEM team. We also changed the bacterial strain from TOP10 to BL21, specialized from cloning to expressing with a strong T7 promoter.

We utilized pET-151/TOPO vector to drive the expression of CgMT coding sequence under T7 promoter inside BL21(DE) *E. coli* strain. Then, the bacteria transformants will be tested inside copper (II) sulphate added culture medium to measure their copper absorption efficiency along time.

![](https://static.igem.org/mediawiki/2019/8/8d/T--Hong_Kong_JSS--newdesign.jpg)

Our improved composite parts design:
using protein expression strain BL21(DE) and expression vector pET-151/TOPO.

New Parts: CgMT / Vanabin2

*Corynebacterium glutamicum* metallothionein (CgMT) protein was reported by literature that shows a strong affinity towards binding divalent ions, such as zinc(II) and lead (II). [1] Furthermore, *Corynebacterium glutamicum* belongs to the same order as *Mycobacterium tuberculosis*. Previous iGEM team Oxford 2016 demonstrated that MT from *M. tuberculosis* ([BBa K1980002](http://parts.igem.org/Part%3ABBa_K1980002)) chelates copper ions in cells effectively, thus we expected CgMT also chelates copper (II) efficiently and it will increase the accumulation of copper ions inside *E. coli* expressing CgMT.

Based on our results, we showed that after IPTG induction, there is a significant reduction in copper level when compared with IPTG- and empty vector transformant control. It indicates *E. coli* strains (BL21) expressing CgMT has increased copper absorption ability.

![](https://static.igem.org/mediawiki/2019/a/a6/T--Hong_Kong_JSS--CgMT_Result.jpeg)

The percentage decrease of copper concentration in the culture medium.

In the graph, the percentage change of copper(II) ion concentration along time is shown. The copper added media were incubated with IPTG+ (IPTG induced CgMT expression *E. coli*), IPTG- (CgMT expression *E. coli* without IPTG added) and Empty (Empty vector control *E. coli*). IPTG+ showed ~30% decrease, IPTG- showed ~18% decrease and empty vector control showed ~14% decrease.

On the other hand, when we compare IPTG- with empty vector control, IPTG- also showed a statistical significant difference. We believe it could be due to the leaky expression of the T7 promoter. Further investigation is required.

Gene Knockout experiments

We aimed to increase the amount of copper accumulation in  *E. coli* so we studied the effect of knocking out some of the major copper exporter genes inside the bacterial genome. The National Institutes of Genetic in Japan has provided us with the 4 *E. coli* knockout strains, *cusA, cusF, CopA* and *cutA*. [3-4]
 All these strains contain a single-gene deletions in *E.coli* K-12. [5] As these genes are responsible for the export of copper ion from the cell, we expect more copper ion will be retained inside of the cell once these genes are knockout.

![](https://static.igem.org/mediawiki/2019/e/e2/T--Hong_Kong_JSS--copper_exporter.jpeg)

Genes related to exporting copper ions from the cell.
 Knockout strains of *cusA, cusF, copA* and *cutA* were tested for the copper absorption ability.

After the knockout strains were tested in the bacterial copper absorption test, *cusA, copA* and *cutA* knockout strains do no show any increased copper absorption ability when compared to the control (normal  *E. coli*). This could be due to the fact that the gene has not been completely nullified or gene redundancy, whereas the existence of multiple genes in the genome performing the same function.

![](https://static.igem.org/mediawiki/2019/4/4c/T--Hong_Kong_JSS--KO_test.jpeg)

Knockout of *cusA, copA* and *cutA* cannot increase copper absorption ability of *E. coli*.

However, for *cusF* knockout strain, we can see the copper level was reduced by 19% after 2 hours of incubation and the reduction increased to 27% after 4 hours. it is significantly higher than that of the control, which is 9% at 2 hours and 17% at 4 hours. This is implicating that after *cusF* was knocked out, the bacteria failed to export copper and thus retaining more copper ions inside the cell.

![](https://static.igem.org/mediawiki/2019/d/da/T--Hong_Kong_JSS--cusF_KO_result.jpeg)

Knockout of *cusF* significantly increase copper absorption ability of *E. coli*.

Limitation and Improvement

In our experiments, we did not monitor the expression level of CgMT. This is because we do not have the equipment and reagents for western blot. Also, due to the extremely hazardous nature of acrylamide, we are not suggested to conduct western blotting in our high school laboratory. We are currently seeking collaboration with local university so that we can use their facilities to perform protein work.

Future Direction

In our experiments, we had demonstrated that ectopic expression of CgMT and knockout of *cusF* in *E. coli* can increase the copper adsorption ability. Compared to the empty vector control, more copper from the solution is removed when mixing with these GM  *E. coli*. However, due to time constraint, we have not compared the effect of having CgMT expressed in the *cusF* knockout background. We believe this could further increase the copper adsorption ability of *E. coli*.

Reference:

1. Jafarian, V., & Ghaffari, F. (2017). A unique metallothionein-engineered in *Escherichia coli* for biosorption of lead, zinc, and cadmium; absorption or adsorption? *Microbiology*, 86(1), 73–81. doi: 10.1134/s0026261717010064
2. Rensing, C., Fan, B., Sharma, R., Mitra, B., & Rosen, B. P. (2000). *CopA*: An *Escherichia coli* Cu(I)-translocating P-type ATPase. *Proceedings of the National Academy of Sciences of the United States of America*, 97(2), 652–656. doi:10.1073/pnas.97.2.652
3. Padilla-Benavides, T., George Thompson, A. M., McEvoy, M. M., & Argüello, J. M. (2014). Mechanism of ATPase-mediated Cu+ export and delivery to periplasmic chaperones: the interaction of *Escherichia coli CopA* and *CusF*. The Journal of biological chemistry, 289(30), 20492–20501. doi:10.1074/jbc.M114.577668
4. Franke, S., Grass, G., Rensing, C., & Nies, D. H. (2003). Molecular analysis of the copper-transporting efflux system CusCFBA of *Escherichia coli.* *Journal of bacteriology*, 185(13), 3804–3812. doi:10.1128/jb.185.13.3804-3812.2003
5. Fong, S. , Camakaris, J. and Lee, B. T. (1995), Molecular genetics of a chromosomal locus involved in copper tolerance in *Escherichia coli* K‐12. *Molecular Microbiology*, 15: 1127-1137. doi:10.1111/j.1365-2958.1995.tb02286.x
6. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Mori, H. (2006). Construction of *Escherichia coli* K-12 in-frame, single-gene knockout mutants: the Keio collection. *Molecular systems biology*, 2, 2006.0008. doi:10.1038/msb4100050