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

Graph 1.2: Copper concentration of water samples from households

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

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

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

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

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

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

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
Concentration of copper solution/mg/L	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

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

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

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

*Error Bar is standard deviation of estimated copper concentration

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

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
Sample	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
Sample	Initial concentration,mg/L	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)

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
Sample	Initial concentration,mg/L	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%
Elsholtzia haichowensis MT1 gene	2.0	-0.0	-0.2	-0.0%	-10.9%
Homo sapiens MT1 gene	10.2	-1.6	-3.1	-15.7%	-30.4%
Homo sapiens MT1 gene	2.7	-1.1	-1.7	-39.4%	-63.0%
Empty Vector(Set A)	10.2	-1.7	-2.1	-16.8%	-21.0%
Empty Vector(Set A)	2.0	-0.3	-0.3	-16.3%	-16.3%
Empty Vector(set B)	10.2	-2.1	-3.8	-21.0%	-37.7%
Empty Vector(set B)	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

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

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

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

Conclusion

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

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

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.

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

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.

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.

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.

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.

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.

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.

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

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Team:Hong Kong JSS/Results

Results

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

Abstract of assay

Aim of Experiment

Raw data link:

Graphed data:

Discussion

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

Abstract of assay

Aim of Experiment

Raw data link:

Graphed data:

Discussion

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

Abstract of assay

Aim of Experiment

Raw data link:

Graphed data:

Discussion

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

Abstract of assay 4A

Results

Abstract of assay 4B

Results

Conclusion

Prospect

Assay 5 : Test for the permeability of dialysis tubing

Abstract of assay

Aim of Experiment

Raw data:

Graphed data:

Assay 6 : Testing the cardboard colorimeter

Abstract of assay

Aim of Experiment

Raw data:

Graphed data:

Plasmid Cloning result

Hong Kong JSS

Contact