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

Add 2 mL of water sample into Salifert Profit test kit test vial.
Add 5 drops of the Salifert Profit Copper reagent to the test vial and swirl gently for 5 seconds.
Allow to stand for 10 minutes .
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.

Colour Chart

Reference

About Salifert Copper Test Kit:

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

Prepare 10 mg/L Copper solution from Copper(II) sulphate powder.
Transfer 5 ml Copper solution to a test tube.
Add 10 drops of API Copper testing solution into the copper solution.
Wait 30 seconds.
Transfer 2.5 ml of the reaction mixture into a clean cuvette. Insert the cuvette into colorimeter.
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

Label four test tubes 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, 10 mg/L.
Add 4.5, 4, 3.5, 3 ml of distilled water into the test tubes respectively.
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.
Wait 30 seconds.
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

Dilute 1000 mg/L copper solution into 100 mg/L.
Label five test tubes 0 mg/L, 2 mg/L, 2.5 mg/L, 5 mg/L, 7.5 mg/L, 10 mg/L.
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

Add 175 ul of API Cu2+ test solution into each tube
Measure the absorbance of each solution by using SMART 2 colorimeter.
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

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.
Dissolve 7.02 g SOB medium in 250 ml distilled water.
Autoclave six 250ml empty conical flask.
Autoclave six flask of 40 ml SOB medium in 250 ml conical flask.
Prepare 1000X ampicillin (100mg/ml) by dissolving 1g ampicillin in 10 ml distilled water.
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)
Wait until the SOB medium to cool down to room temperature.
Label the two flask with A1/B1/C1 and E1 respectively.
Add 40 uL of 1000X ampicillin into each flask with 40 ml SOB medium.
Inoculate respective cell culture into 2 conical flask with SOB medium.
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

Rinse the test tubes with distilled water then autoclave it.
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	2nd incubation period	3rd incubation period
A1/B1/C1, 10 mg/L	2 hours	4 hours
A1/B1/C1, 2 mg/L	2 hours	4 hours
E1, 10 mg/L	2 hours	4 hours
E1, 2 mg/L	2 hours	4 hours

Transfer 4.5ml of cell culture in 250ml conical flask into respective test tubes.
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.
Transfer 0.5ml of 100 mg/L copper solution into all respective test tubes.
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.
Transfer 0.5ml of 20 mg/L copper solution into all respective test tubes.
Incubate all 0hrs, 2hrs, 4hrs test tubes at 37’°Cat its respective time.
After incubation, transfer cell culture and copper mixture to two 1.5ml tubes.
Centrifuge all tubes at 6000g for 3 mins at 4’C.
Extract 2.5ml of supernatant into a cuvette.
Transfer 175ul of API copper test solution into each.
Wait for 1 minute. Measure the result with a colorimeter using water as blank (440nm).
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

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

Protocols

Testing if copper ion can pass through the dialysis tubing

100mg/L copper (II) solution was prepared using anhydrous copper (II) sulphate.
10mg/L copper (II) solution was prepared by diluting the 100mg/L copper (II) solution.
180mL of 10mg/L copper (II) solution was added to a 500ml beaker.
20mL of distilled water was added into a 20cm long dialysis tubing.
Two knots were tied at two ends of the dialysis tubing.
The dialysis tubing was immersed into the beaker as shown in the below figure.
After 2 hours of immersion, the dialysis tubing was removed from the beaker and rinsed with distilled water .
The dialysis tubing was cut open and the solution inside was isolated.
To tested for copper concentration of the solution, 2.5 mL of the solution was added to a cuvette.
175uL of the API copper test solution was added to the cuvette.
After 1 minute, the absorbance was measured with a colorimeter, the result was then compared to standard curve to calculate the copper concentration.
The experiment was repeated with 8 and 24 hours of immersion.

Testing if bacteria can pass through the dialysis tubing.

180mL of SOB solution and 180uL of Ampicillin (100mg/mL) were added to the beaker.
20mL of SOB solution and 20uL of Ampicillin (100mg/mL) were added into a 20cm long dialysis tubing.
Two knots were tied at two ends of the dialysis tubing.
The dialysis tubing was immersed into the beaker as shown in the below figure.
The SOB with ampicillin in the beaker was inoculated with Ampicillin-resistance E. coli.
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

100mg/L copper (II) solution was prepared using anhydrous copper (II) sulphate.
2.5mg/L, 5mg/L, 7.5mg/L and 10mg/L were prepared by diluting the 100mg/L copper (II) solution.
2.5mL of each copper (II) solution were added to a labeled test tubes.
2.5mL of distilled water was added to a labeled test tube as blank.
175uL of API copper test solution was added to each of the labeled test tubes in step 3-4.
After 1 minute, the blue intensity of each of the solution was measured by the smartphone using a color identification Apps “ColorMeter”.
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.
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

Grow transformed E. coli(TOP10) ( BBa_K2578810, BBa_K2578610, BBa_K2578410 ) in 1–5 mL LB medium overnight.

Purification Procedure Using Centrifugation

Centrifuge 5 mL of the overnight LB-culture. Remove all medium.
Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous.
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.
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.
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.
(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.
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.
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.
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

Prepare one 8-tube strip, BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul), dH2O, NEB Buffer 2, BSA and Restriction Enzymes: EcoRI, PstI.
Add 250 ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul to every reaction.
Add 2.5ul of NEBuffer 2 to every reaction.
Add 0.5ul of BSA to every reaction.
Add 0.5ul of EcoRI to every reaction.
Add 0.5ul of PstI to every reaction.
There should be a total volume of 20 ul of solution in every reaction. Mix well and spin down briefly.
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

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

Loading The Gel

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.
Add 2 ul loading buffer on parafilm. (Same number as sample)
Carefully pipette 10 ul of each sample and mix with loading buffer.
Load the mixed sample to the corresponding well.
Pipette 10 ul of the DNA ladder marker into at least one well of each row on the gel.

Running The Gel

Place the lid on the gel box, connecting the electrodes.
Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected. (Remember – “Run to Red”)
Turn on the power supply to about 120 volts.
Check to make sure the current is running through the buffer by looking for bubbles forming on each electrode.
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).
Let the power run until the blue dye approaches the end of the gel.
Turn off the power.
Disconnect the wires from the power supply.
Remove the lid of the electrophoresis chamber.
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

Equilibrate a water bath or heat block to 50°C.
Excise a minimal area of gel containing the DNA fragment of interest.
Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g.
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)

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.
After the gel slice appears dissolved, incubate the tube for an additional 5 minutes .
For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
Purify the DNA using a Centrifuge.

Purifying DNA Using a Centrifuge

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.
Add 500 µL Wash Buffer (W1) containing 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 maximum speed for 1–2 minutes . Discard the flow-through.
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.
Centrifuge the tube at >12,000 × g for 1 minute.
Store the purified DNA in elution tube at 4°C for immediate use or at −20°C for long-term storage.

Ligation

Add 2 ul of digested plasmid backbone (25 ng).
>Add 2 ul of EcoRI-HF PstI digested fragment.
Add 1 ul T4 DNA ligase buffer.
Note: Do not use quick ligase.
Add 0.5 ul T4 DNA ligase.
Add water to 10 ul.
Ligate 16C/30 min, heat kill 80C/20 min.
Transform with 1-2 ul of product.

Single tube transformation

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.
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.
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.
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.
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.
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.
Close 1.5ml tubes, incubate on ice for 30 minutes, tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
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.
Incubate on ice for 5 minutes, Return transformation tubes to ice bucket.
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.
Incubate at 37°C for 1 hours, shaking at 200-300rpm
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.
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.
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.
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.