With the rapid development of many industries like mining, energy and fuel producing, fertilizer, pesticide, electroplating, electrolysis, electric appliance manufacturing and atomic energy installations, wastes containing metals are directly or indirectly being discharged into the environment causing serious environmental pollution and even threatening human life[1].

Our project aim is to control heavy metals pollution in water by a synthetic biological approach with E. coli containing heavy metal-binding metallothionein. Our idea is inspired by the heavy metal poisoning incidents around the world. For instance, the lead water incident brought a panic to Hong Kong residents. People were afraid of drinking lead-polluted water into their bodies as the welder used lead solder when soldering the water pipes[2]. Many children were affected and their growth might be hindered if lead accumulated in toxic amounts in their bodies. Their brain may be damaged and they may experience learning or behavioral problems. Another example of heavy metal pollution is the cadmium(Cd) pollution in Japan. In 20th century, many Japanese were having a disease called itai itai disease. It was believed that the river that used for irrigating the rice fields was polluted with waste that contained cadmium(Cd) from factory. High level of cadmium(Cd) was accumulated in their bodies when they ate the Cd-polluted rice. This may cause softening of the bones and bring a lot of pain to the patients as cadmium(Cd) replace the Calcium(Ca) in human bones. What's more, arsenic pollution is also common in many places. It is estimated that more than 200 million people globally are exposed to higher than safe levels of arsenic through drinking water and the most affected area are Bangladesh and West Bengal. Long-term exposure to arsenic can result in different skin lesions and skin cancer.

However, the above cases is only the tip of the iceberg. Therefore, to deal with this problem, we decided to create a device that could reduce heavy metal ions in water, which is a major category of heavy metal pollution.

Aquaponics: a co-exist system for bacteria, fish and plant

Aquaponics is a system that promote the coexistence between aquatic animals, plants and nitrobacter. The idea has been developing since the Tang Dynasty of China[3]. Until now, the idea of aquaponics is well known and wide-spreaded all over the world.

In an aquaponics cycle, the waste from the fish is converted into nutrients by bacteria suitable for plants to absorb for growth. When fishes produce waste, ammonia will be released[4], and this substance will be transferred into useful and harmless nutrients nitrate for plants by nitrifying bacteria[5][6].

Certain species of freshwater fish, such as tilapia and carp are the most common aquatic animal raised using aquaponics due to their ability to tolerate crowding. Although freshwater crayfish and prawns are also sometimes used.

Apart from fishes, plants and nitrifying bacteria, there is another important element in aquaponic cycle, which is water. Water provides an aqueous environment for fishes to live, supports the survival of fishes and plant and also act as a transporting agent of wastes and nutrients. As the water in this system can be reused for several times, the system is really environmentally friendly.

As we have mentioned before, both metal water pipes and fish pellet are source of heavy metal pollution presents in water, which water is closely related to aquaponics. Therefore, in this project, we shrink the area covered in our project, only aiming to cope with the heavy metal pollution problem in the aquaponic system.

Design

As we have mentioned in the description part, we hope to create a device that could reduce heavy metal ions in water, after research, we found that Metallothionein is a solution for us to allay the issue in a synthetically biological way as it is ubiquitous and can be found in all animal phyla examined to date and also in certain fungi and plants, as well as Homo sapiens[9]. Also, metallothionein can bind to various metal ions like cadmium (Cd), lead (Pb), mercury (Hg), copper (Cu), zinc (Zn), and nickel (Ni) at the same time which makes it a good metal ion binder[7].

As there are a large amount of different kinds of metallothionein from all 16 phylogenetic family, we decided to choose several of them each from five species with distinctive features for our project, which the selected gene are MymT(from Mycobacterium tuberculosis), CUP1(from Saccharomyces cerevisiae), EhMT1(from Elsholtzia haichowensis), Mt1, Mt2(from Mus musculus), MT1A and MT2A(from Homo sapiens).

Metallothionein

Metallothionein are a group of ubiquitous low molecular mass cysteine-rich intracellular metal binding proteins which are characterized by their unusual high cysteine content (30%) and lack of aromatic amino acids.

Mechanism of metallothionein

As what has been mentioned about, metallothionein are rich in cysteine content, which this is the reason why they can bind to several metal ions[8].

Cysteine is a type of amino acid with a side chain containing a thiol group.

Fig. 1 Structure of cysteine.

Fig. 2 Structure of thiol.

Thiol is believed to have the ligand behaviour[10] such that they can bind to metal ion by dative covalent bond a form a metal thiolate complex.

Fig. 3 Structure of metal thiolate complex. Where the hydrogen on thiol is replaced by a metal
Atom in cysteine.

With the aid of thiol group in cysteine of metallothionein, metallothionein can bind to several metal ion such as cadmium (Cd), lead (Pb), mercury (Hg), copper (Cu), zinc (Zn), and nickel (Ni), and also protect cells and tissues against heavy metal toxicity[7].

Fig. 4 Saccharomyces cerevisiae metallothionein bound to copper ions. Cysteines in yellow, copper in brown.

Composite Part design

All the five above metallothionein expressing gene are added a constitutive promoter, J23100, a RBS, B0034, a double terminator, B0015.

J23100 is chosen as it is one of the strongest relative constitutive promoter in the iGEM promoter catalog, that the highest amount of metallothionein can be produced constitutively with the aid of J23100, increasing the copper ion absorbing ability of the engineered E. coli.

All the five above metallothionein expressing gene are added a constitutive promoter, J23100, a RBS, B0034, a double terminator, B0015.

B0032 is chosen as it is one of the RBS with the highest relative strength in the iGEM ribosome binding site catalog, that the production of metallothionein can be secured to be translated fully, guaranteeing the enhanced copper ion absorbing ability of the engineered E. coli.

B0010 and B0012 are used together as a double terminator B0015. This is the most commonly used terminator, which we believe that it is reliable to terminate the coding process in the most accurate time.

The figure below is the sampling model of our composite part design.

Process

We ordered five different DNA from IDT to perform our assays, below shows the process of how we used the ordered DNA and what problems we faced during handling the gene.

For Mycobacterium tuberculosis MymT coding gene, we first get the sequence from IGEM library, which this gene was designed by IGEM16_Oxford team. Then, we designed a composite part, which was registered as BBa_K2578410, for the expression of Mycobacterium tuberculosis metallothionein. After then, we ordered the plasmid containing BBa_K2578410 from IDT. At the time the DNA was shipped, we start our cloning and assay process. We transformed the plasmid to the E.coli, then cultivate the bacteria to have enough amount for performing the assays of the effect of transformed E.coli to the concentration of copper in water, which will be detailedly explain on the experiment page. At the end, we did restriction enzyme digestion and ligate the part with pSB1C3 backbone for shipping.

For Saccharomyces cerevisiae CUP1 coding gene, we obtained its sequence from NCBI genomic sequence library. After obtaining the sequence, we immediately designed a composite part for expression, which was registered as BBa_K2578510. Then, we ordered the plasmid containingBBa_K2578510 from IDT. However, unfortunately, we failed to obtain the gene due to IDT failing to synthesize the DNA, which the reason of the failed synthesis is insertion mutation.

For Elsholtzia haichowensis EhMT1 gene, we first obtained its sequence by reversing its mRNA sequence into DNA sequence. After that, we designed a composite part for its expression, which was registered as BBa_K2578610. Then, we ordered the plasmid containing BBa_K2578610 from IDT. When this ordered DNA was shipped, we started our cloning process. We transformed the plasmid to the E.coli, then cultivate the bacteria to have enough amount for performing the assays of the effect of transformed E.coli to the concentration of copper in water, which will be detailedly explain on the experiment page. At the end, we did restriction enzyme digestion and ligate the part with pSB1C3 backbone for shipping.

For the Mus musculus Mt1 coding gene, we first obtained its sequence from NCBI genomic sequence library. Then, we designed a composite part for its expression, which was registered as BBa_K2578710. However, unfortunately again, we found that we are unable to use the gene due to multiple restriction enzyme sites and introns in the functional gene of BBa_K2578710.

For the Mus musculus Mt2 coding gene, we first obtained its sequence from NCBI genomic sequence library. Then, we designed a composite part for its expression, which was registered as BBa_K2578720. However, we found out that the number of nucleotides in the functional gene can’t be divided by 3, which means several introns may present in the gene. Since E. coli naturally doesn't have the ability to cut introns from a mammalian gene, therefore, we decided not to use Mus musculus Mt2 coding gene.

For Homo sapiens MT1A gene, we first obtained its sequence from NCBI genomic sequence library. After that, we designed a composite part for its expression, which was registered as BBa_K2578810. Then, we ordered the plasmid containing BBa_K2578810 from IDT. When this ordered DNA was shipped, we started our cloning process. We transformed the plasmid to the E.coli, then cultivate the bacteria to have enough amount for performing the assays of the effect of transformed E.coli to the concentration of copper in water, which will be detailedly explain on the experiment page. At the end, we did restriction enzyme digestion and ligate the part with pSB1C3 backbone for shipping.

For Homo sapiens MT2A gene, we first obtained its sequence from NCBI genomic sequence library. After that, we designed a composite part for its expression, which was registered as BBa_K2578820. However, due to lacking of time and budget, and considering the risk of cross species gene engineering, we decided not to use Homo sapiens MT2A gene in our project this year.

Summary

Application

Bacterial copper filtration device

Description

The bacterial copper filtration device is composed of a reservoir containing bacterial culture, a peristaltic pump for circulating the bacteria and a cage for housing the dialysis tubing. The pump is connected to the reservoir and the dialysis tubing. The dialysis tubing is connected to the reservoir. They form a loop of bacteria culture that is transferred from the reservoir to the dialysis tubing then back to the reservoir as shown in the figure below.

Principle

Based on the results of our experiments, we had confirmed that copper ion in water can pass through the dialysis tubing while bacteria cannot. Therefore, this device allow copper ion to be removed from the water while engineered E. coli will not escape to the environment.

Details of design

The figures below show the design and 3D-printed product of the cage containing the dialysis tubing. This part of the device will be submerged in water, allowing the bacteria to absorb copper ion diffused into the tubing. The two holes on the lid allow the dialysis tubing in and out of the cage, there are 7 hooks for holding the tubing in place in water.

The figures below show the design and the 3D-printed product of the reservoir for containing the bacterial culture. Two holes on two sides of the reservoir allow bacterial culture moving out of the reservoir to the dialysis tubing, and vice versa. The base of the reservoir is levitated on one end, allowing it to have an inclination to prevent bacteria accumulating at the bottom.

Cardboard colorimeter

Description

Using cardboard, duct tape, test tubes and a smartphone, we had made a low-cost colorimeter as shown below. The advantages of this colorimeter are 1) cost-effective 2) portable 3) does not require energy, allowing us to take accurate measurement of copper concentration in field studies.

During our interview with local fish farms and aquaponics owners, 40% of them are interested in trying our bacterial copper filtration device. Yet, none of them have a colorimeter for measuring the copper concentration in water. As a result, this low-cost colorimeter may come in handy.

Principle

The structure of a colorimeter is basically constituted by a light source, a monochromator, a detector and, of course, a sample solution. A laboratory colorimeter can measure the absorbance of wavelengths of light at a particular frequency (color) by a sample. When a sample solution is placed into the sample compartment of the colorimeter, the built-in light source emits light and it passes through the collimator which is a device narrowing beams of light. Owing to the nature of white light (that consists of a combination of lights of different wavelengths), a monochromator is embedded in front of the collimator so as to select a designated frequency of light. Ultimately, the transmitted light wave is passed through the sample solution and received by a detector. The absorbance is calculated by an analogue or digital meter by the application of the Beer-Lambert law[11].

Details of design

Inspired by this configuration[12], we tried to replace the light source simply by sunlight / table lamp and the monochromator by an A4 color card (in our experiment we need blue light so a blue card is used). When sunlight scatters on the cardboard, any color apart from blue is filtered out. As regards the detector, we use an iPhone X with a color identification App ‘ColorMeter’ to record the blue Intensity of the RGB value as shown below.

To minimize the side color effect among the area between the color card and detector, it is surmountable by making a cardboard box to have the area encircled. Placing the sample solution amid the cardboard box, the absorbance and hence concentration can both be computed by measuring the incident Blue intensity and the transmitted Blue intensity. The figure below shows the design of our “cardboard colorimeter”.