Description

Inspiration

Heavy metal poisoning has become a serious problem and one of the most common topics around the world. Heavy metals are high atomic weight elements that can result in environmental contamination when accumulating to a certain level. Once these metals are released into the environment (e.g., soil, air and water) from industrial plants at high concentrations, they can lead to considerably adverse effects on human health. In particular, overexposure of cobalt and arsenic usually causes neurological disorders, respiratory and cardiovascular diseases, as such metals can compete and replace with certain molecules that are significant for organisms.

Cobalt

Cobalt comprises only about 0.001% of the Earth’s crust, but more widely distributed in compounds with nickel and arsenic [1]. Since cobalt is relatively unreactive and high-temperature resistant, it plays an important role not only in industrial applications, such as manufacturing alloys but also in human and animal nutrition as a constituent of vitamin B12. In addition, cobalt is widely used for rechargeable batteries because of its ability to make magnets. However, it can be toxic to the environment and humans. According to EPA, the toxicity reference value (TRV) of cobalt is 23 μg/L [2]. When people are exposed to the concentration that exceeds this value, normal functions of the body may be affected, causing some health problems and even death.

Common Symptoms

Cardiomyopathy
Lung cancer
Deafness
Thyroid problems

Cases

High concentrations of cobalt are usually found near ore deposits and in soils and water contaminated by airport traffic and industrial pollution. A coal power plant of San Antonio in Texas was reported leaking more than 600 micrograms per liter cobalt in the groundwater through the coal ash. This is 100 times higher than safe levels [3]. The pollution was also found over 4600 groundwater wells of other 265 coal plants in the United States. Although some measures have taken to clean up coal ash, the cost is high. According to a member of the Virginia State Corporation Commission, it could cost between $2.4 billion and $5.6 billion to clean up Virginia-based Dominion Energy’s 11 coal ash ponds and six ash landfills in the state [4].

Arsenic

Arsenic is highly toxic for humans and other life forms. Due to the arsenic pollution, it was estimated that around 200 million people worldwide are exposed to arsenic in drinking water above 50 µg/L, which is significantly higher than the restriction of 10 µg/L [5]. Drinking water with exceeding arsenic concentration would cause the development of cancers and skin lesions. Thus, arsenic has been classified as a group 1 human carcinogenic substance by WHO.

Common Symptoms

Drowsiness
Stomach cramps
Liver disease
Diabetes

Cases

A 50-year-old man of Pakistan who visited his relatives in Saudi Arabia for three months died after he was admitted to a local hospital. His blood report shows that the concentrations of lead, mercury, and thallium are within range of normal. However, the concentration of arsenic is shown to be 7 µmol/L (reference range < 0.135 µmol/L). In other examinations, the arsenic concentration is 64.5 µmol/L (reference range < 0.25 µmol/L) in urine, 11 µg/g (reference range < 0.5 µg/g) in hair sample [6].

Current Techniques

To alleviate harmful effects to ecosystems, certain measures of absorbing and filtrating heavy metals have been examined. One of the most commonly used methods is the oxidation techniques, which convert the soluble arsenite to arsenate, followed by adsorption, filtration, or ion exchange. Another technique is the flocculation, where positively charged ions are used to reduce the negative charge of the colloids, resulting in the particles colliding and growing larger, then filtering them out [5]. However, there is a risk of secondary contamination associated with these methods due to the formation of sludge. In this case, membrane technology has been identified as an appropriate way to deal with water contamination since it is environmentally friendly and needs relatively low energy. The problem is that highly selective membranes are required to prevent specific metals across. In addition, to improve the filtering efficiency, high pressure is needed as well to push water molecules across the membrane.

Heavy metal detection and bioremediation is always a hot topic in iGEM competition. Arsenic is one of the heavy metals that we are focusing on. There are several proteins have been studied by several teams: 2006 Edinburgh, 2009 Groningen, 2009 Virginia, 2010 UIUC-Illinois, 2012 Cornell, 2012 Gaston Day School, 2012 IvyTech-South Bend, 2012 UANL Mty-Mexico, 2013 Buenos Aires, 2017 Cadets2Vets, 2017 Peshawar, 2017 FAFU-China, 2018 HongKong JSS team. Most teams used ArsR with other proteins, like LamB, ArsB, GFP, and MTs.

Other iGEM teams have also worked on the heavy metal bioremediation. There are several proteins have been proved by previous teams’ work that are ideal for binding heavy metal ions. One of them is metallothionein, which was used by 2009 Groningen team, 2011 Tokyo-NoKoGen, 2012 UANL Mty-Mexico, 2014 Cornell team, 2014 Nagahama team, 2014 UFAM Brazil team, 2015 UMBC-Maryland team, and 2016 Newcastle team.

In 2014, the Cornell team designed a filtration system for nickel, mercury, and lead absorption by expressing the heavy metal transport proteins and metallothioneins for each metal [7]. Although this system successfully decreased the heavy metal concentration in the water, the cell growth was inhibited as shown in the results. The same problem was also shown in the project of the 2009 team Groningen, who used the human metallothionein (rh-MT) and Fucus vesiculosis metallothionein (fMT) for arsenate biosorption. Our team reviewed their project and decided to improve their work targeting mostly at arsenic and cobalt.Rather than relying on intracellular capture, another team from INSA Lyon designed a bacteria with nano-sponge surfaces for the water filtration system of nickel. Specifically, they modified the curli structure of bacteria by adding His-Tag motifs to chelate nickel. In addition, the bacteria are engineered to produce biofilms, allowing it to stick on the filter matrix and exposed to the polluted water. According to the results, thick biofilms were formed and the nickel was successfully chelated, but the nickel chelation capacity was relatively low and needs to be improved [8].

Our Goal

This year our team aims to develop a heavy metal biosorption system for arsenic and cobalt remediation by implementing the metallothionein gene and transporter protein for arsenic and cobalt into the DH5-α and BL21 DE3 E. coli bacteria. Moreover, we decided to incorporate SOD genes that should decrease the sensitivity of heavy metal poisoning effects and thus, extend the lifecycle of bacteria. In this case, we believe the arsenic and cobalt level of water and soil can be controlled under the standard limit.

What is Metallothionein?

Metallothionein (MT) is a class of small metal-binding proteins that exists in bacteria, plants and animals. These proteins depending on their amino acid compositions have a high binding affinity with different bivalent metal ions. Once MT detects the corresponding metal, it binds the goal through covalent bonds, which are composed of sulfhydryl cysteine residues and stores the metal by tightly chelating the metal. Typically, it is assumed that MTs have two binding domains, one of which is the C-terminal part (α-domain) with three binding sites. The other one is the N-terminal part (β-domain) with four divalent binding sites [9]. Therefore, MTs are important for protecting the cell against heavy metal toxicity and maintaining cellular homeostasis.

What is SOD?

The word SOD stands for superoxide dismutase. It is an enzyme that attaches to molecules of copper and zinc to break down toxic. When bacteria face oxidative stress, superoxide radicals will be accumulated in the bacteria which may cause cell damage. SOD works as an enzyme to reduce the number of superoxide radicals and produce hydrogen peroxide or oxygen, which are less toxic and can be spontaneously processed out of the bacteria by itself. SODs can be found in both prokaryotes and eukaryotes. There are several types of SODs, and the most well-known ones are: CuZnSOD, FeSOD, MnSOD, and NiSOD.

References

[1] Russell, D., & Kemmitt, R. (n.d.). Cobalt. Retrieved from https://www.sciencedirect.com/topics/chemistry/cobalt

[2] AQUATIC TOXICITY REFERENCE VALUES (TRVs). (1999). Retrieved June 20, 2019, from https://clu-in.org/download/contaminantfocus/dnapl/Toxicology/DOE_SW_tox_valuep76.pdf.

[3] Collier, K. (2019, January 17). Report: Texas coal power plants leaching toxic pollutants into groundwater. Retrieved from https://www.texastribune.org/2019/01/17/report-texas-coal-power-plants-leaching-toxic-pollutants-groundwater/

[4] Mufson, S., & Dennis, B. (2019, March 04). Report finds widespread contamination at the nation's coal ash sites. Retrieved from https://www.washingtonpost.com/national/health-science/report-finds-widespread-contamination-at-nations-coal-ash-sites/2019/03/03/d80c82e6-3ac8-11e9-aaae-69364b2ed137_story.html?utm_term=.06c33ff3ec91

[5] Nicomel, N. R., Leus, K., Folens, K., Van Der Voort, P., & Du Laing, G. (2015, December 22). Technologies for Arsenic Removal from Water: Current Status and Future Perspectives. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730453/

[6] Duncan, A., Taylor, A., Leese, E., Allen, S., Morton, J., & McAdam, J. (n.d.). Homicidal arsenic poisoning - Andrew Duncan, Andrew Taylor, Elizabeth Leese, Sam Allen, Jackie Morton, Julie McAdam, 2015. Retrieved from https://journals.sagepub.com/doi/full/10.1177/0004563214559222

[7] (n.d.). Retrieved June 11, 2019, from https://2014.igem.org/Team:Cornell/project

[8] (n.d.). Retrieved from https://2014.igem.org/Team:INSA-Lyon

[9] Ruttkay-Nedecky, B., Nejdl, L., Gumulec, J., Zitka, O., Masarik, M., Eckschlager, T., … Kizek, R. (2013). The role of metallothionein in oxidative stress. International journal of molecular sciences, 14(3), 6044–6066. doi:10.3390/ijms14036044

Team:RHIT/description