Team:SCAU-China/description
Problems
Over the past century, global industrialization has developed rapidly. In the process of industrial development, various environmental problems also arise. As one of them, heavy metal pollution is also endangering our health in many ways. The so-called heavy metals refer to metal elements with relatively high density compared with water (Chaney, 1991). Nowadays, the term "heavy metal" has been used to describe metal chemical elements and quasi metals that are toxic to the environment and human beings (Briffa et al., 2020). Heavy metals can cause toxicity to various organelles of cell, damage of DNA, apoptosis or carcinogenesis (Tchounwou et al., 2012).
Figure 1: Heavy metal pollution and biological toxicity (Godwell et al., 2019).
There are many forms of heavy metal pollution, but the main ones still exist in water and soil.
| Heavy Metal | Upper Crust/mg kg−1 | Surface Soil/mg kg−1 | River Water/μg L−1 |
|---|---|---|---|
| Arsenic | 1.8 | 4.7 | 0.13-2.71 |
| Cadmium | 0.1 | 0.41 | 0.06 × 10−2 – 0.61 |
| Chromium | 35 | 42 | 0.29–11.46 |
| Copper | 14 | 14 | 0.23–2.59 |
| Lead | 15 | 25 | 0.007–308 |
| Mercury | 0.07 | 0.07 | - |
| Nickel | 19 | 18 | 0.35–5.06 |
| Zinc | 52 | 62 | 0.27–27 |
Table 1: Background values of heavy metals in global soil and water (Vareda et al., 2019).
Solvation
At present, there are many methods to deal with heavy metal pollution. The commonly used methods mainly include physical methods, chemical methods and bioremediation methods. For heavy metals in soil, leaching is the most widely used physical remediation. Heavy metals are dissolved by leaching agent. And a chemical method of mixing chemical additives into the soil through chemical reaction to remove heavy metals in the soil or reduce the content of heavy metals in the soil. In addition, there are bioremediation methods, that is, through plants that can absorb heavy metals, such as reed Phragmites australis ,reed mace Typha latifolia or microorganisms that adsorb heavy metals (Jiang Yongbin, 2007; Ding Yuqiao and Liu Xiaoguang, 2021). Heavy metal pollution in water can be treated by electrolysis, chemical precipitation and other chemical methods. It can also be treated by physical methods such as ion exchange resin adsorption, inert adsorbent (such as activated carbon), coagulation, flocculation and membrane filtration (Fu and Wang, 2011). However, neither physical nor chemical methods can solve the problem of heavy metal environmental pollution as gently as biological methods, which has a small impact on the environment. Biological methods will have a broader development space and application prospect in the future.
What do we think？--Intelligent Molecular magnet
In previous years, the iGEM teams made great achievements in bioremediation. In 2019, Hubei University of technology let yeast absorb Ni ions by expressing Ni ion channels. Some teams also use heavy metal binding proteins (metallothionein) to achieve their goals, such as 2015-UMBC-Maryland and 2014-Penn. We will use the autophagy pathway which never been used in the history of iGEM to help deal with the problem of heavy metal pollution. This year, we will use an kind of algae- Chlamydomonas reinhardtii , which has been used in bioremediation, to treat three representative heavy metal ions of cadmium, copper and zinc in water. Chlamydomonas reinhardtii can adsorb heavy metal ions through several groups on the extracellular wall, such as carboxyl, hydroxyl, amino, sulfhydryl and phosphate (Kong Xiangxue et al., 2017). However, it is inevitable that heavy metal ions can still enter cells through ion channels on cell membrane and cause stress to Chlamydomonas reinhardtii , shortening its service life for bioremediation (Jiang Yongbin, 2007).
Figure 2: Interaction between heavy metal ions and Chlamydomonas reinhardtii
Therefore, we think that if there is an intelligent molecular magnet, it can not only adsorb heavy metal ions, but also transport them to storage places such as vacuoles, then it can reduce the toxicity of heavy metal ions to cells and enrich heavy metals.
Autophagy can selectively degrade damaged components.，which can be understood as intelligent orientation. If we can couple autophagy with molecular magnet-metallothionein (polycysteine proteins that can bind heavy metals), we can use the vacuoles of Chlamydomonas reinhardtii to delay the damage of heavy metals to cells and assist in the storage of heavy metal ions. So this year, we intend to construct a fusion protein consists of AIM (Atg8 interacting motif), metallothionein and fluorescent protein, and use the autophagy pathway of Chlamydomonas reinhardtii to help deal with the problem of heavy metal pollution. Please see design for specific project principles.
Model
Fusion protein
The construction of fusion protein is not just like a simple A + B. In our project, we need to determine the appropriate order of the three proteins and whether linker peptide needed to ensure the normal function of the fusion protein. The construction of fusion protein requires two factors: constituent protein and linker peptide. The constituent protein is selected according to the function of the desired fusion protein product, which is relatively simple in most cases. However, it is difficult to select the appropriate linker peptide, and it is easy to be ignored in the design of fusion protein. If the functional domain is fused directly without linker peptides, it will lead to some adverse results, such as misfolding, low yield or impaired activity of the fusion protein. Therefore, the rational design and selection of linker peptides are very important for the construction of fusion proteins (Li Jianfang et al., 2015). Combined with wet test, we established the molecular model of fusion protein in continuous learning. The appropriate protein sequence and the types of linker peptides were determined. Please see model for details.
Figure 3: Schematic diagram of the fusion protein constructed in the project
MIBP represents heavy metal ion binding protein (metallothionein), aim represents Atg8 interacting motif, and mCherry is a red fluorescent protein.
Recovery time
We hope that eventually our engineered Chlamydomonas reinhardtii can be applied to the real environment. In order to facilitate recovery, we need to know what indicators indicate the absorption of heavy metal ions in our algae cells have been saturated which means we should recover our device for further treatment. This is important because the recovery time will affect the working effect of the whole device.Please see model for details.
Biosafety
As igemer, we always pay attention to biosafety. Gene leakage may change the gene exchange process in nature. This will lead to unexpected results. We also do not want such a biosafety accident happens. This year, we will learn from the results of other projects, using sodium alginate + PVA cell fixation and embedding method and semi permeable membrane. We will also design a device to comprehensively use physical methods to prevent the leakage of engineered algae cells. Please see the hardware and safety page for details.
Innovation
We are the first team in iGEM history to use autophagy to deal with harmful substances. Theoretically, any kind of harmful substance, even pathogen, can be degraded or stored through autophagy by AIM structure, as long as there are specific intracellular proteins that can bind to it. There will be endless potential. We hope that autophagy can be more widely and deeply applied in synthetic biology.
References
- Briffa, J., Sinagra, E., and Blundell, R. (2020). Heavy metal pollution in the environment and their toxicological effects on humans. Heliyon 6, e4691.
- Chaney, R.L. (1991). The Heavy Elements: Chemistry, Environmental Impact, and Health Effects. J ENVIRON QUAL 20, 876.
- Fu, F., and Wang, Q. (2011). Removal of heavy metal ions from wastewaters: A review. J ENVIRON MANAGE 92, 407-418.
- Tchounwou, P.B., Yedjou, C.G., Patlolla, A.K., and Sutton, D.J. (2012). Heavy metal toxicity and the environment. Experientia supplementum (2012) 101, 133-164.
- Vareda, J.P., Valente, A.J.M., and Durães, L. (2019). Assessment of heavy metal pollution from anthropogenic activities and remediation strategies: A review. J ENVIRON MANAGE 246, 101-118.
- 丁禺乔, and 柳晓光. (2021). 土壤重金属污染修复技术及展望. 资源节约与环保, 77-78
- 江用彬. (2007). 莱茵衣藻（Chlamydomonas reinhardtii）对镉污染水体的生物修复研究 (首都师范大学).
- 孔祥雪, 李宝珍, and 杨金水. (2017). 微藻去除重金属镉的抗性机理研究进展. 微生物学通报 44, 1980-1987.
- Godwill Azeh Engwa, Paschaline Udoka Ferdinand, Friday Nweke Nwalo and Marian N. Unachukwu (June 19th 2019). Mechanism and Health Effects of Heavy Metal Toxicity in Humans, Poisoning in the Modern World - New Tricks for an Old Dog?, Ozgur Karcioglu and Banu Arslan, IntechOpen
