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Hong Kong JSS

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Design

Current Solutions to Copper pollution

Currently, there are 4 major ways to treat copper pollution in water:
  1. Reverse osmosis
    97 - 98% of copper in water will be removed. Reverse osmosis works by forcing water through a membrane that allows water to pass through but blocks ions such as copper ion.
  2. Distillation system
    It can remove most dissolved solids from water. However, these systems are limited to produce small amounts of water per day and the cost of using distillation systems is higher than other methods.
  3. Ion exchange filters
    It removes copper ions by adsorbing them onto mineral particles or resins. This takes place in filter cartridges which needed to be replaced periodically to maintain their effectiveness over time.
  4. Activated carbon filtration
    It will also remove copper by adsorption and the cost is lower than other methods. However, the production of activated carbon usually requires the burning of fuels and thus not very environmentally friendly.

However, these methods are either too expensive for fish farms and aquaponics, or the production of filtering materials cause pollution to the environment. Therefore, we recognize this as an opportunity for us to innovate and make some impact. Our project aimed to create a synthetic bacteria that can adsorb copper from its surrounding environment. Since bacteria can reproduce by itself, using them as an absorbent can lower both the cost and environmental impact when compared to current methods.

Reference:

  1. Copper toxicity. (2019, September 17). Retrieved from https://en.m.wikipedia.org/wiki/Copper_toxicity
  2. Kiprop, J. (2018, May 31). What Are The Sources And Effects Of Copper Pollution In The Environment? Retrieved from https://www.worldatlas.com/articles/what-are-the-sources-and-effects-of-copper-pollution-in-the-environment.html.
  3. HyperTEK Corporation. (n.d.). Aqua Pure Water Filters & Systems. Retrieved from https://www.aquapurefilters.com/contaminants/124/copper.html.
  4. Itai-itai disease. (2019, September 22). Retrieved from https://en.m.wikipedia.org/wiki/Itai-itai_disease.
  5. Abdulrazak, S., Hussaini, K., & Sani, H. M. (2016). Evaluation of removal efficiency of heavy metals by low-cost activated carbon prepared from African palm fruit. Applied Water Science, 7(6), 3151–3155. doi: 10.1007/s13201-016-0460-x



How Bacteria Handle Copper

How bacteria handle copper?
By studying how bacteria protect itself from metal-induced damage. We identified two major systems and some proteins related to the binding and exporting of copper ions.

  1. Cus system
    Cus system is a copper transport system mainly included CusA, CusB, CusC and CusF. CusABC is a complex responsible for transporting copper from the cytoplasm to outside of the cell. CusF is a periplasmic chaperone which can interact with and deliver Copper ion to CusABC.

  2. Pco system
    The plasmid-encoded Pco determinant can help bind copper with the copper ions in the cell and detoxify copper (II) to copper (I). PcoC and PcoD are membrane proteins responsible for shuttling copper ions to PcoA while PcoA can oxidate copper (II) to copper (I).

The Pco and Cus system in bacteria, they are responsible for detoxify or export of copper ions.
image source: Magnani, David & Solioz, Marc. (1970). How Bacteria Handle Copper. 10.1007/7171_2006_081.

  1. CopA
    CopA is a Cu(I)-translocating P-type ATPase which is a ubiquitous group of protein. In order to lower the copper accumulation in the cell, CopA acts as a copper pump to transport the Cu(I) to periplasm.

  2. CutA
    CutA is a copper binding protein that is also required for copper tolerance and maybe responsible for assisting the export of copper ions. It can increase copper sensitivity. It can handle many metal ions such as zinc, nickel and cobalt.

  3. Metallothionein (MT)
    MT is a family of cysteine-rich, low molecular weight proteins. They have the capacity to bind both heavy metals ion through the thiol group of its cysteine residues.

The structure of Metallothionein. The metal ions bind property mainly comes from the Cysteines.

Reference:

  1. Magnani, D., & Solioz, M. (n.d.). How Bacteria Handle Copper. Molecular Microbiology of Heavy Metals Microbiology Monographs, 259–285. doi: 10.1007/7171_2006_081
  2. Rensing, C., & Grass, G. (2003). Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS Microbiology Reviews, 27(2-3), 197–213. doi: 10.1016/s0168-6445(03)00049-4
  3. Rensing, C., Fan, B., Sharma, R., Mitra, B., & Rosen, B. P. (2000). CopA: An Escherichia coli Cu(I)-translocating P-type ATPase. Proceedings of the National Academy of Sciences, 97(2), 652–656. doi: 10.1073/pnas.97.2.652
  4. Gold, B., Deng, H., Bryk, R., Vargas, D., Eliezer, D., Roberts, J., … Nathan, C. (2008). Identification of a copper-binding metallothionein in pathogenic mycobacteria. Nature chemical biology, 4(10), 609–616. doi:10.1038/nchembio.109
  5. Argüello, J. M., Raimunda, D., & Padilla-Benavides, T. (2013). Mechanisms of copper homeostasis in bacteria. Frontiers in cellular and infection microbiology, 3, 73. doi:10.3389/fcimb.2013.00073




Using E. coli as an Absorbent

This is our plan. In order to make E. coli a good biological copper absorbent, we have to enhance its copper absorption ability. This can be done in two ways:

  1. Overexpression of copper-binding protein
    In our project, we aimed to ectopically overexpress copper binding proteins, Corynebacterium glutamicum metallothionein (CgMT) and Ascidia sydneiensis samea vanabin2, in a high-level protein expression E. coli strain BL21(DE3) in order to enhance the copper binding ability of E. coli.

  2. Knockout of copper exporter gene
    We ordered 4 E. coli gene knockout strains, CusA, CusF, CopA and CutA, from The National Institutes of Genetic in Japan. All these strains contain a single-gene deletions in E.coli K-12. As these genes are responsible for the export of copper ion from the cell, we expected that the bacteria will retain more copper ion inside the cell once these genes are knockout.

Our project plan. We aimed to increase the copper absorption ability of E. coli
so that it can be used as an cheap and environmentally friendly heavy metal absorbent.