Project Description

The how and why we chose our project.

Background

Anthropogenic activities such as mining sites, industrial sites, waste treatment plants, fertilisers and pesticides create enormous amounts of heavy metal pollution, which has an important ecological impact on biodiversity, causing detrimental health effects in both humans and animals (Azhar et al., 2022; Kapahi & Sachdeva, 2019). Heavy-metals can enter drinking water and the agro-ecosystem, and accumulate through the food-chain; in humans, arsenic poisoning can lead to multi-organ damage, cancer, and neurological disorders (Satyapal et al., 2018). Hazard management of this waste is extremely costly with $1.5 million USD worth of damages caused by heavy metal pollution annually in ASEAN countries (Ding, 2019).

Current remediation techniques are inadequate in effectiveness and largely limited in scope. Physical remediation techniques are commonly used methods and aim to confine polluted areas, but these are highly expensive and are often destructive to the environment putting ecosystems at risk. Chemical remediation techniques are not much better; these methods are much too expensive, the reagents and byproducts are often toxic, and the method requires reapplication every few years, limiting its effectiveness (Satyapal et al., 2018).

 

Our Solution: a modular bioaccumulation platform for heavy-metal bioremediation

To develop a more environmentally friendly and cost-effective method, we propose a synthetic biology solution consisting of a modular bioaccumulation platform for heavy-metal bioremediation. In our project, we engineer heavy-metal binding proteins (MBPs) localised to encapsulin-nanocompartments delivered by bacteriophages into bacteria to capture and sequester toxic arsenic compounds. Our vision is to have engineered phages that we may intentionally release into the environment, causing bacteria to produce the necessary bioremediation tools and clean up any heavy metal waste, not just arsenic. This strategy will be cost-effective, non-toxic and non-invasive.

 
 
Figure 1. General design of heavy metal sequestering compartment.
 

Encapsulins

Encapsulins are a class of large cage-like proteins present in bacteria and archaea; they are generally expressed as subunits that assemble into homo-oligomers, and assemble around cargo proteins like a cage (Figure 2.)(Wiryaman & Toor, 2022).

 
 
Figure 2. Model for encapsulin nanocompartment assembly in bacteria. The modular nature and ease of targeting cargo to encapsulins facilitates the combinatorial testing of many metal binding proteins (MBPs), provided they bear the appropriate C-terminal cargo-loading peptide (CLP).
 

In order for these to capture our MBPs and heavy metal ions, encapsulin genes must be transformed, expressed, and purified.

 

Metal Binding Proteins

Trivalent arsenic compounds have a high affinity for reduced cysteine residues, the typical arsenic binding proteins such as metallothionein therefore have clusters of cysteine residues (Shen et al., 2013). Preparation of the MBPs required PCR of the pET21b backbone, transformation of MBPs into the pet21 backbone, expression, purification, and testing of arsenic binding using isothermal titration calorimetry (ITC) mass spectrometry and a differential scanning fluorimetry assay (DSF).

After successful expression of the encapsulins and MBPs, they are transformed into a pet21b backbone together for coexpression. The coexpressed proteins are then run on a gel and are tested for binding with a DSF.

 

Bacteriophages as a delivery system

Bacteriophages are viruses that specifically infect bacteria. These biological agents hold genetic information within a protein capsule, their capsid, to deliver into bacteria (Clokie et al., 2011). With this function, we use CRISPR/Cas9 to engineer genes encoding our encapsulins and MBPs into the phage genome, producing a phage that will inject its genome along with the genetic information of our proteins into bacteria. Bacteria infected by our engineered phages will express our encapsulins and MBPs which will self assemble into heavy metal sequestering protein structures.

This work consisted of culturing bacteriophages and designing guide RNAs to insert our genes into non-essential regions of the phage genome to retain infectious activity of engineered bacteriophages. We transformed bacteria with our CRISPR/Cas9 system to engineer successful encapsulin and MBP constructs into phages.

 

References

  1. "Azhar, U., Ahmad, H., Shafgat, H., Babar, M., Munir, H. M. S., Sagir, M., Arif, M., Hassan, A., Rachmadona, N., Rajendran, S., Mubashir, M. & Khoo, K. S. 2022. Remediation techniques for elimination of heavy metal pollutants from soil: A review. Environmental Research, 214."
  2. "Clokie, M. R., Millard, A. D., Letarov, A. V. & Heaphy, S. 2011. Phages in nature. Bacteriophage, 1, 31-45."
  3. "Ding, Y. 2019. Heavy metal pollution and transboundary issues in ASEAN countries. Water Policy, 21, 1096-1106."
  4. "Kapahi, M. & Sachedeva, S. 2019. Bioremediation Options for Heavy Metal Pollution. Journal of Health and Pollution, 9."
  5. "Satyapal, G. K., Mishra, S. K., Srivastava, A., Ranjan, R. K., Prakash, K., Haque, R. & Kumar, N. 2018. Possible bioremediation of arsenic toxicity by isolating indigenous bacteria from the middle Gangetic plain of Bihar, India. Biotechnology Reports, 17, 117-125."
  6. "Shen, S., Li, X., Cullen, W. R., Weinfeld, M. & Le, X. C. 2013. Arsenic Binding to Proteins. Chemical Reviews, 113, 7769-7792."
  7. "Wiryaman, T. & Toor, N. 2022. Recent advances in the structural biology of encapsulin bacterial nanocompartments. Journal of Structural Biology: X, 6."