Individual Final Project - Transgenic Phytomining: Lemna minEr

pre-fire	post-fire

Figure 1. Proof‑of‑principle results showing phytoremediation by Lemna minor cultured in magnesium, chromium, and copper solutions. The resulting color residues indicate metal contamination and are demonstrated through the use of the biomass as an underglaze dye on ceramic tiles for visual reference.

Section 1: Abstract

Environmental pollution caused by anthropogenic activity, particularly heavy metal contamination, has created an increasing need for sustainable and cost-effective methods of environmental monitoring and remediation. Lemna minEr, a project supervised by Dr. Andrew Scarpelli, explores the use of phytoremediation to address water scarcity and contamination caused by industrial metal pollution. The project focuses on engineering a transgenic Lemna minor (duckweed) system with enhanced copper uptake, tolerance, and accumulation capabilities.

The overall objective of this project is to improve copper phytomining and remediation efficiency in Lemna minor through transgenic expression of metal transport and detoxification proteins. The project tests the hypothesis that expression of the copper transporter AtCOPT1 from Arabidopsis thaliana will enhance copper uptake, while co-expression of phytochelatin synthase (PCS) will improve intracellular detoxification and sequestration of copper ions. A red fluorescent protein (RFP) reporter is additionally incorporated to enable visual confirmation of successful transformation and gene expression.

To achieve this objective, the project first aims to design and assemble a multi-cassette plant expression plasmid containing COPT1, PCS, and RFP expression modules. The plasmid is constructed using an E. coli backbone and initially propagated through heat-shock transformation in E. coli. The amplified plasmid is then transferred into Agrobacterium tumefaciens through electroporation. Finally, Agrobacterium-mediated transformation is performed by co-cultivating the bacteria with Lemna minor fronds, enabling DNA transfer into plant cells through the bacterium’s natural infection pathway.

Following successful transformation, the project will evaluate whether transgenic Lemna minor demonstrates enhanced copper uptake and improved metal tolerance compared to wild-type plants. In the long term, this work explores the scalability of engineered duckweed as a sustainable phytoremediation platform for environmental cleanup and water treatment applications.

Section 2: Project Aims

Aim 1: Experimental Aim

The first aim of my final project is to design and assemble a multi-cassette plant expression plasmid capable of expressing AtCOPT1, phytochelatin synthase (PCS), and a red fluorescent protein (RFP) reporter in Lemna minor by utilizing molecular cloning, bacterial transformation, and Agrobacterium-mediated plant transformation protocols.

Aim 2: Development Aim

The second aim of this project is to evaluate whether transgenic Lemna minor overexpressing COPT1 and PCS demonstrates improved copper uptake, accumulation, and detoxification compared to wild-type plants through comparative copper remediation assays and tissue stress analysis.

Aim 3: Visionary Aim

The third aim of this project is to develop a scalable phytoremediation platform using engineered Lemna minor for sustainable environmental monitoring, heavy metal cleanup, and water treatment applications in polluted aquatic environments.

Section 3: Background

paper 1	paper 2

Dr. Andrew Scarpelli referred me to several papers that explored the use of synthetic biology as a way to address environmental issues through transgenic expression systems. These studies provided both conceptual and technical foundations for this project by demonstrating that plants can be genetically engineered for specialized biological functions beyond their native capabilities.

In “Programmable Ligand Detection System in Plants through a Synthetic Signal Transduction Pathway,” (Antunes et al., 2011), demonstrated that plants can be genetically engineered with synthetic signaling pathways capable of detecting environmental ligands with high specificity. The study utilized computationally-redesigned periplasmic binding proteins (PBPs) coupled with transmembrane histidine kinase (HK) signaling systems to create programmable biosensing capabilities in plants. This work demonstrated the successful design of synthetic biological pathways that can function within plant systems while maintaining environmental responsiveness. The study is significant because it showed that plants can be engineered beyond their native biological functions to perform targeted sensing and response behaviors.

Additional research has reported successful transgenic engineering experiments specifically in duckweed species, providing valuable technical references for genetically engineering Lemna plants. In “Engineering triacylglycerol accumulation in duckweed (Lemna japonica),” (Liang et al., 2023), successfully enhanced triacylglycerol (TAG) accumulation through Agrobacterium-mediated multi-cassette transformation. The study demonstrated stable transgenic expression in duckweed callus cultures and highlighted the potential of duckweed as an engineered biological platform for biofuel production through increased lipid accumulation in vegetative tissues. Other studies, including (Boehm et al., 2001; Wang et al., 2021), further established reproducible Agrobacterium-mediated transformation systems across multiple duckweed species. Together, these studies demonstrated both the feasibility of transgenic duckweed engineering and the compatible methodological approach via agrobacterium-mediated transformation.

Seeing the successful applications of transgenic expression in synthetic signaling systems and metabolic engineering, Dr. Andrew Scarpelli proposed extending similar synthetic biology approaches toward environmental phytoremediation. While previous studies have focused primarily on biosensing or biofuel production, our project adapts these methods to address heavy‑metal contamination by engineering duckweed for enhanced uptake and detoxification. We are particularly motivated by the feasibility of this system: Lemna species are distributed across five continents, supported by extensive genetic and ecological research, and offer scalable potential when paired with region‑appropriate species lines. At the same time, this broad distribution underscores the ethical importance of non‑maleficence, especially regarding ecological risks, unintended gene flow, and the possibility of bioinvasion if engineered lines were released without proper containment.

To ensure the project remains ethically responsible, the research is structured in two stages. Stage one focuses on the design and engineering of the transgenic line under controlled laboratory conditions, with strict containment to prevent environmental exposure. Stage two involves cultivation experiments to monitor growth patterns, ecological behavior, and potential risks before any consideration of applied use. These measures directly address concerns related to non‑maleficence by evaluating ecological safety, minimizing unintended consequences, and preventing gene flow or invasive behavior. Together, this staged approach supports responsible innovation while ensuring that engineered duckweed contributes to environmental remediation without introducing new ecological harm.

SECTION 4: EXPERIMENTAL DESIGN, TECHNIQUES, TOOLS, AND TECHNOLOGY

Create a detailed experimental plan for your final project. Include a timeline for each part of your experimental plan (i.e., how long you would expect each step in your final project to take). (min. 15 lines/sentences—a numbered list is acceptable)

SECTION 5: Results & Quantitative Expectations

w/ RFP	w/out RFP

For my project validation, I used UniProt and Addgene to identify an appropriate copper‑transporter (COPT1) protein sequence, and then used Benchling to virtually prototype a plasmid design. The design process involved selecting an organism‑specific promoter and evaluating whether a synthetic promoter would be necessary to fine‑tune expression efficiency. I incorporated the gene of interest, COPT1, along with a complementary red fluorescent protein (RFP) reporter to confirm successful translation and expression of the synthetic construct.

Building on the design proposal described in earlier sections, Dr. Andrew Scarpelli at the ChiTownBio laboratory node had ordered the synthetic plasmid through Twist Bioscience and are preparing for Agrobacterium‑mediated enhancement of gene expression. This preparation is intended to support the eventual transfer of the engineered Agrobacterium construct into Lemna minor for downstream phytoremediation testing.

Section 6: Reference

• Antunes et al., 2011
  Antunes, M. S., Ha, S. B., Tewari‑Singh, N., Morey, K. J., Trofka, A. M., Kugrens, P., Deyholos, M. K., & Medford, J. I. (2011). Programmable ligand detection system in plants through a synthetic signal transduction pathway. PLOS ONE, 6(1), e16292. https://doi.org/10.1371/journal.pone.0016292

• Liang et al., 2023
  Liang, Y., Zhang, Y., Zhang, J., Wang, Y., Li, Y., & Zhao, H. (2023). Engineering triacylglycerol accumulation in duckweed (Lemna japonica). Plant Biotechnology Journal, 21(2), 317–330. https://doi.org/10.1111/pbi.13943

• Boehm et al., 2001
  Boehm, R., Kruse, C., Voeste, D., Barth, S., & Schnabl, H. (2001). A transient transformation system for duckweed (Wolffia columbiana) using Agrobacterium‑mediated gene transfer. Journal of Applied Botany, 75, 107–111.

• Wang et al., 2021
  Wang, K.‑T., Hong, M.‑C., Wu, Y.‑S., & Wu, T.‑M. (2021). Agrobacterium‑mediated genetic transformation of Taiwanese isolates of Lemna aequinoctialis. Plants, 10, 1576. https://doi.org/10.3390/plants10081576