Glossary
This glossary compiles technical and project-related terms to help readers understand the methods, tools, and concepts used in our iGEM project.
Agrobacterium
Definition: A genus of bacteria known for transferring DNA into plants via its Ti plasmid.
Context: Widely used to genetically modify plants such as Nicotiana benthamiana for synthetic biology applications, including PETase expression.
Agroinfiltration
Definition: A transient gene delivery technique where Agrobacterium tumefaciens carrying a genetic construct is gently pressed into the underside of plant leaves to introduce T-DNA without stable genome integration.
Context: Used to quickly express PETase in Nicotiana benthamiana for proof-of-concept secretion tests.
Alpha-Helices
Definition: A common coiled structure within proteins, stabilized by hydrogen bonds.
Context: Many enzymes, including PETase, have alpha-helices that contribute to their shape and catalytic activity.
AutoDock Vina / PyRx
Definition: Software tools for performing molecular docking and virtual screening of enzyme–substrate interactions.
Context: Used in the dry lab to test PETase binding to PET fragments and inform construct optimization.
Bakelite
Definition: One of the first synthetic plastics, made by polymerizing phenol and formaldehyde.
Context: Bakelite shows how synthetic polymers can persist in the environment and highlights the need for biodegradable alternatives.
Binary Vector System
Definition: A two-part system separating the T-DNA (gene of interest) from the virulence genes that mediate DNA transfer.
Context: Enabled safe and efficient Agrobacterium-mediated transformation using vectors like pCAMBIA1302 and pGWB2.
CaMV 35S Promoter
Definition: A strong, constitutive promoter from cauliflower mosaic virus widely used in plant biotechnology to drive gene expression.
Context: Used to ensure PETase is consistently produced in plant tissues.
ccdB Gene
Definition: A bacterial toxin gene used as a negative selection marker in cloning vectors.
Context: Removed during cloning to allow successful propagation of the PETase construct.
Codon Optimization
Definition: Altering a gene’s DNA sequence to match the preferred codon usage of the target host organism for better expression.
Context: PETase was codon-optimized for Nicotiana benthamiana to maximize enzyme production.
DBTL Cycle (Design–Build–Test–Learn)
Definition: An iterative engineering workflow used in synthetic biology to optimize genetic constructs and systems.
Context: Guided the entire PETase expression project from vector design to plant testing.
Disulfide Bridges
Definition: Covalent bonds between sulfur atoms in cysteine residues of a protein that stabilize its structure.
Context: Disulfide bridges affect the stability and activity of enzymes such as PETase when expressed in plants or engineered systems.
Electroporation
Definition: A method that uses electric pulses to temporarily open cell membranes, allowing DNA to enter.
Context: Used to introduce PETase vectors into Agrobacterium after initial cloning difficulties due to vector size.
Ethylene Glycol (EG)
Definition: A colorless, odorless compound commonly used as an antifreeze and industrial raw material.
Context: When PET is degraded by PETase, one of the products released is ethylene glycol, which can be further metabolized by certain microorganisms.
Gastrointestinal Obstruction
Definition: A blockage in the digestive tract that prevents normal passage of food or fluids.
Context: Microplastics ingested by aquatic animals can accumulate and cause gastrointestinal obstruction, harming ecosystems and potentially entering the human food chain.
Green Fluorescent Protein (GFP)
Definition: A protein that glows green under UV light, used as a reporter to confirm gene expression.
Context: Added to PETase constructs to visualize protein localization in plant cells.
GROMACS
Definition: A molecular dynamics simulation package for modeling how proteins behave over time.
Context: Considered for analyzing PETase stability and secretion efficiency in silico.
HMMER
Definition: A bioinformatics tool that searches sequence databases for homologous proteins using hidden Markov models.
Context: Useful in synthetic biology for identifying potential PETase-like enzymes or analyzing engineered protein sequences.
HPLC (High-Performance Liquid Chromatography)
Definition: An analytical technique that separates, identifies, and quantifies compounds in a mixture.
Context: Used to measure PETase activity by detecting PET degradation products (TPA and EG) in plant extracts.
Ideonella sakaiensis
Definition: A bacterium capable of producing an enzyme called PETase, which can break down polyethylene terephthalate (PET) plastics.
Context: Discovered in a Japanese recycling plant, this bacterium is important in synthetic biology research because it naturally degrades PET, a major contributor to plastic pollution.
In-Fusion Cloning
Definition: A seamless DNA cloning technique that joins fragments with short overlapping ends without needing restriction sites.
Context: Used to assemble PETase constructs into plant expression vectors.
Kanamycin Resistance Gene
Definition: A selectable marker that allows only cells containing the desired plasmid to survive on antibiotic media.
Context: Used to confirm successful Agrobacterium transformation with the PETase construct.
Kill Switch
Definition: A genetic safety mechanism designed to trigger cell or organism death under specific conditions, preventing uncontrolled spread.
Context: Proposed for future large-scale use of engineered plants to ensure biosafety compliance.
Molecular Docking
Definition: A computational method for predicting how molecules, such as enzymes and substrates, interact at the atomic level.
Context: Used to model PETase’s interaction with PET and validate secretion designs before lab testing.
Mutagenesis
Definition: The process of creating genetic mutations to study or improve protein function.
Context: Used in synthetic biology to modify enzymes like PETase, enhancing their ability to degrade plastics.
Nicotiana benthamiana
Definition: A plant closely related to tobacco, widely used in synthetic biology due to its high susceptibility to pathogens and ease of genetic modification.
Context: Commonly used as a “proof-of-concept” host for expressing engineered proteins, including enzymes like PETase, before scaling to aquatic plants such as water hyacinth.
pCAMBIA1302 / pGWB2
Definition: Common plant transformation vectors; pCAMBIA1302 carries selectable markers and GFP reporter genes, while pGWB2 provides a CaMV 35S promoter for high expression.
Context: Used as backbones for the PETase construct to ensure strong expression and easy detection in plants.
PETase
Definition: An enzyme that breaks down PET into its building blocks, terephthalic acid (TPA) and ethylene glycol (EG).
Context: PETase is a key tool for engineering biological systems that degrade plastics, such as in this iGEM project’s exploration of plant-based biofilters.
Phenolic Resins
Definition: A group of synthetic polymers formed by reacting phenol with formaldehyde.
Context: Phenolic resins, like Bakelite, are examples of durable plastics that do not break down naturally, inspiring modern plastic-degradation research.
Polyethylene Terephthalate (PET)
Definition: A strong, lightweight polyester used in synthetic fibers, beverage bottles, food containers, and packaging.
Context: PET is a major target for biodegradation because it is one of the most widely produced and persistent plastics found in waterways.
Polymer
Definition: A very large molecule composed of repeating subunits (monomers) linked in long chains.
Context: Plastics, including PET, are synthetic polymers that persist in water systems, driving the need for bio-based degradation strategies.
PyMOL
Definition: A molecular visualization program used to inspect and refine 3D protein structures.
Context: Helped visualize PETase structure and signal peptide placement in design stages.
Repolymerization
Definition: The chemical process of linking monomers back into a polymer chain.
Context: After breaking PET into TPA and EG, industries can use repolymerization to create new PET products from recycled building blocks.
Restriction Enzymes (XbaI, SacI)
Definition: Proteins that cut DNA at specific sequences, creating sticky or blunt ends for cloning.
Context: Used to prepare pGWB2 and other vectors for inserting PETase and signal peptide sequences.
Signal Peptide (Amy3SP)
Definition: A short amino acid sequence at the start of a protein that directs it to the plant’s secretory pathway.
Context: The alpha-amylase 3 signal peptide from rice was fused to PETase to ensure it is secreted to the apoplast where microplastics accumulate.
Stable Transformation
Definition: A permanent method of plant genetic engineering where T-DNA integrates into the plant genome, making modifications inheritable.
Context: Planned for later stages to create fully transgenic plants that continuously produce and secrete PETase.
Terephthalic Acid (TPA)
Definition: An aromatic compound used to produce polyester fibers and plastics.
Context: PETase activity on PET releases TPA, which can potentially be recycled to make new plastics or other materials.
Ti Plasmid
Definition: A natural tumor-inducing plasmid from Agrobacterium tumefaciens that can deliver DNA into plants.
Context: Disarmed Ti plasmids were used as vectors for inserting PETase constructs into N. benthamiana.
Transient Transformation
Definition: Temporary gene expression in plants where T-DNA does not integrate into the genome, enabling rapid testing of constructs.
Context: Chosen for the initial phase of this project to test PETase secretion before attempting stable plant lines.