Contents

    Project

    Plant SynBio

    Conventional Plant Breeding

    • Definition: Since the practice of agriculture began, eight to ten thousand years ago, farmers have been altering the genetic makeup of the crops they grow. Early farmers selected the best-looking plants and seeds and saved them to plant for the next season. Then, once the science of genetics became better understood, plant breeders used what they knew about the genes of a plant to select for specific desirable traits to develop improved varieties.
    • Context and Importance: The global population is projected to surpass 9 billion by 2050, leading to an estimated 100-110% increase in the demand for crops compared to 2005. To meet the food requirements of this rapidly growing population, especially in the context of uncertain climate change and diminishing arable land, it is essential to enhance food production while reducing inputs. Traditional plant breeding methods are often labor-intensive and time-consuming, whereas genetic engineering offers a promising approach to boost crop productivity.

    Nuclear Genome Transformation

    • Introduction: Nuclear genome transformation has become a widely adopted technique in many economically important plant species. However, it comes with several limitations, such as unpredictable expression of the gene of interest and gene silencing due to the random integration of T-DNA.
    • Alternative Approaches: In addition to nuclear transformation, plants provide another avenue for genetic modification through the transformation of the small genomes of two DNA-containing cell organelles: plastids (chloroplasts) and mitochondria, which are derived from cyanobacteria and α-proteobacteria, respectively.

    Advantages of Plastid Transformation

    • Highly Precise Transgene Insertion: Efficient homologous recombination allows for precise and targeted insertion of transgenes into the plastid genome.
    • High Levels of Protein Expression: The potential for expressing foreign proteins at extremely high levels, up to 75% of the total soluble protein, makes plastid transformation highly attractive for producing recombinant proteins.
    • Multigene Engineering: The ability to stack multiple transgenes in synthetic operons in a single transformation event facilitates the engineering of complex traits.
    • Absence of Epigenetic Effects: Plastid transformation avoids issues such as gene silencing and position effects, which are common in nuclear transformation.
    • Increased Biosafety: Since plastids are generally not transmitted through pollen in most crops, there is a reduced risk of transgene spread, enhancing biosafety.

    Applications of Plastid Transformation

    • Production of Recombinant Proteins and Enzymes: Plastids can be used to produce pharmaceutical proteins and industrial enzymes.
    • Resistance to Biotic and Abiotic Stresses: Enhanced resistance against insect pests, viral, fungal, and bacterial diseases, as well as tolerance to abiotic stresses like salt, drought, and cold.
    • Herbicide Resistance: Improved resistance to herbicides, facilitating weed management.

    Historical Development and Current Status

    • First Successes: The first successful plastid transformation was achieved in Chlamydomonas reinhardtii, a unicellular green alga, followed by Nicotiana tabacum (tobacco), a dicotyledonous flowering plant.
    • Expansion to Other Species: Since then, plastid transformation technology has been extended to over 20 species of flowering plants. Reproducible protocols for achieving homoplastic offspring are currently limited to a few species, including tobacco, potato, tomato, cabbage, soybean, lettuce, poplar, and licorice weed.
    • Challenges with Cereals: Cereals, the world's most important food crops, have proven to be recalcitrant to chloroplast transformation. This is primarily due to the lack of suitable selectable systems, low shoot regeneration frequencies, and the absence of effective cis-elements for transgene expression in non-green plastids.
    • Recent Advances: A high-efficiency plastid transformation protocol for the model plant Arabidopsis thaliana has been developed, relying on a root-based selection and regeneration system, along with the use of acc2 knockout lines, which significantly enhances the efficiency of plastid transformation.

    Stable Plastid Transformation in Kiwifruit (Actinidia chinensis)

    • Introduction: Kiwifruit is a valuable crop with a high content of vitamin C and other beneficial metabolites. Although nuclear transformation in kiwifruit has been routine for several years, plastid transformation has not been reported until now.
    • Potential Benefits: The development of a plastid transformation system in kiwifruit would enable efficient production of recombinant proteins, edible vaccines, and biopharmaceuticals, and provide a tool to study the complex inheritance patterns of plastids in this species.

    Methods

    • Optimization of Plant Regeneration and Selection System: PLASTID PESTICIDES™ tested various media compositions to develop an effective leaf-based regeneration and selection system. The optimal medium, AcReM3, containing 1 mg L-1 thidiazuron (TDZ), 2 mg L-1 6-benzyladenine (6-BA), and 1 mg L-1 α-naphthalene acetic acid (NAA), was selected based on its ability to induce the maximum number of shoots from young leaf explants.
    • Selection of Plastid-Transformed Kiwifruit: The spectinomycin-resistance gene aadA was used as a selectable marker, and 300 mg L-1 spectinomycin was found to be effective in suppressing background growth while allowing the growth of transformants.
    • Construction of the Plastid Transformation Vector: The vector pQQC7 was constructed to carry both the aadA selectable marker and a green fluorescent protein (GFP) reporter gene. Both cassettes are flanked by kiwifruit-specific sequences to allow targeted insertion into the plastid genome between the trnfM and trnG genes.
    • Biolistic DNA Delivery and Selection of Transplastomic Plants: Leaf explants were bombarded with the pQQC7 plasmid using optimized parameters. Primary spectinomycin-resistant calli appeared after three months, and six independent transplastomic lines were obtained from 12 plates. These lines were further propagated and regenerated to achieve homoplasmy, and then transferred to soil.

    Results

    • Optimization of Plant Regeneration and Selection System for Kiwifruit: Different hormone combinations were tested, and AcReM3 was found to be the most effective for inducing shoots from young leaf explants.
    • Selection of Plastid-Transformed Kiwifruit: The use of 300 mg L-1 spectinomycin was effective for selecting plastid-transformed kiwifruit, resulting in the establishment of stable transplastomic lines.

    Backcrossing

    • Definition: Backcrossing is a breeding method where a hybrid offspring is crossed back to one of its parents, often the parent that has the desired trait. This process is repeated over several generations to introduce a specific trait from the donor parent into the genetic background of the recurrent parent.
    • Usage: It is commonly used in plant breeding to incorporate a single gene or a few genes controlling a desirable trait, such as disease resistance, into an otherwise well-adapted variety.

    Marker-Assisted Selection (MAS)

    • Definition: Marker-assisted selection involves using molecular markers linked to traits of interest to select individuals for breeding. These markers can be DNA sequences that are associated with the expression of a particular phenotype.
    • Usage: MAS allows breeders to identify and select plants carrying the desired traits at an early stage, without having to wait for the trait to be expressed, thus accelerating the breeding process.

    Genome Editing

    • Definition: Genome editing refers to a set of technologies that enable scientists to change an organism's DNA by adding, removing, or altering genetic material at particular locations in the genome.
    • Usage: Techniques like CRISPR-Cas9 have revolutionized the field, allowing for precise modifications to plant genomes, which can lead to improved crop varieties with enhanced characteristics.

    Cisgenesis

    • Definition: Cisgenesis is a form of genetic engineering where only genes from the same species or closely related species are transferred. This approach aims to mimic traditional breeding but with the precision and speed of genetic engineering.
    • Usage: Cisgenic crops are designed to address consumer concerns about transgenic (GMO) crops, as they contain no foreign DNA and may be more acceptable to the public.

    Transgenesis

    • Definition: Transgenesis is the process of introducing foreign DNA (transgenes) into an organism, resulting in the stable inheritance of the introduced genetic material.
    • Usage: In agriculture, transgenic plants (often called GMOs) are developed to exhibit traits such as herbicide tolerance, pest resistance, or enhanced nutritional value.

    T-DNA (Transfer DNA)

    • Definition: T-DNA is a segment of DNA that is transferred from Agrobacterium tumefaciens to the plant cell during the natural infection process. In genetic engineering, it is used as a vector to deliver the desired gene(s) into the plant genome.
    • Usage: The T-DNA region of a binary vector is engineered to include the gene of interest along with regulatory elements to ensure proper expression in the host plant.

    Selectable Markers

    • Definition: Selectable markers are genes that confer a trait that can be easily identified, usually antibiotic or herbicide resistance, which allows for the selection of transformed cells or organisms.
    • Usage: In genetic engineering, selectable markers help researchers to distinguish between successfully transformed cells and those that have not taken up the new genetic material.

    Reporter Genes

    • Definition: Reporter genes are used to study the regulation and function of other genes. They encode proteins that produce a measurable signal, such as fluorescence or luminescence, when expressed.
    • Usage: Common reporter genes include green fluorescent protein (GFP), β-glucuronidase (GUS), and luciferase. They are often included in transformation vectors to track the expression of the gene of interest.

    Homologous Recombination (HR)

    • Definition: Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA.
    • Usage: In plastid transformation, HR is exploited to insert transgenes precisely into the plastid genome, avoiding random integration and potential disruption of endogenous genes.

    Gene Silencing

    • Definition: Gene silencing is the process by which the expression of a gene is reduced or completely suppressed. It can occur through various mechanisms, including RNA interference (RNAi) and transcriptional gene silencing.
    • Usage: Gene silencing is a concern in nuclear transformation because the random integration of T-DNA can lead to the inactivation of the introduced gene. Plastid transformation avoids this issue due to the absence of epigenetic effects.

    Transformation Efficiency

    • Definition: Transformation efficiency refers to the proportion of cells that successfully take up and integrate the exogenous DNA.
    • Usage: High transformation efficiency is desirable in genetic engineering to minimize the amount of work and resources needed to generate and screen transformants.

    Biomass Production

    • Definition: Biomass production refers to the total mass of living biological organisms in a given area or volume at a given time.
    • Usage: Increasing biomass production is a goal in many agricultural and bioenergy applications, as it can lead to higher yields of food, feed, or raw materials for energy production.

    Sustainable Agriculture

    • Definition: Sustainable agriculture is farming in sustainable ways, meeting society's present food and textile needs, without compromising the ability of future generations to meet their own needs.
    • Usage: Genetic engineering and advanced plant breeding techniques play a role in sustainable agriculture by developing crops that require fewer inputs, such as water and pesticides, while maintaining or increasing yields.

    Description

    • Agricultural Productivity: The measure of the efficiency and effectiveness with which agricultural inputs (such as land, labor, capital, and technology) are used to produce agricultural outputs. It is often expressed in terms of yield per unit area or per unit of input. Agricultural productivity can be affected by a range of factors, including climate, soil quality, farming practices, and the use of fertilizers and pesticides.
    • Environmental Stimuli: External factors that can cause a response or change in an organism's behavior or physiology. These stimuli can include abiotic factors such as temperature, light, water, and atmospheric gases, as well as biotic factors like interactions with other organisms, including predators, competitors, and pathogens. In agriculture, environmental stimuli can affect plant growth, development, and crop yields, as well as the activity and survival of pests and beneficial organisms.
    • Pests and Pathogens: Organisms that can cause damage or disease to plants, animals, or humans. Pests typically refer to insects, mites, nematodes, and other animals that feed on crops or stored products, while pathogens are microorganisms, such as bacteria, viruses, fungi, and oomycetes, that cause diseases. Both can significantly reduce agricultural productivity and have economic, social, and ecological impacts.
    • Food and Agriculture Organization (FAO): A specialized agency of the United Nations that leads international efforts to defeat hunger and improve nutrition and food security. The FAO provides technical assistance, policy advice, and knowledge exchange to help countries make informed decisions about agriculture, forestry, fisheries, and rural development. It also monitors global food supplies and works to prevent and mitigate food crises.
    • Chemical Insecticides: Substances designed to kill, repel, or control insect populations. They may act through various mechanisms, such as disrupting the nervous system, inhibiting growth, or preventing reproduction. Chemical insecticides have been widely used in agriculture for pest control, but their overuse has led to issues such as resistance, environmental contamination, and negative effects on non-target organisms, including pollinators and natural enemies of pests.
    • Resistance to Insecticides: The ability of an insect population to survive and reproduce after exposure to a pesticide that would normally be lethal. Resistance develops due to genetic changes within the population, often as a result of selection pressure from repeated applications of the same or similar chemicals. Over time, the resistant individuals become more common, leading to a decrease in the efficacy of the pesticide.
    • Lepidoptera: An order of insects that includes butterflies and moths. Lepidopterans are characterized by large, scale-covered wings, a coiled proboscis for feeding, and a complete metamorphosis life cycle. Many species are important pollinators, but some, particularly certain moth larvae, are significant agricultural pests.
    • Spodoptera litura: Also known as the tobacco cutworm or cluster caterpillar, S. litura is a polyphagous pest that attacks a wide variety of crops across Asia, Africa, and Oceania. The larvae can cause extensive damage by defoliating plants, and they are difficult to control due to their rapid development and the emergence of resistance to conventional pesticides.
    • Synthetic Biology: An interdisciplinary branch of biology and engineering that involves the design and construction of new biological parts, devices, and systems, as well as the redesign of existing, natural biological systems for useful purposes. Synthetic biology combines elements from molecular biology, genetics, biotechnology, and bioinformatics, with the goal of creating novel biological functions and solving complex problems in areas such as medicine, energy, and the environment.
    • RNA Interference (RNAi): A cellular mechanism by which small RNA molecules, such as small interfering RNAs (siRNAs) and microRNAs (miRNAs), silence gene expression. This process can occur at the transcriptional level, where it affects DNA methylation and chromatin structure, or at the post-transcriptional level, where it leads to the degradation of mRNA or blocks its translation into proteins. RNAi is a powerful tool for studying gene function and has potential applications in pest control, therapy, and functional genomics.
    • Chitin Synthase (CHS1): An enzyme that catalyzes the polymerization of N-acetylglucosamine units to form chitin, a long-chain polysaccharide that is a major component of the exoskeleton in arthropods, including insects, and the cell walls of fungi. Chitin synthases are essential for the development and integrity of these structures, making them attractive targets for pest control strategies.
    • Artificial MicroRNA (amiRNA): A type of small RNA molecule that is engineered to mimic the structure and function of endogenous microRNAs. AmiRNAs can be designed to target specific genes for downregulation through the RNA interference pathway. By binding to complementary sequences in the target mRNA, amiRNAs can inhibit gene expression, leading to reduced levels of the corresponding protein.
    • MS2 Coat Protein (CP): A structural protein that forms the capsid, or outer shell, of the bacteriophage MS2. The MS2 CP self-assembles into icosahedral particles that can encapsulate and protect RNA. When used in synthetic biology, the MS2 CP can be modified to display peptides or other molecules on the surface of the virus-like particles (VLPs) it forms, allowing for targeted delivery of RNA to cells.
    • HIV TAT47-57 (TAT): A peptide derived from the human immunodeficiency virus (HIV) trans-activator of transcription (TAT) protein. The TAT47-57 region is part of the protein transduction domain and possesses the ability to cross cell membranes, facilitating the intracellular delivery of macromolecules, including nucleic acids and proteins. This property makes TAT47-57 a valuable tool for developing drug delivery systems.
    • Virus-Like Particles (VLPs): Non-infectious, self-assembling particles that resemble viruses but lack viral genetic material. VLPs are composed of one or more viral structural proteins and can be used to deliver vaccines, therapeutic agents, or genetic material. They are considered safer than live or attenuated viruses because they cannot replicate, and they can induce strong immune responses, making them useful in vaccine development and gene therapy.
    • Plastid Engineering: The genetic modification of plastids, such as chloroplasts, which are organelles found in plant cells responsible for photosynthesis. Plastid engineering allows for the introduction of foreign genes into the plastid genome, resulting in high-level expression of the introduced genes, stable inheritance, and the potential for enhanced biosafety, since plastid genomes are generally not transferred through pollen.
    • Transplastomic Plants: Plants that have been genetically modified by introducing foreign DNA into their plastid genomes. Transplastomic plants can express the introduced genes at very high levels, and the genetic modifications are usually maternally inherited, reducing the risk of gene flow to wild relatives. This approach can be used to confer traits such as herbicide tolerance, pathogen resistance, or the production of pharmaceuticals and industrial compounds.
    • Epigenetic Effects: Changes in gene function that do not involve alterations to the underlying DNA sequence. Epigenetic mechanisms, such as DNA methylation, histone modification, and chromatin remodeling, can influence how genes are expressed. In the context of genetic engineering, epigenetic effects can lead to unintended silencing or activation of genes, which may affect the stability and predictability of the engineered traits.
    • Biosafety: The application of measures to ensure the safe handling, storage, transport, and use of living modified organisms (LMOs) and their products. Biosafety aims to minimize the potential risks to human health and the environment, including the spread of genetically modified organisms (GMOs) to non-target areas, the creation of superweeds, and the unintended consequences of gene transfer. Regulations and guidelines for biosafety vary by country and are designed to assess and manage the risks associated with GMOs.
    • siRNA (small interfering RNA): Short, double-stranded RNA molecules, typically 20-25 base pairs in length, that are involved in the RNA interference (RNAi) pathway. siRNAs can be generated from longer dsRNA precursors and guide the RNA-induced silencing complex (RISC) to cleave or repress the expression of complementary mRNA, thereby silencing the target gene. siRNAs are used in research and have therapeutic potential for treating diseases caused by aberrant gene expression.

    Design

    • Plastid Plastids are a group of organelles found in plant and algal cells. They are involved in the synthesis and storage of food, as well as in the production of pigments. The most well-known plastid is the chloroplast, which is responsible for photosynthesis, converting light energy into chemical energy stored in glucose. Other types of plastids include chromoplasts, which store pigments such as carotenoids, and leucoplasts, which store starch, lipids, and proteins. Plastids have their own DNA, known as the plastome, and they can replicate independently of the cell's nucleus.
    • RNAi (RNA Interference) RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA (messenger RNA) molecules. This process is used by cells to regulate gene activity and defend against viruses. In research and biotechnology, RNAi has become a powerful tool for studying the function of genes and developing potential therapeutic agents. It involves the use of small interfering RNAs (siRNAs) or microRNAs (miRNAs) that target complementary mRNA sequences, leading to their degradation or blocking translation, thus preventing the production of the corresponding protein.
    • Tobacco (Nicotiana tabacum) Tobacco is an annual herbaceous plant, widely cultivated for its leaves, which contain nicotine, a potent alkaloid. The species Nicotiana tabacum is the primary source of commercial tobacco. While it is best known for its use in smoking products, such as cigarettes and cigars, tobacco is also a model organism in plant biology due to its ease of genetic manipulation and transformation. In biotechnological applications, tobacco plants can be engineered to produce pharmaceuticals, enzymes, and other valuable compounds.
    • Pollen Pollen is a fine, powdery substance consisting of microscopic grains (pollen grains) that are produced by the male parts of seed plants, specifically the anthers of angiosperms (flowering plants) and the microsporangia of gymnosperms (cone-bearing plants). Each pollen grain contains the male gametophyte, which includes the male reproductive cells necessary for fertilization. Pollen plays a critical role in the sexual reproduction of plants, and it is dispersed by various means, including wind, water, and animals, to reach the female part of the flower (the stigma) where it can germinate and initiate the formation of a pollen tube, leading to fertilization.
    • Backbone (in Genetic Engineering) In genetic engineering, a backbone refers to the core structure of a vector, such as a plasmid, viral vector, or bacteriophage, into which foreign DNA fragments can be inserted. The backbone provides essential elements for the propagation and maintenance of the construct within host cells, including origin of replication (ori), antibiotic resistance genes for selection, and sometimes regulatory elements. By selecting appropriate backbones, researchers can control the expression of the inserted genes and ensure that the genetic material is stably inherited during cell division.
    • Chitin Chitin is a long-chain polymer of N-acetylglucosamine, a derivative of glucose. It is a structural polysaccharide that forms a key component of the exoskeletons of arthropods, such as insects, crustaceans, and arachnids, as well as the cell walls of fungi. Chitin is second only to cellulose in abundance among naturally occurring organic polymers. Its properties, such as strength, flexibility, and resistance to many chemicals, make it an important material in nature. In biotechnology, chitin and its derivatives, like chitosan, have various applications, including in wound healing, drug delivery, and water treatment.
    • Chitin Synthase (CHS) Chitin synthase is an enzyme that catalyzes the formation of chitin from its monomer, N-acetylglucosamine (NAG). This enzyme is crucial for the development and maintenance of the exoskeleton in arthropods and the cell walls in fungi. There are multiple isoforms of chitin synthase, each with specific roles in different stages of development and in response to environmental conditions. In pest management, chitin synthase is a target for the development of insecticides, as inhibiting this enzyme can disrupt the growth and survival of pests.

    Contribution

    • Enriching the Part Registry Enriching the Part Registry refers to the process of adding new and useful genetic parts and devices to a database, such as the iGEM Parts Registry. This helps to expand the available resources for synthetic biology projects, allowing researchers to build more complex and innovative biological systems.
    • Biological Contributions Biological contributions in the context of research and development refer to the novel or improved biological materials, methods, or knowledge that are provided to the scientific community. These can include new genes, proteins, organisms, or techniques that advance the field.
    • Novel RNAi Constructs RNAi constructs are designed genetic elements that produce double-stranded RNA (dsRNA) to trigger the RNA interference pathway, leading to the silencing of specific target genes. Novel RNAi constructs are newly developed versions that may offer enhanced specificity, stability, or delivery efficiency.
    • Chalcone Synthase 1 (CHS1) Chalcone synthase 1 (CHS1) is an enzyme involved in the biosynthesis of chalcones, which are precursors to various flavonoids. In plants, these compounds play roles in defense, pigmentation, and signaling. Silencing CHS1 in pests can disrupt their development and survival by interfering with the production of essential metabolites.
    • Essential Genes Essential genes are those that are necessary for the survival of an organism. Targeting these genes through genetic engineering, such as RNAi, can have a significant impact on the pest population by disrupting critical biological processes like molting, digestion, and reproduction.
    • Double-Stranded RNA (dsRNA) Expression Cassettes Double-stranded RNA (dsRNA) expression cassettes are genetic constructs designed to produce dsRNA within a cell. The dsRNA is then processed into small interfering RNAs (siRNAs) that mediate gene silencing via the RNA interference (RNAi) pathway.
    • Intron-Spliced Hairpin RNA (ihpRNA) Intron-spliced hairpin RNA (ihpRNA) is a design strategy for creating stable dsRNA molecules. The ihpRNA contains introns that, when spliced out, form a hairpin structure, which is then processed into siRNAs. This approach enhances the stability and processing efficiency of the dsRNA.
    • Sequence Optimization Sequence optimization involves using bioinformatics tools to select and modify nucleotide sequences to achieve desired properties, such as high specificity and minimal off-target effects. This is crucial for designing effective RNAi constructs that efficiently silence the target gene without affecting other genes.
    • MS2 Virus-Like Particles (VLPs) MS2 virus-like particles (VLPs) are non-infectious, self-assembling protein structures derived from the MS2 bacteriophage. They can encapsulate and protect RNA molecules, enhancing their stability and facilitating their delivery into cells.
    • Transmembrane Peptides (TAT) Transmembrane peptides (TAT), such as the TAT peptide, are short amino acid sequences that can penetrate cell membranes, enabling the delivery of attached or encapsulated molecules into cells. Fusing TAT to the MS2 coat protein (MCP) can significantly improve the cellular uptake of VLPs.
    • Biolistic Transformation Biolistic transformation, also known as particle bombardment, is a method used to introduce foreign DNA into cells. It involves using a gene gun to deliver DNA-coated microprojectiles (usually gold or tungsten) into plant cells, where the DNA can integrate into the genome.
    • Fluorescence Microscopy and Flow Cytometry Fluorescence microscopy and flow cytometry are analytical techniques used to study and quantify the presence and distribution of fluorescently labeled molecules within cells. These methods are often used to assess the efficiency of cellular uptake and the localization of delivered molecules, such as VLPs containing dsRNA.

    Notebook

    • Spodoptera litura Spodoptera litura, also known as the tobacco cutworm or cotton leafworm, is a species of moth in the family Noctuidae. It is a significant agricultural pest, causing damage to a wide range of crops.
    • Chitin Synthase (CHS1 and CHS2) Chitin synthases are enzymes that catalyze the synthesis of chitin, a key component of the exoskeletons of arthropods, including insects. In the context of Spodoptera litura, SlCHS1 and SlCHS2 refer to two specific chitin synthase genes. SlCHS1 is highly expressed in the outer epidermis and related ectodermal cells, while SlCHS2 is primarily expressed in the midgut, playing roles in protection against mechanical disruption and pathogen invasion.
    • Double-Stranded RNA (dsRNA) Double-stranded RNA (dsRNA) is a form of RNA that consists of two complementary strands. In the context of gene silencing, dsRNA can be used to trigger the RNA interference (RNAi) pathway, leading to the degradation of specific messenger RNAs (mRNAs) and the subsequent inhibition of gene expression.
    • Artificial microRNA (amiRNA) Artificial microRNAs (amiRNAs) are synthetic small RNA molecules designed to mimic natural microRNAs. They are engineered to target and silence specific genes through the endogenous miRNA pathway. The backbone of an amiRNA refers to the sequence structure that is used to carry the guide sequence for targeting the mRNA of interest.
    • Backbone Selection Backbone selection in the context of amiRNA design involves choosing the most effective pre-microRNA (pre-miR) structure from different organisms. The choice of backbone can influence the efficiency of gene silencing, with some backbones being more effective than others.
    • Bioinformatics Tools Bioinformatics tools are computational programs and databases used to analyze biological data, such as DNA, RNA, and protein sequences. These tools can help predict the most effective fragments for designing amiRNAs, ensuring high specificity and minimal off-target effects.
    • Polymerase Chain Reaction (PCR) Polymerase chain reaction (PCR) is a technique used to amplify specific DNA segments. It involves repeated cycles of heating and cooling to denature, anneal, and extend DNA, resulting in the exponential amplification of the target DNA sequence.
    • Agarose Gel Electrophoresis Agarose gel electrophoresis is a method used to separate DNA, RNA, or proteins based on their size. The sample is loaded into wells in an agarose gel, and an electric current is applied, causing the molecules to migrate through the gel. Smaller molecules move faster and travel farther than larger ones.
    • DNA Extraction and Purification DNA extraction and purification involve isolating DNA from a sample, such as a bacterial culture, and removing contaminants. This process typically includes cell lysis, followed by the addition of buffers to precipitate and purify the DNA.
    • Plasmid Transformation Plasmid transformation is the process of introducing plasmid DNA into bacteria, such as E. coli. This is often done to propagate the plasmid, express a gene of interest, or perform genetic manipulations.
    • Restriction Enzymes (XhoI, XbaI) Restriction enzymes are enzymes that recognize and cut DNA at specific sequences, known as restriction sites. XhoI and XbaI are examples of restriction enzymes that create sticky ends, which are useful for cloning and ligating DNA fragments.
    • T4 DNA Ligase T4 DNA ligase is an enzyme that catalyzes the formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate groups of two DNA fragments, joining them together. This enzyme is commonly used in molecular biology for the construction of recombinant DNA.
    • Colony PCR Colony PCR is a method used to screen bacterial colonies for the presence of a specific DNA insert. A small portion of each colony is used as a template for PCR, and the resulting products are analyzed to determine if the desired DNA fragment has been successfully cloned.
    • Gene Silencing Gene silencing is a process by which the expression of a gene is reduced or completely inhibited. This can be achieved through various mechanisms, such as RNA interference (RNAi), where double-stranded RNA (dsRNA) triggers the degradation of specific messenger RNAs (mRNAs).
    • Insecticides Insecticides are substances used to kill or control insects. They can be natural or synthetic and are commonly used in agriculture to protect crops from pests. Insects, like Spodoptera litura, may develop resistance to insecticides, necessitating the development of alternative control methods.
    • Resistance Resistance in the context of pest management refers to the ability of an organism to withstand the effects of a substance that is normally toxic to it. For example, Spodoptera litura has developed a high degree of resistance to many insecticides, making traditional control methods less effective.
    • Epidermis The epidermis is the outermost layer of cells covering an organism, including the skin of animals and the cuticle of plants. In insects, the epidermis is part of the exoskeleton and plays a crucial role in protection and support.
    • Midgut The midgut is a part of the digestive tract in many animals, including insects. It is involved in the digestion and absorption of nutrients. In Spodoptera litura, the midgut is protected by chitin synthase 2 (SlCHS2), which helps maintain the integrity of the intestinal epithelium.
    • Mechanical Disruption Mechanical disruption refers to physical damage to tissues or organs, which can occur due to external forces or internal processes. In the context of the midgut, mechanical disruption could result from the passage of food or the presence of pathogens.
    • Pathogens Pathogens are organisms that cause disease. In the context of Spodoptera litura, pathogens could include bacteria, viruses, or fungi that invade the midgut and potentially cause harm to the insect.
    • Molting Molting is the process by which an insect sheds its old exoskeleton to allow for growth and the formation of a new one. This process is critical for the development of larvae into adults and is influenced by the activity of genes like SlCHS1.
    • Electrophoresis Electrophoresis is a laboratory technique used to separate molecules based on their size and charge. In the context of molecular biology, agarose gel electrophoresis is commonly used to separate DNA fragments.
    • Agarose Gel Agarose is a polysaccharide extracted from seaweed and is used to make gels for electrophoresis. An agarose gel is used to visualize and analyze DNA fragments after electrophoresis.
    • DNA Quantification DNA quantification is the process of measuring the concentration of DNA in a sample. This is important for ensuring that there is enough DNA for downstream applications, such as PCR, cloning, or sequencing.
    • Transformation Mix A transformation mix is a solution containing plasmid DNA and other reagents that is used to introduce the DNA into bacterial cells. The mix is often prepared with a buffer and sometimes includes heat shock to facilitate the uptake of the plasmid by the bacteria.
    • Heat Shock Heat shock is a method used in bacterial transformation where the cells are briefly exposed to high temperatures (typically 42°C) to increase the permeability of the cell membrane, allowing the uptake of plasmid DNA.
    • Luria-Bertani (LB) Medium Luria-Bertani (LB) medium is a nutrient-rich broth or agar used for the growth of bacteria. It contains tryptone, yeast extract, and sodium chloride, and is often used in microbiology and molecular biology for culturing E. coli and other bacteria.
    • Kanamycin Kanamycin is an antibiotic used to select for bacteria that have been successfully transformed with a plasmid carrying a kanamycin resistance gene. Only those bacteria that have taken up the plasmid will survive in the presence of kanamycin.
    • Plasmid A plasmid is a small, circular, double-stranded DNA molecule that is separate from the chromosomal DNA and can replicate independently. Plasmids are commonly used in molecular biology as vectors to clone and express genes of interest.
    • Recombinant DNA Recombinant DNA is DNA that has been created by combining DNA sequences from different sources, often using restriction enzymes and DNA ligases. Recombinant DNA technology is used to create new genetic combinations that do not exist naturally.
    • Expression Vector An expression vector is a type of plasmid or virus that is designed to carry a gene of interest into a host cell and promote the expression of that gene. Expression vectors typically contain regulatory elements, such as promoters and enhancers, to control the level and timing of gene expression.

    Engineering

    Safety

    Protocol

    Wet lab

    Experiments & Results

    Parts

    Dry lab

    Model

    Software

    Hardware

    Human Practices

    Education &

    Communication

    Collaboration &

    Partnership

    Intergrated

    Human Practices

    Entrepreneurship

    Bigger Picture

    Inclusivity

    Team

    MEMBER(TEAM)

    Attributions

    Plasmid Transformation Vectors

    • Definition: Plasmid transformation vectors are small circular DNA molecules used to introduce foreign DNA into host cells (such as bacterial or plant cells). They typically carry one or more selectable marker genes to facilitate the identification of successfully transformed cells.
    • Usage: In synthetic biology and genetic engineering, plasmid vectors are used to deliver specific gene sequences, conferring new functions or characteristics to the host cells.

    Particle Bombardment (Gene Gun Technology)

    • Definition: Particle bombardment is a physical method that uses high-pressure gas to shoot tiny metal particles coated with DNA into cells, thereby introducing the DNA into the cell.
    • Usage: Commonly used for gene transfer in plant cells, especially in species that are difficult to transform using other methods, such as Agrobacterium-mediated transformation.

    Homoplasmy

    • Definition: Homoplasmy is the condition in which all the plastids (e.g., chloroplasts) or mitochondria in a cell or organism contain the same genetic material.
    • Importance: In transgenic plants, ensuring that all cells achieve homoplasmy is crucial for maintaining a stable phenotype, as it ensures that all cells have the introduced genetic material.

    Insect Bioassays

    • Definition: Insect bioassays are an experimental method used to evaluate the effects of chemicals, microorganisms, or other treatments on insects.
    • Application: In this project, insect bioassays are used to test the efficacy of genetically modified plants against targeted insect pests.

    Electrophoresis

    • Definition: Electrophoresis is a technique used to separate and analyze DNA, RNA, or proteins based on their size and charge. The molecules are moved through a gel matrix by an electric field.
    • Usage: Used to confirm the size, purity, and quality of DNA fragments, as well as the products of PCR amplification.

    PCR (Polymerase Chain Reaction)

    • Definition: PCR (Polymerase Chain Reaction) is a molecular biology technique used to amplify specific DNA segments. It involves repeated cycles of denaturation, annealing, and extension, resulting in the exponential amplification of the target DNA.
    • Usage: PCR is used to generate large quantities of DNA for various applications, including cloning, sequencing, and genetic analysis.

    Gel Extraction

    • Definition: Gel extraction is a process used to isolate and purify specific DNA fragments from an agarose or polyacrylamide gel after electrophoresis.
    • Usage: This technique is often used to prepare DNA for further manipulation, such as cloning or sequencing.

    Plasmid Extraction

    • Definition: Plasmid extraction is a method used to isolate plasmid DNA from bacterial cells. This can be done using various techniques, including alkaline lysis, which disrupts the bacterial cell wall and membrane, releasing the plasmid DNA.
    • Usage: Plasmid extraction is a fundamental step in molecular cloning and genetic engineering, providing the necessary DNA for subsequent experiments.

    Background Research

    • Definition: Background research is the process of gathering and analyzing existing information and data related to a specific topic or problem. This often involves reviewing scientific literature, market data, and other relevant sources.
    • Usage: In this project, background research was conducted to understand the current state of agricultural pest control and the potential of synthetic biology in this field.

    Project Administration

    • Definition: Project administration is the management and coordination of all aspects of a project, including planning, task assignment, resource allocation, and progress tracking.
    • Usage: Project administration involves ensuring that all team members are informed about the project's goals, protocols, and procedures, and that the project is carried out efficiently and effectively.

    Public Engagement

    • Definition: Public engagement includes activities and efforts aimed at involving and informing the public about a project or research. This can include educational outreach, community events, and communication with stakeholders.
    • Usage: Public engagement activities in this project included educating high school students about genetically modified plants, participating in conferences, and collaborating with other iGEM teams.

    Safety Form

    • Definition: A safety form is a document that outlines the safety measures and protocols for a project, including risk assessments, handling procedures, and emergency response plans.
    • Usage: The safety form is a critical component of the project, ensuring that all team members are aware of the potential risks and how to mitigate them.

    Synthetic Biology Open Conference (SBx.0)

    • Definition: The Synthetic Biology Open Conference (SBx.0) is an annual international conference focused on synthetic biology, where researchers, students, and industry professionals gather to share their work, discuss advancements, and network.
    • Usage: Participation in the Synthetic Biology Open Conference provided an opportunity for the team to present their project, receive feedback, and learn from others in the field.

    Modeling

    • Definition: Modeling is the process of creating mathematical or computational models to simulate and predict the behavior of biological systems.
    • Usage: Modeling was used in this project to predict the effectiveness of the genetically modified plants and to optimize the design of the experiments.

    Spodoptera exigua (Beet Armyworm)

    • Definition: Spodoptera exigua is a species of moth in the family Noctuidae, known for its larval stage, which is a significant agricultural pest. The larvae feed on a wide range of crops, causing substantial damage.
    • Usage: Understanding the growth cycle and resistance patterns of Spodoptera exigua was important for the project, as it helped in designing effective pest control strategies.

    RNA Interference (RNAi)

    • Definition: RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules.
    • Usage: RNAi is a key technology in this project, used to create genetically modified plants that can produce RNA molecules capable of silencing specific genes in pest insects, thereby controlling their population.

    Virus-Like Particles (VLPs)

    • Definition: Virus-like particles (VLPs) are non-infectious, self-assembling protein structures that mimic the organization and conformation of authentic viruses but do not contain any viral genetic material.
    • Usage: VLPs were used in this project to deliver RNA molecules into the target pest insects, facilitating the RNAi mechanism.

    Notebook/Record Keeping

    • Definition: Notebook/record keeping is the practice of documenting all experimental procedures, results, and observations in a systematic and organized manner.
    • Usage: Maintaining a detailed and accurate notebook is essential for reproducibility, accountability, and the overall success of the project.

    Fundraising

    • Definition: Fundraising is the process of soliciting and gathering financial support for a project or organization.
    • Usage: Fundraising was a crucial aspect of the project, enabling the team to secure the resources needed to conduct their research and participate in the iGEM competition.

    Conceptualization

    • Definition: Conceptualization is the process of forming a concept or idea, often involving brainstorming, discussion, and the development of a clear vision or plan.
    • Usage: Conceptualization was a key early phase of the project, where the team discussed and decided on the focus and direction of their research.

    Biopedia

    An encyclopedia page dedicated to explaining terms and allusions mentioned in wikis.