Develop a spider plant that changes color when flu particles are in the air, thereby detecting if someone in the house is sick - Your Published Bio Team Output
**Pre-Project**To achieve the goal of developing a spider plant that changes color when flu particles are in the air, we should consider adding protein coding sequences to the plant. Specifically, we could introduce a protein that binds to the flu particles and triggers a color change in the plant.
One potential protein to consider using is the CRISPR-Cas13 protein. This protein has been shown to have the ability to detect specific RNA sequences and trigger a fluorescence response. We could engineer the spider plant to produce the Cas13 protein and modify it so that it targets the RNA of the flu virus. If the virus is present in the air, the Cas13 protein will detect it and cause the spider plant to change color.
Another approach would be to import whole metabolic pathways into the spider plant that respond to flu particles. For instance, we could look at metabolic pathways in other plants that respond to pathogens and adapt them to detect flu particles. However, this approach may be less precise than using a specific protein target, as it may also trigger false positives.
Considering the species selection, Chlorophytum comosum (aka spider plant), would be an excellent choice for this application. It is a hardy and common indoor plant that can tolerate various conditions. Additionally, its variegated leaves already exhibit different colors, providing a great platform to incorporate a color-change mechanism.
Overall, we should investigate the modification of the spider plant by adding CRISPR-Cas13 protein or metabolic pathways that respond to flu particles. Testing the modified spider plant in a controlled environment would be the next step to ensure that it is sensitive to flu particles while not triggering false positives. If successful, this could be a cost-effective and easy-to-use home sensor for flu detection.
**Genes:** Genes to add: 1. Cas13 protein (e.g. from Leptotrichia shahii) - This protein has been shown to have RNA-targeting and fluorescent properties, making it a prime target for detecting flu particles. 2. Fluorescent protein (e.g. mCherry) - Adding a fluorescent protein to the spider plant will allow for the color change to be easily visible. 3. Pathogen recognition receptor (e.g. FLS2) - Introducing a pathogen recognition receptor could enhance the sensitivity of the spider plant to flu particles. 4. Promoter for gene expression in response to pathogen detection (e.g. PR1 promoter) - Adding a promoter that is activated when a pathogen is detected will ensure that the Cas13 protein and fluorescent protein are produced upon flu detection. 5. Chlorophyll degradation gene (e.g. pheophytinase) - This gene could be added to allow for the breakdown of the green pigment in the spider plant's leaves, allowing for a more visible color change.
Genes to tweak expression of: 1. Photosynthesis genes - Modifying the expression of photosynthesis genes could impact the overall growth and health of the spider plant, potentially shifting its balance towards producing the fluorescent protein. 2. Protein degradation genes - By reducing the expression of protein degradation genes, the spider plant could maintain the Cas13 and fluorescent proteins for longer periods of time, increasing the sensitivity of the color change. 3. Protein transport genes - Tweaking the expression of genes involved in protein transport could allow for efficient movement of the Cas13 and fluorescent proteins within the plant. 4. Defense response genes - Activating the plant's defense response pathway upon flu particle detection could enhance the sensitivity of the spider plant to flu particles. 5. Stress response genes - Modifying the expression of stress response genes could help the spider plant cope with any potential negative effects from the gene modifications or color change mechanism.
**Regulatory Elements:** Promoters: 1. CaMV 35S Promoter - This promoter has been extensively used for gene expression in plants, including spider plants. It is known to have strong constitutive activity, which makes it an ideal candidate for driving the expression of the genes for fluorescent protein, Cas13, and the pathogen recognition receptor. 2. PR10 Promoter - This promoter has been shown to be activated specifically in response to viral infections, making it an ideal candidate for driving the expression of the pathogen recognition receptor gene specifically in response to flu particles. 3. PsaD Promoter - This promoter is involved in photosynthesis and could be utilized to drive the expression of the photosynthesis genes in a manner that can subtly shift the balance of carbon fixation towards the production of the fluorescent protein.
Enhancers: 1. Wounding-responsive element (WRE) - This enhancer has been shown to be activated upon mechanical damage to the plant tissue, which could be utilized to enhance the expression of the defense response genes in response to the introduction of flu particles. 2. Heat shock element (HSE) - This enhancer is known to be activated under conditions of heat stress, and could potentially enhance the expression of the stress response genes in response to the gene modifications or color change mechanism. 3. G-Box - This enhancer is activated in response to pathogen infection and could potentially enhance the expression driven by the PR10 promoter.
Terminators: 1. Nopaline synthase terminator - This terminator has been used extensively as a 3' end for gene expression cassettes in plants and is a solid choice for the genes in question. 2. T35S Terminator - This terminator is derived from the terminator region of the CaMV 35S promoter and could be utilized for transcription termination in the case of the PR10 and PsaD promoters. 3. rbcS-E9 Terminator - This terminator has been shown to be effective in terminating transcription in plants and could be used as a secondary termination signal if necessary.
The PR1 promoter and FLS2 gene will enhance the sensitivity of the spider plant to flu particles, while the Cas13 and fluorescent proteins will allow for easy detection of the flu presence in the air. The chlorophyll degradation gene can be used to enhance the color change effect, and the photosynthesis, protein degradation, and transport genes can ensure that the Cas13 and fluorescent proteins are expressed and maintained effectively in the plant. The chosen promoters, enhancers, and terminators will ensure effective expression and regulation of the genes in question.
**Vector & Delivery:** To deliver the genetic modifications to the spider plant, Agrobacterium tumefaciens can be used as a vector. This soil-dwelling bacterium is known to transfer DNA to plants and can therefore be used to introduce the modified genetic material into the spider plant. The Cas13 and fluorescent protein genes can be delivered together within one T-DNA cassette, while the FLS2 gene can be introduced on a separate T-DNA cassette.
To optimize the desired genetic modifications, the Agrobacterium-mediated transformation can be carried out on young spider plants, as they are more amenable to regeneration and shoot proliferation than mature plants. The spider plants can be grown in vitro and hormonally induced to regenerate into callus tissue before being co-cultivated with the Agrobacterium carrying the T-DNA cassettes. The spider plant callus tissue can then be selected for containing the introduced genetic modifications and can be induced to regenerate into shoot tissue.
In conclusion, using Agrobacterium tumefaciens as a vector, along with in vitro plant transformation techniques, can optimize the desired genetic modifications for spider plants that change color when flu particles are in the air. The chosen vector and delivery method are specific to the spider plant species and the desired genetic modifications, allowing for efficient and effective gene delivery and expression.
**Selection Marker:** If a selection marker is deemed needed for this project, we will use a herbicide resistance gene, such as the bar gene. The introduction of the herbicide resistance gene will enable the selection of plants that have successfully integrated the modified genetic material. This will allow for the efficient production of spider plants that have the ability to detect flu particles in the air, while minimizing the likelihood of producing non-modified plants.
**Transformation Protocol:** Protocol for Transformation of Spider Plants to Detect Flu Particles:
Materials: - Spider plant callus tissue - Agrobacterium tumefaciens containing the desired T-DNA cassettes - Murashige and Skoog (MS) medium with appropriate hormones and supplements - Herbicide-resistant selection medium (if using selection marker) - Sterile instruments (scalpel, forceps, etc.) - Sterilized Petri dishes - Sterilized plant growth chamber
1. In vitro growth of spider plant callus tissue: - Sterilize the spider plant cuttings by washing them with 70% ethanol and then soaking them in a 10% bleach solution for 10 minutes. - Rinse the cuttings with sterile water and then dry them with sterile paper towels. - Cut the sterilized spider plant cuttings into small pieces (less than 1cm). - Place the cuttings onto MS medium supplemented with appropriate growth hormones (e.g. 1 mg/L 6-benzylaminopurine, 2 mg/L naphthalene acetic acid) to induce callus formation. - Incubate the callus tissue in a sterile growth chamber at 25°C under continuous light (~100 µmol/m2/s).
2. Agrobacterium-mediated transformation: - Prepare the Agrobacterium containing the desired T-DNA cassettes by growing it in liquid culture medium to mid-log phase. - Pellet the Agrobacterium cells by centrifugation and re-suspend them in an appropriate buffer (e.g. MS medium). - Add the spider plant callus tissue to the Agrobacterium suspension and co-culture them for 1-3 days on a shaker (e.g. at 100 rpm) in the dark. - After co-cultivation, wash the spider plant callus tissue with a sterilized buffer (e.g. MS medium with 250 mg/L cefotaxime). - Allow the spider plant callus tissue to recover for 3-5 days on selection medium (if using selection marker).
3. Selection of transformed spider plant tissue: - Select the spider plant callus tissue that has successfully integrated the desired genetic modifications using the selection medium (if using selection marker). - Transfer the selected spider plant callus tissue onto fresh MS medium without antibiotics, but with the appropriate growth hormones (e.g. 1 mg/L 6-benzylaminopurine, 2 mg/L naphthalene acetic acid) to induce regeneration.
4. Regeneration of spider plant shoots: - Incubate the selected spider plant callus tissue in a sterile growth chamber at 25°C under continuous light (~100 µmol/m2/s). - After a few weeks, shoots will start to emerge from the selected spider plant callus tissue. - Transfer the regenerated spider plant shoots onto fresh MS medium without antibiotics, but with the appropriate growth hormones (e.g. 0.5 mg/L gibberellic acid, 0.1 mg/L indole-3-butyric acid) to promote root growth. - After root growth has occurred, transfer the spider plant seedlings to soil in a greenhouse or other appropriate growing environment.
5. Characterization of transformed spider plants: - Analyze the expression of the introduced genes in the transformed spider plants using techniques such as RT-PCR or Western blotting. - Confirm that the Cas13 protein is functional by testing its RNA-targeting capability and fluorescent properties. - Determine the sensitivity of the transformed spider plant to flu particles by exposing them to different concentrations of flu particles and observing the color change effect. - Optimize the balance of
**Gene cassette**: The 5' to 3' list of elements in the multi-purpose cassette suitable for cloning into the spider plant using Agrobacterium tumefaciens as a vector are as follows:
The cassette will contain the Cas13 and fluorescent protein genes together within one T-DNA cassette while the FLS2 gene can be introduced on a separate T-DNA cassette. To optimize the desired genetic modifications, the Agrobacterium-mediated transformation can be carried on young spider plants. If a selection marker is deemed necessary, a herbicide resistance gene like Bar gene can be used.
The cassette elements can be synthesized by a reputable supplier such as GenScript or Bio Basic.
**Paper Abstract:** The main objective of this project is to modify spider plants to change color in response to the presence of flu particles in the air, providing an easy and affordable way to detect the flu. This can be achieved by introducing genes such as Cas13 protein, fluorescent protein, pathogen recognition receptor, promoter for gene expression, and chlorophyll degradation genes. The expression of photosynthesis, protein degradation, protein transport, defense response, and stress response genes will also be modulated to optimize the genetic modifications.
The regulatory elements such as promoters, enhancers, and terminators, including CaMV 35S promoter, PR10 promoter, Wounding-responsive element, and Nopaline synthase terminator, will be used to ensure the proper regulation of gene expression. Agrobacterium tumefaciens will serve as a vector for introducing modified genetic material, and in vitro plant transformation techniques will be used to optimize the desired genetic modifications for spider plants.
If necessary, the bar gene can be used as a selection marker to identify plants that have successfully integrated the modified genetic material. This project has significant implications in providing an affordable and accessible alternative to flu detection, with the potential for further applications in the detection of other airborne pathogens.
**Growth, Selection & Stabilization:** Optimal Conditions for Growth and Selection: Spider plants have specific requirements for growth, so it is important to provide them with the optimal conditions to successfully express the introduced genetic modifications. Spider plants have a preference for bright, indirect sunlight and moderate temperatures, ideally between 60-75°F (15-24°C). Soil should be well-draining and kept evenly moist, without being too wet or too dry. The growth medium should also provide essential nutrients, such as nitrogen, phosphorous, and potassium.
To select for the successful integration of the introduced genetic modifications, the selection marker, such as the herbicide resistance gene, can be introduced alongside the desired genes. The selection process can be done by exposing the transformed callus tissue to the herbicide and selecting for the plants that successfully survived the exposure.
Stabilization Protocol: To ensure the modified spider plants remain stable and viable, it is important to carefully monitor their growth and health over time. The plants can be grown under controlled conditions and monitored for any signs of stress, improper growth, or color changes. The Cas13 and fluorescent proteins are susceptible to degradation over time, so it is important to monitor the expression of these proteins and possibly adjust the expression levels of the protein degradation genes as needed. Additionally, any changes in the expression of the introduced genes, or symptoms of stress or instability should be investigated and addressed promptly.
Overall, establishing the optimal conditions for growth and selection, and monitoring the expression of introduced genes, will enhance the stability of the modified spider plants and ensure their success as an effective tool for detecting flu particles in the air.
**Proliferation Method:** Once the spider plants have been transformed and selected for the desired genetic modifications, the method of proliferating the final transformants can be through a process called micropropagation. Micropropagation involves the use of tissue culture techniques to generate large numbers of identical plantlets from a small piece of tissue.
To begin micropropagation, a small piece of the transformed spider plant tissue can be excised and sterilized to avoid contamination. The tissue can then be placed onto a sterile nutrient medium containing plant growth regulators, such as cytokinins and auxins, which promote cell division and shoot formation. The resulting shoots can then be excised and transferred to fresh media to promote further growth.
The newly generated plantlets can then be transferred to soil and grown in controlled environments under the appropriate lighting and temperature conditions. The use of micropropagation ensures that the resulting population of spider plants contains the desired genetic modifications and that these modifications are maintained from generation to generation.
Overall, the use of micropropagation ensures efficient proliferation of the final transformants while maintaining the desired genetic modifications in the resulting population. This method allows for the production of a large number of identical plants that have the ability to detect flu particles in the air, which can be immensely helpful in detecting and preventing flu outbreaks.
**Conclusion:** In conclusion, this project presents a transformation protocol for spider plants to detect flu particles, which can provide an accessible and affordable alternative to traditional methods of flu detection. The introduction of genes encoding Cas13 protein, fluorescent protein, pathogen recognition receptor, promoter for gene expression, and chlorophyll degradation genes, along with the modulation of expression of various genes, can optimize the desired genetic modifications in the spider plants. The use of regulatory elements such as promoters, enhancers, and terminators can ensure proper regulation of gene expression. The protocol for growth, selection, and stabilization of the modified spider plants can enhance the stability of the plants and ensure their success as an effective tool for detecting flu particles. Micropropagation can be used to ensure efficient proliferation of the desired transformants. The successful application of this project can serve as a model for developing similar cost-effective diagnostic tools for other airborne pathogens. This work provides immense potential and future directions for research and development in the field of plant biotechnology.