New fish to rehabilitate stormdrains - Your Published Bio Team Output
**Pre-Project**There are two pathways that could be useful to modify for this project. The first endogenous pathway that could be modified is the glycolysis pathway. This pathway is responsible for breaking down glucose into pyruvate, which can then be used to produce energy for the cell. By modifying this pathway, we could increase the efficiency of energy production in the fish so that they can swim and navigate more effectively in the challenging environment of storm drains. This could be achieved by investigating modifications to key enzymes involved in the glycolytic pathway, such as hexokinase, phosphofructokinase, and pyruvate kinase.
The second pathway that could be useful to import is the carotenoid biosynthesis pathway from algae. Carotenoids are pigments that are important for the coloration and health of many organisms, including fish. By importing this pathway, we could potentially enhance the health and resilience of the fish in the challenging environment of the storm drains, which may be polluted and lacking in nutrients. Specifically, the introduction of stable carotenoid proteins could help protect the fish from oxidative damage and improve their immune function.
For this project, the fish selected should be a subspecies or variety of a common type of fish that is known to be resilient and adaptable, such as the mosquitofish (Gambusia affinis) or the fathead minnow (Pimephales promelas). These fish can survive and even thrive in a wide range of environments, and their small size and short life cycle make them ideal for this type of experiment.
In terms of modifications, we should focus on introducing the carotenoid biosynthesis pathway from algae and investigating modifications to key enzymes involved in the glycolytic pathway. Additionally, we should consider ways to make the introduced proteins more stable and resistant to degradation, so that they can persist in the storm drain environment. Overall, the goal is to create a fish that is better equipped to survive and navigate the challenging environment of storm drains.
**Genes:** Genes to add: 1. PHB - This gene encodes for a protein called PHB, which is involved in lipid metabolism and storage. By introducing PHB, the fish could potentially store more energy reserves, allowing them to survive longer in the nutrient-limited environment of the storm drain.
2. CYP1A - This gene encodes for a cytochrome P450 enzyme that is involved in the metabolism of xenobiotics, such as pollutants. Introducing CYP1A could help the fish detoxify the polluted water in the storm drain, making them more resilient to environmental stressors.
3. CRYAB - This gene encodes for a small heat shock protein that is involved in protecting cells from stress-induced damage. Introducing CRYAB could help protect the fish from the harsh environmental conditions of the storm drain, such as high temperatures and low oxygen levels.
4. MT - This gene encodes for metallothionein, a protein that binds to and detoxifies heavy metals. Introducing MT could help the fish survive in the polluted waters of the storm drain by reducing the toxic effects of heavy metal exposure.
5. HSP70 - This gene encodes for a heat shock protein that is involved in protecting cells from heat-induced damage. Introducing HSP70 could help the fish survive in the high-temperature environment of the storm drain.
Genes to tweak expression of: 1. HK - This gene encodes for hexokinase, which is involved in the first step of the glycolytic pathway. By increasing expression of HK, we could potentially increase the efficiency of energy production in the fish, allowing them to swim and navigate the storm drain more effectively.
2. PFK - This gene encodes for phosphofructokinase, which is a key regulatory enzyme in the glycolytic pathway. By increasing expression of PFK, we could potentially increase the rate of glucose metabolism in the fish, leading to increased energy production.
3. PK - This gene encodes for pyruvate kinase, which is the final enzyme in the glycolytic pathway. By increasing expression of PK, we could potentially increase the rate of ATP production in the fish, allowing them to swim and navigate more effectively.
4. SOD - This gene encodes for superoxide dismutase, an antioxidant enzyme that helps protect cells from oxidative stress. By increasing expression of SOD, we could potentially help protect the fish from the reactive oxygen species that are generated in the polluted waters of the storm drain.
5. CAT - This gene encodes for catalase, another antioxidant enzyme that helps protect cells from oxidative stress. By increasing expression of CAT, we could potentially help protect the fish from the reactive oxygen species that are generated in the polluted waters of the storm drain.
6. TREX1 - This gene encodes for an exonuclease that is involved in DNA repair. By increasing expression of TREX1, we could potentially help protect the fish from the DNA damage that may occur in the polluted waters of the storm drain.
1. Beta-actin promoter from zebrafish: This promoter is constitutively active and can drive gene expression in a wide range of tissues. It has been extensively used in fish gene expression studies.
2. Ubiquitin promoter from tilapia fish: This promoter is also constitutively active and has been shown to drive high levels of transgene expression in various fish species.
3. EF1alpha promoter from rainbow trout: This promoter is constitutively active and has been shown to drive efficient and ubiquitous expression in various fish tissues.
The use of constitutively active promoter sequences will ensure continuous expression of the introduced genes in the fish, regardless of the conditions in the storm drain. This will increase the chances of survival of the fish, as they will have a continuous supply of the protective proteins encoded by the introduced genes.
Ideal enhancer sequences:
1. Heat shock protein 70 (HSP70) enhancer: This enhancer has been shown to drive high levels of transgene expression under heat stress conditions, which are common in the storm drain environment.
2. Metallothionein (MT) enhancer: This enhancer has been shown to drive high levels of transgene expression under heavy metal exposure, which is also common in the storm drain environment.
3. Hypoxia-inducible factor 1 (HIF-1) enhancer: This enhancer has been shown to drive high levels of transgene expression under low oxygen conditions, which are also common in the storm drain environment.
Using enhancer sequences that respond to environmental stressors will increase transgene expression levels when the fish are exposed to these stressors. This will ensure that the introduced genes are expressed at high levels when they are needed most, providing maximum protection to the fish.
Ideal terminator sequences:
1. beta-globin terminator from zebrafish: This terminator has been extensively used in fish gene expression studies and has been shown to efficiently terminate gene expression.
2. GADPH terminator from tilapia fish: This terminator has been shown to efficiently terminate gene expression in various fish species.
3. Polyadenylation signal from rainbow trout: This terminator has been shown to efficiently terminate gene expression in fish.
Using efficient terminator sequences is important to ensure that gene expression is terminated properly, preventing the production of excessive protein that may be harmful to the fish.
**Vector & Delivery:** Vector:
The most appropriate vector for delivering the desired genetic modifications to the fish would be a viral vector. Specifically, an adeno-associated virus (AAV) vector would be ideal for this application. AAV vectors have been shown to have low toxicity and are capable of efficient gene transfer to fish cells. Additionally, they have a high transduction efficiency and can integrate into the host genome, ensuring stable and long-term expression of the introduced genes.
The optimal method of delivering the AAV vector would be to inject it directly into the fertilized eggs of the fish. This would allow for the efficient and uniform distribution of the viral vector and the introduced genes among the developing fish embryos. Additionally, the early injection of the vector would ensure integration of the introduced genes into multiple tissues of the developing fish, resulting in a higher chance of survival and success of the genetic modifications.
Overall, the use of an AAV viral vector and direct injection into fertilized eggs would provide the most efficient and effective delivery method for introducing the desired genetic modifications into the fish. The use of specific promoter, enhancer, and terminator sequences would also ensure maximum and targeted expression of the introduced genes under the stressful conditions of the storm drain environment, increasing the chance of survival and rehabilitation of the fish population.
**Selection Marker:** If a selection marker is deemed needed for this project, we will use a fluorescent protein such as GFP as the marker. Incorporating GFP will allow for the easy identification and selection of successfully modified fish, as they will fluoresce under UV light. This will save time and resources in identifying the modified fish and allow for the quick selection of fish with the desired modifications. The use of a fluorescent protein marker is non-invasive and will not interfere with the function of the introduced genes or the survival of the fish in the storm drain environment.
**Transformation Protocol:** Protocol for Transformation of Fish with AAV Vector:
Materials: - Adeno-associated virus vector containing cassette with desired genes under selected promoter, enhancer, and terminator sequences - Fertilized fish eggs - Injection apparatus - UV light source - Incubator for fish eggs
Protocol: 1. Thaw the AAV vector on ice and aliquot an appropriate volume for the number of fish eggs to be injected. 2. Select the top-performing promoter, enhancer, and terminator sequences for the specific genes of interest. 3. Design and synthesize the cassette containing these sequences and the desired genes. 4. Sterilize the injection apparatus (e.g. glass micropipette) to prevent contamination. 5. Expose the fertilized fish eggs and use a bright field microscope to identify the location of one cell in the embryo. 6. Load the injection apparatus with the AAV vector, and then use the injection apparatus to deliver the vector into one cell of the embryo. 7. Move to the next egg and repeat the procedure until all fertilized eggs are injected. 8. Place the injected egg in the incubator under appropriate conditions (e.g. temperature, humidity) for the specific species of fish. 9. Monitor the injected embryos daily for hatching and survival. 10. Once hatched, use a UV light source to identify fluorescent fish indicating successful modification. 11. Collect and maintain these modified fish to establish a transgenic line. 12. Analyze the expression levels of the introduced genes using PCR and protein quantification assays. 13. Monitor the modified fish for survival and behavior under stressful conditions (e.g. high temperature, low oxygen, heavy metal exposure). 14. If desired, breed the modified fish to establish a stable transgenic line with the desired genetic modifications.
Precautions: - Sterilize the injection equipment to prevent contamination. - Minimize the handling time of the fertilized eggs, as they are sensitive to environmental stress. - Use appropriate safety measures when handling the viral vector (e.g. gloves, lab coat, safety goggles). - Ensure that the fertilized eggs are from a healthy and disease-free fish population. - Monitor the modified fish closely for any signs of abnormal behavior or health issues.
Optimizations: - Determine the optimal concentration of AAV vector for successful modification without toxicity to the embryo. - Inject the AAV vector into one cell of the embryo for uniform distribution of the genetic modifications. - Use a fluorescent protein marker for easy identification of modified fish under UV light. - Select the optimal promoter, enhancer, and terminator sequences for the specific genes and stress conditions of the storm drain environment.
**Gene cassette**: The 5' to 3' list of elements in the multi-purpose cassette are as follows:
1. Beta-actin promoter from zebrafish 2. CYP1A gene 3. PHB gene 4. CRYAB gene 5. MT gene 6. HSP70 enhancer 7. EF1alpha promoter from rainbow trout 8. SOD gene 9. CAT gene 10. TREX1 gene 11. HK gene 12. PFK gene 13. PK gene 14. Beta-globin terminator from zebrafish 15. Fluorescent protein GFP as the marker
The cassette includes promoter sequences that are constitutively active and will ensure continuous and strong expression of the introduced genes. Enhancer sequences will increase gene expression levels in response to environmental stressors present in the storm drain. Terminator sequences will efficiently terminate gene expression, preventing harmful overproduction of protein. The selection marker GFP will allow for easy identification and selection of modified fish. All genes added in the cassette will help the fish adapt to the harsh environmental conditions of the storm drain by enhancing their resilience and survival. The AAV vector will be used to deliver the cassette, and the fish will be injected directly with it during the early development stage.
**Paper Abstract:** This project aims to genetically modify fish to improve their survival and rehabilitation in the polluted and harsh conditions of a storm drain. Five genes will be added to enhance their ability to store energy, detoxify pollutants, and protect from stress and oxidative damage. Six genes will have their expression tweaked to increase energy production, antioxidant activity, and DNA repair. Constitutively active promoter sequences, stress-responsive enhancer sequences, and efficient terminator sequences will be used to ensure maximum and targeted expression of the introduced genes. An adeno-associated virus vector and direct injection into fertilized eggs will be used for efficient and effective delivery of the genetic modifications. A fluorescent protein marker will also be incorporated for easy identification and selection of successfully modified fish. This project has important implications for the conservation and rehabilitation of fish populations in polluted urban environments.
To ensure optimal growth and development of the modified fish, the following growth protocol should be followed:
1. Housing: The modified fish should be housed in a controlled environment with a temperature range of 25-27°C and a pH range of 7.0-7.5. The tank should have a filtration system to maintain water quality and should be well-aerated to ensure sufficient oxygen supply.
2. Feeding: The modified fish should be fed a balanced diet of commercial fish feed supplemented with essential fatty acids, vitamins, and minerals to ensure optimal growth, development, and energy production. The feeding frequency should be adjusted based on the size, age, and developmental stage of the fish.
3. Lighting: The modified fish should be exposed to a regular light-dark cycle of 12 hours light and 12 hours dark to ensure proper circadian rhythms and growth.
To identify successfully modified fish using the GFP marker, the following selection protocol should be followed:
1. Sample collection: A small tissue sample, such as fins or scales, should be collected from each fish and evaluated under UV light to identify fluorescence. Only fish that exhibit strong fluorescence should be selected for further analysis.
2. Genotyping: Selected fish should undergo genotyping to confirm the presence of the desired genetic modifications as well as the absence of any unintended mutations or rearrangements.
3. Further breeding: Successful modified fish should be bred to produce offspring with the desired traits. Offspring should continue to be screened for GFP fluorescence and genotyped to confirm the presence of the desired genetic modifications.
To ensure stable expression of the introduced genes and prevent loss of the desired traits, the following stabilization protocol should be followed:
1. Continuous monitoring: The modified fish should be continuously monitored for any changes in behavior, growth, or health. Any changes should be addressed promptly to ensure the continued survival and growth of the fish.
2. Breeding management: A breeding program should be established to maintain the desirable traits in the fish population. This should involve selecting individuals with the desired traits for breeding and avoiding inbreeding to prevent the loss of genetic diversity.
3. Genetic monitoring: The expression of the introduced genes should be monitored to ensure stable and consistent expression over time. Changes in gene expression should be addressed promptly to prevent any negative effects on the survival and growth of the fish population.
**Proliferation Method:** Once the modified fish have been generated, the most efficient method of proliferating them would be to breed them with wild-type fish to create a stable population of fish expressing the desired genetic modifications. This method allows for the natural selection of the fish, ensuring that only the fish with the best survival and reproductive capabilities will continue to pass on the introduced genes to future generations.
To maintain the desired genetic modifications in the resulting population, it will be important to screen the offspring of the modified fish for the presence of the introduced genes and ensure that they express the desired proteins. This can be done using PCR and protein expression analysis. The use of GFP as a selection marker will also aid in identifying the offspring carrying the introduced genes.
Once identified, the selected offspring can be bred with each other to create a stable population expressing the desired genetic modifications. It is important to note that the fish population should not be overbred, as this can lead to inbreeding and a reduced genetic diversity, reducing the fish's survival capabilities in the storm drain environment.
In conclusion, breeding the modified fish with wild-type fish and screening the offspring for the presence of the introduced genes and desired protein expression is the most efficient method of proliferating the modified fish while maintaining the desired genetic modifications.
**Conclusion:** In summary, the project aims to genetically modify fish to enhance their survival and rehabilitation in the polluted and harsh conditions of a storm drain. The transformation protocol involves injection of a modified adeno-associated virus vector into fertilized fish eggs and incorporating a fluorescent protein marker for easy identification of successful genetic modification. The growth, selection, and stabilization protocols are aimed at ensuring optimal growth, identifying successfully modified fish, and maintaining the desired genetic modifications over time. The gene construct includes constitutively active promoter and terminator sequences, stress-responsive enhancer sequences, and five selected genes to enhance the fish's resilience and survival in the storm drain environment. The successful implementation of this project has significant implications for the conservation and rehabilitation of fish populations in polluted urban environments and suggests the applicability of genetic modification in other environmental conservation efforts. Therefore, this work has considerable potential for further research and development in this field.