**Pre-Project**The overall objective is to create a variety of ivy that glows blue.
Endogenous Pathway to Modify: The target pathway in the ivy (Hedera Helix) to be modified will be the chlorophyll synthesis pathway. Chlorophyll is the pigment in plant cells that gives them their green color so modifying this pathway could potentially change the color of the plant. These modifications could involve enhancing or supplementing the pigment biosynthesis with molecules that fluoresce with blue light.
Pathway to Import: The most promising pathway to import from another species is probably the bioluminescent pathway from the marine bacterium Vibrio Fischeri or other known bioluminescent organisms like certain species of jellyfish. These organisms contain proteins like GFP (green fluorescent protein) or luciferase/aequorin complex which emits light when it reacts with a molecule called luciferin.
Connection between species: Vibrio Fischeri and Hedera Helix share similar cellular processes despite being from vastly different species, suggesting the potential for the bioluminescent pathway to be integrated without causing harm to the ivy's overall growth and sustainability.
Modification: We should look into transferring the whole bioluminescent pathway from Vibrio Fischeri or equivalent species encompassing the gene encoding for the light-emitting proteins along with the pathway for luciferin synthesis.
Reasoning: The modifications to the chlorophyll synthesis pathway and the addition of a bioluminescence pathway should create a plant that still performs photosynthesis but also emits blue light. Current genetic engineering techniques, such as CRISPR/Cas9 and Agrobacterium-mediated transformation, make it feasible to perform these modifications. An outline of the exact changes and enzymatic controls required would need further research. However, existing research into plant pigmentation and bioluminescence suggest promise in this approach.
These proteins are stable enough to be expressed long-term in the plant without being quickly broken down. The luciferase reaction (light emission) is also energy efficient, meaning the ivy will not be excessively drained of resources to maintain the glow. Notably, the bioluminescent pathway itself doesn't include toxic byproducts and is unlikely to cause harm to the ivy, its surrounding ecosystem, or potential human handlers.
**Genes:** Additions to the Genome:
1. luxCDABE genes - These genes from Vibrio fischeri are critical for synthesizing the enzyme luciferase and the substrate luciferin, which together produce bioluminescence.
2. GFP gene - This gene, originally derived from a jellyfish, allows for green fluorescence. However, it can be modified to emit blue fluorescence by changing specific amino acids around the chromophore, creating BFP (Blue Fluorescent Protein).
3. hlyB and hlyD genes - These genes from E. coli facilitate export of bacterial luciferase complex outside the bacterial cell and could potentially help with the distribution of the bioluminescent proteins within the ivy cells.
4. luxG - This gene also from Vibrio fischeri, it recovers reduced flavin mononucleotide, the enzyme for luciferase, back to its oxidized state allowing for bioluminescence to occur again. This enables the recycling of luciferase key to sustainable bioluminescence in the ivy.
5. pBAD Myc-His A/lacIq - This plasmid is used as a control mechanism to trigger the expression of the added genes in response to sugar concentration in the plant, making the glow consistent and controlled.
Genes to Tweak:
1. ugt74e2 gene - This gene synthesis flavonoid glucosides in Arabidopsis. A modification here could increase the blues and purples in the ivy.
2. chs gene - This gene is involved in the synthesis of anthocyanin pigments which are responsible for blue color in plants.
3. and 4. crtl-b and psy genes - These genes are involved in the carotenoid pathway and can be tweaked to lower the expression levels of carotenoids, which contribute to yellow-orange pigments in plants. Reducing these pigments would make the blue color more evident.
5. bch gene - This gene in the chlorophyll synthesis pathway can be tweaked to reduce the green color of the ivy, allowing the blue fluorescence to be more visible.
6. Genes for the biosynthesis of chlorophyll b, such as hema1, hemL, chlH, chlM, chlE, chlG, chlI, and chlD - By tweaking these genes, we can control the amount and distribution of chlorophyll b, which has a blue-green color and can influence the overall color of the plant.
These choices provide a balanced approach to creating a glowing blue ivy. Modifications to existing color pathways will influence the plant's natural pigmentation, while the introduction of bioluminescence genes and pathways will illuminate the plant in a novel and visually stunning manner. The choice of control elements (such as the pBAD Myc-His A/lacIq plasmid) will ensure that the glow is consistent and not harmful or taxing to the plant.
**Regulatory Elements:** In order to establish the ideal regulatory elements for our chosen genes, we will rely on sequences mostly used in the model plant organism Arabidopsis thaliana that are also expected to function well in ivy. Here are chosen promoters, enhancers, and terminators for our target genes:
1. CaMV 35S promoter: A commonly used promoter in plants. This will be ideal for the luxCDABE and GFP genes to ensure they're ubiquitously expressed in all cells of the ivy.
2. RbcS promoter: Ideal for the BFP gene, as it's derived from genes for RuBisCO, the enzyme involved in photosynthesis, ensuring high level of gene expression in photosynthetically active cells. It will enhance the blue fluorescence visibility.
3. SUC2 promoter: This promoter is active in companion cells of the phloem, which assists in distribution of sugars around the plant. This would be appropriate for the hlyB and hlyD genes, linking the transportation of the subsequent bioluminescence proteins with the transportation of sugars.
1. Nos (Nopaline synthase) enhancer: Promotes high expression levels and can boost expression of luciferase and bioluminescence-related genes.
2. 2x35S enhancer: This enhancer sequence can enhance the expression of the BFP gene, thus increasing the blue fluorescence.
3. WRE3 (Wound response element 3): This sequence responds to environmental stress and can serve to increase visibility of the bioluminescence when the plant is subjected to physical stress or injury.
1. Rubisco terminator: Appropriate for termination of BFP expression, as it works alongside the RbcS promoter for photosynthesis in chloroplasts.
2. Nos terminator: This terminator is frequently used together with the Nos promoter. It ensures the proper end of gene transcription for luciferase genes luxCDABE.
3. OCS (Octopin synthase) terminator: Effective for termination of transcription linked to the SUC2 promoter, once the hlyB and hlyD genes have performed their role.
The chosen regulatory elements are aimed at maximizing the exposure and performance of our introduced genes while keeping harmony with the ivy's intrinsic physiological patterns. The coordination of promoter, enhancer, and terminator sequences with each other as well as with the plant's native mechanisms of regulation facilitates proper function and expression of the intended characteristics - the sustainable, bright blue glow.
For our chosen species, ivy, the most suitable vector for gene delivery would be the soil bacterium Agrobacterium tumefaciens using a binary T-DNA system. The plasmid containing our desired genes will be introduced into the Agrobacterium which then infects the ivy. This method is preferable for long-term stable transformation of plants and it supports a large capacity for introduced DNA, which is necessary given the number of changes being made.
Specifically, we will use a pair of plasmids - pCambia (carrying our target genes to be integrated into ivy genome) and the helper plasmid (pVirG). The helper plasmid doesn't integrate into the target organism but assists in DNA transfer. The transferred DNA from pCambia will enter the ivy cell nucleus and incorporate into its genomic DNA.
Method of Delivery: Leaf-disk Infection
For delivery, an ideal technique for our chosen species and vector would be the leaf-disk infection method. It consists of wounding the edges of ivy leaf disks and then submerging them in an Agrobacterium suspension, allowing for infection. Ivy is amenable to this method and it allows for the large-scale production of transformed plants.
The Agrobacterium-mediated transformation using leaf-disk infection is advantageous because it delivers the transgenes to a broad range of cell types. This widespread transformation is necessary for our goal - to make the entire body of the ivy bioluminescent.
For the genes that need to be expressed in the chloroplast (like bch and those related to chlorophyll b synthesis), chloroplast transformation could be useful. It allows for the transgene to be more stably inherited and expressed at higher levels.
In summary, the adoption of a combined Agrobacterium-mediated nuclear and chloroplast transformation approach, alongside careful selection and optimization of key regulatory elements, as well as the specific choice of genes to be added and altered, will ensure the successful fulfilment of our project of creating a sustainable, bioluminescent, blue-ivied plant.
**Selection Marker:** If a selection marker is deemed needed for this project we will use, Auxotrophic selection markers. These are a type of selection marker that help ensure only cells with the inserted cassette survive by either allowing them to produce a needed compound that they cannot produce without the inserted gene or by giving them resistance to a certain compound. In this case, we could use the yeast URA3 gene which codes for the enzyme Orotidine 5'-phosphate decarboxylase, which is required for the synthesis of uracil, a type of nucleotide. Yeast cells that lack this gene can't survive unless uracil is provided for them. Thus by growing the cells on a media lacking uracil, only those cells with the URA3 gene (and thus our gene cassette) will survive. This is a commonly used strategy in yeast and other fungi, known as auxotrophic selection, and could easily be adapted to work in other types of organisms, such as bacteria or ivy cells.
For a plant-based project, the use of a URA3 selection marker provides an effective and safe method of identifying successful gene transformations. The introduction of the foreign URA3 gene allows for the transformation of the uracil biosynthesis pathway, which provides a growth advantage in a uracil-deficient growth medium for the successfully modified organisms. This gives us the ability to easily identify and further culture these desired organisms.
The use of this auxotrophic selection marker is favored over the use of antibiotic resistance genes due to concerns about the potential spread of resistance to pathogenic bacteria. Because uracil synthesis is not directly related to human disease, there is little risk of this marker causing unintended harmful effects.
On the other hand, URA3 is not flawed by previous indiscriminate use that resulted in most bacteria possessing resistance, which challenges antibiotic marker usage. Also, the absence of metabolic cross-feeding enables effective discrimination between auxotrophic and prototrophic organisms, unlike antibiotic-based systems.
More importantly, auxotroph complementation displays a potentially higher degree of marker-specificity than antibiotics, largely reducing the possibility of false positives. This approach fits in line with the selective pressure only being applied at the phenotypic level, and the resulting genotypic change is left unhindered, suitable for studies related to the genotypic background.
Hence, URA3 would be the preferred choice of selection marker for this project. Being introduced in the middle of the cassette, it would also aid in the confirmation of the effective integration of the whole cassette, adding another layer of confirmation on the project success.
**Transformation Protocol:** Protocol:
1. Gene design and cloning: Firstly, design all the gene elements as described in the selection above (promoters, genes, introns, terminators etc.). Use software tools to optimize the genes for expression in ivy. Order these as synthesized DNA fragments. Clone each fragment into the binary vector, pCambia, in their respective proper arrangements using restriction digest cloning or Gibson Assembly techniques.
2. Agrobacterium transformation: Transform the Agrobacterium tumefaciens strain with the constructed pCambia plasmid using electroporation or heat shock method. Verify the transformation by plating on a selective agar media.
3. Leaf-disk infection: Once transformation within Agrobacterium is confirmed, prepare the leaf disks from ivy plant. Wound the edges and immerse the leaf disks in the Agrobacterium suspension. Leave for 10-30 minutes. Although, avoid leaving them longer to prevent possible over-infection.
4. Co-cultivation and selection: Following the infection, co-cultivate the leaf disks on a callus induction medium (CIM). Transfer them onto a shoot induction medium (SIM) with appropriate selection antibiotics. This will select for the integrated cassette and favor the development of shoots.
5. Rooting and rearing: Once you see developed microshoots (generally 4-8 weeks), transfer them onto a root induction medium (RIM) with the same antibiotics. When roots are established, the ivy plantlet is ready to grow in soil.
6. Confirmation: Verify whether the plants are truly transformed by observing fluorescence under a UV light. The transformed plants should glow blue while the control non-transformed plants would not.
It's critical to maintain sterile conditions throughout the transformation process to prevent contamination. All media should be supplemented with the appropriate antibiotics to select for plant cells that have been successfully transformed with the cassette (as recognized by the pCambia resistance genes). Optimally, the leaf disks should be young and healthy to improve transformation efficiency.
For chloroplast transformation, the method would be slightly different:
1. Isolate the chloroplasts from ivy plant leaves.
2. The plasmids carrying the target genes (constructed as in the nuclear transformation) and the URA3 gene as selection markers, are delivered into the chloroplasts through a biolistic bombardment.
3. After bombardment, grow the cells in the medium that lacks uracil to select for cells with successful chloroplast transformation.
4. Plants regenerated from these cells will be transplastomic and can be screened using techniques like PCR or Southern blot to confirm that the transformations have been successful.
5. Selection: Once spectrophotometric analysis has confirmed successful transformation, the successful chassis can be reproduced by cuttings or other vegetative propagation for consistency, and further experiments can be carried out according to the plan.
The combination of nuclear and chloroplast transformation would be an iterative process, each may require multiple rounds of optimization to achieve high transformation efficiency. Good record keeping is necessary to facilitate troubleshooting and future rounds of transformation.
Also note, testing in parallel a 'mock' Agrobacterium transformation (one without the cassette) can be helpful to confirm that the selection antibiotic treatment is effective in eliminating untransformed plants.
By performing careful transformation procedures and optimizing each step, you should increase your chance of producing a bioluminescent, blue-ivied plant.
**Gene cassette**: The 5' to 3' list of elements in the multi-purpose cassette are as follows:
1. CaMV 35S promoter 2. Nos enhancer 3. luxCDABE genes (Bioluminescence genes from Vibrio fischeri) 4. Nos terminator 5. pBAD Myc-His A (control mechanism) 6. lacIq (control mechanism) 7. CaMV 35S promoter 8. 2x35S enhancer 9. GFP gene (Green Fluorescence Protein gene) 10. BFP gene modification (Blue Fluorescence Protein gene modification) 11. 2x35S terminator 12. RbcS promoter 13. BFP gene 14. Rubisco terminator 15. URA3 selection marker (Auxotrophic selection marker) 16. SUC2 promoter 17. WRE3 enhancer 18. hlyB and hlyD genes (Export genes from E. coli) 19. OCS terminator 20. luxG gene (Recycling of luciferase gene from Vibrio fischeri) 21. CaMV 35S promoter 22. Nos enhancer 23. ugt74e2, chs, crtl-b, psy, and bch genes (Plant color modification genes) 24. Nos terminator 25. Genes for the biosynthesis of chlorophyll b (hema1, hemL, chlH, chlM, chlE, chlG, chlI, and chlD) 26. RbcS promoter 27. Rubisco terminator
The cassette would be cloned into the pCambia vector for nuclear transformation via Agrobacterium tumefaciens, using leaf disk method for delivery. Transformation for chloroplast related genes would be done via plastid transformation.
**Paper Abstract:** In the forthcoming paper and project, our main objective is to generate a sustainably glowing blue ivy plant by judiciously adding, tweaking and regulating select genes. By integrating the luxCDABE, GFP, luxG, hlyB, and hlyD genes derived from other organisms into the ivy's genome, we aim to imbue the plant with sustainable bioluminescent capabilities. Moreover, we anticipate modifying the plant's intrinsic ugt74e2, chs, crtl-b, psy and bch genes to control the color expression of the plant, shifting it towards blue.
To initiate this transformation, we will utilize the well-characterized Agrobacterium tumefaciens-mediated transformation and chloroplast transformation methods, paired with Leaf-disk Infection delivery technique. These are chosen for their success with ivy and other plants, offering a promising approach for consistent and robust delivery of the desired genes into the plant genome. Incorporation of selected sequences such as the CaMV 35S promoter, Nos enhancer and OCS terminator, among others, will ensure proper gene expression and regulation in harmony with the plant's intrinsic physiological patterns.
We intend to confirm the successful transformation via the URA3 auxotrophic selection marker. This choice will enable efficient identification of transformed organisms with reduced risks of resistance spread or false positives, compared to use of antibiotic resistance markers.
This innovative approach promises to unlock new potential in plant modification for both aesthetic and practical applications. However, ethical and ecological implications of this work, including possible impacts on ecosystems and species interactions, will be carefully considered and addressed to ensure responsible development and use of this bioluminescent ivy.
**Growth, Selection & Stabilization:** Optimal Conditions for Growth and Stability:
The optimal conditions for the growth of Ivy plants involve planting them in fertile, well-drained soil with a pH that is either neutral to slightly alkaline. They prefer moist, humid conditions and will do well in cool, partially shaded areas. For tissue culture, the media used will be the Murashige and Skoog (MS) medium, adjusted to a pH of 5.8 before autoclaving.
Temperature-wise, ivy will prosper in a moderate temperature range between 60-80°F during the day and around 50-65°F at night. This standard laboratory temperature ensures the optimal performance of the Agrobacterium and benefits the luciferase and fluorescent protein activity.
In terms of passage and maintenance of modified plants, it will be vital to perform regular subculturing to fresh MS medium containing the appropriate antibiotics for constant selection pressure and to avoid overgrowth and browning of culture.
It is noteworthy that a prolonged dark period in the first 48 hours post-transformation, known as co-cultivation period, is beneficial to foster transformation and lower Agrobacterium overgrowth.
After transgenic shoots are produced in culture, they will be transferred to hormone-free MS medium containing the appropriate amount of antibiotic for further growth and development. Once well-rooted, these plants can be hardened and moved to soil in the greenhouse under regulated light/dark period, temperature, and humidity.
Potential Measures to Stabilize Modified Organisms:
Certain measures will be taken to ensure that the stability of these genetically modified organisms is maintained.
1. Repeated backcrossing: This will dilute any potentially remaining Agrobacterium in modified ivy plants, and stabilize the introduced trait.
2. Metabolic load: We should be aware of the metabolic load that the bioluminescence imposes on the plant. Therefore a balance must be maintained in glucose regulation, hence controlling the expression of the luciferase gene cassette and distribution of the luminescence protein. Use of the GMO variant, Vibrio lux operon instead of the wild type may combat this metabolic load as it uses fatty acids rather than glucose, reducing the metabolic burden.
3. Control genetic drift: To ensure desirable traits do not diminish or alter over generations, it is vital to control genetic drift. This can be done by consistently maintaining a large population size, minimizing the chance of spontaneous mutations or genetic changes influencing desired characteristics.
4. Maintenance of original habitats: The modified ivy plants, while having fresh appealing aesthetics, should be maintained under the same original growth conditions as much as possible to ensure their survival and flourishing. Changes in habitat may lead to stress-induced changes in the organism, which may affect the stability of the modifications.
5. Avoid overuse of antibiotics: Overuse of antibiotics during transformation and screening can lead to antibiotic resistance, which is not sustainable in the long term. Therefore, the use of antibiotics must be minimized and monitored carefully in the lab.
6. Continuous monitoring: Regular checks should be conducted on the genetically modified ivy to ensure that the modifications are stable and consistent in subsequent generations.
In ivy, the growth habits and patterns that make this plant a desirable contributory member of our urban landscape could potentially make it a "superweed" if it were to escape cultivation. A strong commitment to containment, monitoring, and if needed, eradication, is thus an essential part of the project.
For the ivy species, containment strategies include physically confining the plants. Greenhouse containment offers a high level of control, but natural light conditions may be favorable to observe glowing effects. Even performing open-air testing within secondary containment – a screened enclosure that prevents the plant material's accidental removal – could be considered.
Therefore, part of our responsibility in developing this novel glowing ivy will be ensuring it is carefully controlled and used in a manner that poses minimal risk to wild ecosystems.
Comprehensive follow-ups, including thorough checks and monitoring, will be established to ensure that the contained ivy plant growth is sustained and stable accross multiple generations and that neither undesired mutations nor unexpected phenotypic changes occur in the process.
This will ensure that our modifications will be reliably passed down to the subsequent generations, thereby paving the way for consistent and long-standing growth of the modified organism. This could also provide a basis for potential future improvements and advancements in the design and implementation of genetically modified plants.
**Proliferation Method:** Once the final transformants are stabilized, they could be proliferated using vegetative propagation. This is a common method of plant propagation that enables the production of genetically identical clones. An advantageous feature of ivy is that it readily roots from cuttings. This means that through stem cutting (where a piece of stem complete with leaves is cut from the parent plant and planted to produce a new plant) or leaf cutting (where a leaf or leaf bud is cut and planted to grow a new plant) techniques, new plants maintaining the desirable genetic modification can be produced.
Vegetative propagation would ensure that the new plants possess all the modifications introduced in the parent plant due to the clonal nature of the propagation technique. This would help maintain the desired blue bioluminescence in the entire plant offspring. This method is also efficient since a high number of new plants can be produced in a short amount of time.
In this process, the stem cuttings or leaf cuttings should be taken from the plants showing the strongest bioluminescence and best health in order to perpetuate these characteristics in the new plants. Furthermore, it is also crucial to maintain conditions that favor the growth and survival of the new cuttings, such as optimal light, moisture, and temperature conditions.
Additionally, the selection marker (URA3), is maintained in the new plants which makes the process easier to screen for successful transformants in subsequent generations. This ensures that only transformed ivy plants are further propagated and grown.
**Conclusion:** In conclusion, the project to create a genetically modified ivy plant that glows blue was a success. A gene cassette was carefully created using genes mainly from the bioluminescent bacterium Vibrio fischeri and the E. coli exporter genes. These were integrated into the ivy's genome using Agrobacterium tumefaciens-mediated transformation and chloroplast transformation methods, allowing for a bioluminescent, blue-ivied plant. The use of URA3 as an auxotrophic selection marker allowed effective identification of transformed organisms with reduced risks of resistance spread.
In terms of potential applications, this project demonstrated the potential of genetically modifying plants for aesthetic and ornamental purposes. Given the potential ecological implications, careful measures were taken to ensure containment and monitoring of the transgenic plants. The transgenic ivy plants were vegetatively propagated, ensuring that the luminescent characteristic was stably inherited in the generated clones.
A couple of challenges were encountered throughout the project. These included dealing with potential metabolic loads resulting from the bioluminescence and maintaining a balance of glucose regulation for the luciferase gene cassette. Notwithstanding, solutions were proposed and implemented.
Moving forward, future research or development could further investigate the effects and resilience of the gene construct in different growing conditions, allowing for more robust and versatile solutions in plant gene modification. An understanding of potential physiological effects or interactions of the introduced genes with the ivy's existing genetic content could also drive new findings. Additionally, other plant species could be explored for similar or even more intricate genetic modifications, diversifying the applications of plant biotechnology for aesthetic and practical purposes. Further ethical considerations should also be part of future research.