Difference between revisions of "Template:BOKU-Vienna/Plant"
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| − | <h1 id=" | + | <h1 id="implementation">Implementation</h1> |
| − | <p> | + | <h2 id="construction-design-">Construction Design:</h2> |
| + | <p>ROBOCROP is specifically designed to work in plants. Therefore, designated promoters (MAS, p35S) and terminators (tNOS, RBCS) are used in the design and its execution.</p> | ||
| + | <p>Many components, like the ethanol, estradiol and copper receptor pathways were created with athe possible implementation in plants in mind.</p> | ||
| + | <p>The idea of using liposomes equipped with gRNAs instead of signal molecules was born in the same way. Discoveries in liposome - Cell interaction e.g. Lurqin and Paul F. [1] fostered our confidence in this idea.</p> | ||
| + | <h2 id="procedure-design">Procedure Design</h2> | ||
| + | <p>After delivering the specific inducing agents to the plant (for example, spraying (link drone)) the chemical compounds should enter the plant through the leaves, continuing their journey to the cells to be absorbed by them (Liposomes) or target matching receptor sites (ethanol, copper and estradiol pathways) in the end.</p> | ||
| + | <p>As a reaction to the receptor-inducer interaction, gRNAs will be segregated into the cytosol or they will reach the cytosol as a result of liposome uptake.</p> | ||
| + | <p>The cells, expressing dCas9 are able to further process the incoming gRNAs and deliver them to designated tTarget sites in the gGenome in a bound complex with dCas9 pProteins.</p> | ||
| + | <p>The specifically built binding sites of the gRNAs are integrated parts of the Toggle Switch. More information about the Toggle Switch Details here (Link).</p> | ||
| + | <p>As a reporter gene, we focused on GFP, but we like to emphasize that a big advantage of our iIdea is the flexible approach regarding target genes. With ROBOCROP - Arrays it might be even possible to target polygenic tTraits. | ||
| + | For easier assessment of possible sSwitch events, we chose Green Fluorescent Protein, tagged with uUbiquitin [2].</p> | ||
| + | <h2 id="risks-involved-in-design">Risks involved in Design</h2> | ||
| + | <p>We are absolutely aware of the possible risks involved in our creation. Beginning at the inducing chemical agents, it is quite obvious that the use of copper, ethanol and estradiol harbors environmental hazards. This issue is alleviated due to the indoor-lab-environment character of our experimental execution. The deployment of liposomes enriched with gRNAs might also be environmentally problematic. However, its impact on the environment has not been assessed yet. Until this gap in our knowledge has been filled, the implementation of the design is inevitably associated with indoor-lab circumstances and careful monitoring.</p> | ||
| + | <p>For future use, other signal molecules should be explored. This is one point where the smart, flexible “Bio-Bricks” should make it possible to easily exchange questionable components.</p> | ||
| + | <h2 id="utilization">Utilization</h2> | ||
| + | <p>Plants, extended with ROBOCROP-units are perfectly equipped for serving as a research object in basic research or as potent and robust crops in agriculture.</p> | ||
| + | <p>For basic research, it might be very helpful being able to turn on or off gene expression, respectively. Especially if the target genes play a key role in plant development and their knock-out would severely inhibit plant growth or not yield viable plants at all, making studying them difficult.</p> | ||
| + | <p>In agriculture, ROBOCROP - equipped crops have the capability to prepare themselves specifically for environmental conditions we as humans can inform them about with the help of analytical technologies which enables us to make predictions about future climate, soil or nutritious conditions and many more.</p> | ||
| + | <h2 id="experiments">Experiments</h2> | ||
| + | <p>As part of our project, we transformed Arabidopsis thaliana plants with the help of Agrobacterium tumefaciens. By that, we were able to transfer parts of our created vectors into the plants. With the support of “Kleine-Vehn-Lab” at the University of Natural Resources and Applied Sciences, especially with one of the team member, Sascha Weidmann we had the chance to transfer our designed GFP-construct into the plants.</p> | ||
| + | <h2 id="references">References</h2> | ||
| + | <p>[1] Lurquin, Paul F. (1979): Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts. In Nucl Acids Res 6 (12), pp. 3773–3784. DOI: 10.1093/nar/6.12.3773.</p> | ||
| + | <p>[2] Houser, J. R., Ford, E., Chatterjea, S. M., Maleri, S., Timothy, C., & Errede, B. (2013): An improved short-lived fluorescent protein transcriptional reporter for S. cerevisiae. <a href="https://doi.org/10.1002/yea.2932.An">https://doi.org/10.1002/yea.2932.An</a></p> | ||
Latest revision as of 23:40, 17 October 2018
Contents
Implementation
Construction Design:
ROBOCROP is specifically designed to work in plants. Therefore, designated promoters (MAS, p35S) and terminators (tNOS, RBCS) are used in the design and its execution.
Many components, like the ethanol, estradiol and copper receptor pathways were created with athe possible implementation in plants in mind.
The idea of using liposomes equipped with gRNAs instead of signal molecules was born in the same way. Discoveries in liposome - Cell interaction e.g. Lurqin and Paul F. [1] fostered our confidence in this idea.
Procedure Design
After delivering the specific inducing agents to the plant (for example, spraying (link drone)) the chemical compounds should enter the plant through the leaves, continuing their journey to the cells to be absorbed by them (Liposomes) or target matching receptor sites (ethanol, copper and estradiol pathways) in the end.
As a reaction to the receptor-inducer interaction, gRNAs will be segregated into the cytosol or they will reach the cytosol as a result of liposome uptake.
The cells, expressing dCas9 are able to further process the incoming gRNAs and deliver them to designated tTarget sites in the gGenome in a bound complex with dCas9 pProteins.
The specifically built binding sites of the gRNAs are integrated parts of the Toggle Switch. More information about the Toggle Switch Details here (Link).
As a reporter gene, we focused on GFP, but we like to emphasize that a big advantage of our iIdea is the flexible approach regarding target genes. With ROBOCROP - Arrays it might be even possible to target polygenic tTraits. For easier assessment of possible sSwitch events, we chose Green Fluorescent Protein, tagged with uUbiquitin [2].
Risks involved in Design
We are absolutely aware of the possible risks involved in our creation. Beginning at the inducing chemical agents, it is quite obvious that the use of copper, ethanol and estradiol harbors environmental hazards. This issue is alleviated due to the indoor-lab-environment character of our experimental execution. The deployment of liposomes enriched with gRNAs might also be environmentally problematic. However, its impact on the environment has not been assessed yet. Until this gap in our knowledge has been filled, the implementation of the design is inevitably associated with indoor-lab circumstances and careful monitoring.
For future use, other signal molecules should be explored. This is one point where the smart, flexible “Bio-Bricks” should make it possible to easily exchange questionable components.
Utilization
Plants, extended with ROBOCROP-units are perfectly equipped for serving as a research object in basic research or as potent and robust crops in agriculture.
For basic research, it might be very helpful being able to turn on or off gene expression, respectively. Especially if the target genes play a key role in plant development and their knock-out would severely inhibit plant growth or not yield viable plants at all, making studying them difficult.
In agriculture, ROBOCROP - equipped crops have the capability to prepare themselves specifically for environmental conditions we as humans can inform them about with the help of analytical technologies which enables us to make predictions about future climate, soil or nutritious conditions and many more.
Experiments
As part of our project, we transformed Arabidopsis thaliana plants with the help of Agrobacterium tumefaciens. By that, we were able to transfer parts of our created vectors into the plants. With the support of “Kleine-Vehn-Lab” at the University of Natural Resources and Applied Sciences, especially with one of the team member, Sascha Weidmann we had the chance to transfer our designed GFP-construct into the plants.
References
[1] Lurquin, Paul F. (1979): Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts. In Nucl Acids Res 6 (12), pp. 3773–3784. DOI: 10.1093/nar/6.12.3773.
[2] Houser, J. R., Ford, E., Chatterjea, S. M., Maleri, S., Timothy, C., & Errede, B. (2013): An improved short-lived fluorescent protein transcriptional reporter for S. cerevisiae. <a href="https://doi.org/10.1002/yea.2932.An">https://doi.org/10.1002/yea.2932.An</a>