Team:Marburg/Cell-Free/Plants Used
Plants we used
Neither the idea of chloroplast engineering nor prototyping in a cell-free context is a novel approach. Important foundational advances have been done by the Lab of Michael C. Jewett from Northwestern University. This research provides the scientific backbone of our project; therefore, we were more than pleased when Lauren Clark agreed to be an Advisor for our project. Lauren Clark is the Lab manager of the Jewett lab and responsible for cell-free chloroplast extracts of Nicotiana tabacum.
When we thought about our project and its application for other iGEM-teams, labs, and companies, we realised the need to expand our scope to different organisms besides Tobacco. We have placed great emphasis on providing protocols for different end-users, so we selected the chassis based on agricultural impact and already existing scientific results. Our decision fell on the model organisms Tobacco (Nicotiana tabacum), Spinach (Spinacia oleracea), Arabidopsis (Arabidopsis thaliana), as well as the single cell green alga Chlamydomonas reinhardtii.
We also decided to focus on highly relevant crops, such as Maize (Zea mays B73), Wheat (Triticum aestivum) and Rice (Oryza sativa), as well as other plants that play a major role in world nutrition like Soybean (Glycine max), Canola (Brassica napus) and Tomato (Solanum lycopersicum).
Additionally we sought to showcase the ability of our project to be applied for chassis rarely used in SynBio Projects. Trees are the species, which would profit the most from chlorplast prototyping. We decided on our native tree species, the Pedunculate Oak, because it is strongly represented in Marburg's forests and therefore, we chose to include this local factor in our project.
Nicotiana tabacum is the model chassis for chloroplast engineering as the protocols on agrobacterium-mediated transformation, as well as on biolistic transformation are the most developed and efficient known today. Therefore, we decided to include Tobacco as a model for our chloroplast cell-free systems in order to characterize our novel parts and compare our in-vitro data to actual in vivo experiments. Nevertheless, this Tobacco species is also a model plant for tissue-culture due to its growth time to the adult stage of only 6 weeks and the generous amount of biomass. Tobacco was thus also the first plant to be planted in the S1 greenhouse, as well as in the phytochambers provided to us. Since we started planting in the greenhouse in November, the temperature and light conditions were not ideal. Due to the light conditions during this time of year, it was not possible to grow the Tobacco plants to an adult stage in 6 weeks. In the meantime, the plants inside the phytochambers grew unhindered.
In a paper from Benjamin J. Miflin et al. from 1974 titled “Isolation of intact plastids from a range of plant tissues” plastids were isolated from Spinacia oleracea, among others. By orienting ourselves on this paper, we also decided to buy Spinach fresh from the weekly market.To make our workflow of using cell-free chloroplast systems as accessible as possible for future iGEM teams, we explored a low resource approach, which would not require any greenhouses or other plant growing capabilities and in which chloroplasts have already been successfully isolated. Thus, Spinach was not incubated in darkness like other plants, due to the starch content being negligible.
We chose Arabidopsis thaliana as another model plant because it is fully sequenced and yields a short generation cycle of 8 weeks. Unfortunately, due to the low leaf mass of the plant, we did not have the capacity to repeatedly generate the 300g of fresh biomass needed for isolation. We did not continue to work on this plant, as it would not have been feasible for us next to our other goals.
Due to the fact that about 821 million people in the world are undernourished, a big focus is on the development of cell-free systems for globally distributed crops and other plants that play a key role in nutrition. An important crop in this context is Maize. Maize also shows high value in the industry as a component of livestock feed, as well as biofuels [1]. Due to the long generation cycle of Maize, we decided to ask other research groups at our university that also work with Zea mays B73 to use part of their biomass. Instead of incubating the potted Maize plants in darkness, they were cut off at the stem and placed in a bucket of water in a dark cold room. For crops such as Maize, Wheat and Barley, it was especially important to carefully cut the leaves into small pieces on the day of harvest.
Wheat is the world's most important cereal in terms of production and use as food and feed for livestock [2]. Therefore, we had to extend our system to Wheat. Instead of planting individual Wheat plants in pots, we used trays that we filled with moist soil, placing the seed directly on top.
Rice is a crop that feeds a large part of the world's population. However, planting Oryza sativa is quite demanding. The plants need a 16h - 8h diurnal rhythm, must be watered daily and require further additions of coconut soil and humus to the soil that is supposed to be drained well permanently. Despite the inconvenient way of growing this crop, the importance of this crop for global nutrition prevailed.
Glycine max, otherwise known as Soybean, is a widely used plant for nutrition, especially in Asian countries. The Soy plant also does not yield a large amount of biomass. Nevertheless, it was possible for us to carry out isolations with Soy. Before planting, the seeds were first stored in a germination glass until small shoots were visible. These were then placed 4cm deep in moist but firm soil.
Canola (Brassica napus) is also a food economically important plant and is used not only as food but also as a textile plant. Due to a nitrogen deficiency, we were initially unable to grow the plant, but after using a 20-20-20 fertilizer the plants had ideal growing conditions.
The Pedunculate Oak (Quercus robur) is ubiquitous in Germany, especially in Marburg and provides a valuable resource of wood, therefore representing an important economic sector. The location of a tree right outside the university building generated some additional value for us, as we were able to harvest leaves within a short distance to our labs. Trees provide naturally a large amount of biomass, which is beneficial for chloroplasts isolation. Due to the time and space requirements, standardising the growth conditions for Oak was not possible, but the leaves were harvested under long day conditions and the tree was growing on soil-type 6.2.4, which is declared as “Soils from loess-loam-poor solifluction blankets with acid rock components” [3]. For Oak, it is advisable to incubate whole branches in a cold and dark room for 7 to 10 days to reduce the starch content in the leaves.
Due to the possibility to obtain phytochambers, as well as places within the greenhouses of the Philipps-University of Marburg, we were able to generate a lot of plant biomass and thus perform weekly isolations of chloroplasts from the previously mentioned plant species. We were not able to generate working cell-free extracts from all plants listed here, but we are certain that it is possible and can be expanded to even more plants! The main hurdle was gathering enough biomass. This is needed to produce enough extract to ensure that we can do at least a couple of different experiments to find the right concentrations of various chemicals and get the system running.
Nonetheless, we managed to get working cell-free systems from chloroplasts of Tobacco, Spinach, Wheat, Rice and from Oak trees! Read more about what we achieved on our results page.
Sources
- https://www.who.int/news/item/12-07-2021-un-report-pandemic-year-marked-by-spike-in-world-hunger
- Hildebrand, D. F., & Yu, K. (2003). GENETIC MODIFICATION OF PRIMARY METABOLISM | Acyl Lipids. In Encyclopedia of Applied Plant Sciences (pp. 464–477). Elsevier. https://doi.org/10.1016/b0-12-227050-9/00173-3
- https://bodenviewer.hessen.de/mapapps/resources/apps/bodenviewer/index.html?lang=de