Best Basic Part
We proudly present our best new basic part: the rrn16 promoter from tobacco. We have extensively characterized this part by showing its functionality in three different species and also demonstrated that it can be easily transferred to other plant chassis. Furthermore, we have established this part as reference to allow future teams to optimize the endogenous machinery in their in-vitro systems.
Our Best Basic Part
Best Composite Part
We proudly present our best new composite part: The Universal test construct 7.0. After seven rounds of the design-build-test-learn cycle, we have identified the most efficient combination of regulatory parts and reporter genes for the expression in chloroplast cell-free extracts. This composite part allowed us to develop the first fully functional cell-free extract of english oak chloroplasts, which would not have been possible without our engineering iterations. We hope that future teams could use our Universal test construct 7.0 to develop complete new project ideas, involving any plant chassis they could think of and directly start troubleshooting their extract preparation without worrying about the optimal DNA construct design in the first place.
Our Best Composite Part
The use of luminescence to measure translation is an emerging method. Unlike for fluorescence, no protocol for normalizing luminescence signals is yet known in the iGEM community. We have therefore developed several protocols to calibrate luminescence measurement signals across measurements. We have taken two different approaches to normalization, one based on chemiluminescence (Luminol normalization) and the second one based on bioluminescence (NanoLuc normalization). Both approaches can be used to obtain a calibration curve that is thereafter used for proper normalization of the obtained data.
In addition, we used a ratiometric approach in our measurements, using two different luciferases. Here, the second luciferase is cloned after the desired construct and always contains the same regulatory elements, ensuring it can be used to normalize to. This allows us to normalize to the standardized expression of one of them, making results accross different batches of our cell-free extracts more comparable.
Our Measurement Efforts
Best Software Tool
We have created a comprehensive chloroplast database that can provide deep insights into the development and engineering for the plants of tomorrow. To this end, we have compiled 6,065 chloroplast genomes into an easy-to-use, freely available database containing well over 2.4 million sequence annotations.
In addition, considerable effort has been invested into reannotating and standardizing the data. In doing so, over 5,000 coding sequences, 77,000 tRNAs, and numerous rRNAs have been annotated to follow a unified naming convention.
The resulting database was successfully used in the design process of both our toolbox as well as the creation of our best basic part.
Our Software Tool
Best Sustainable Development Impact
The establishment of crops adapting to unforeseeable climate change impacts takes approximately 10 years, being too long to counteract its exacerbations. By developing optimized cell-free systems from chloroplasts of diverse plant species with high agricultural utility and fully sequenced model plants, we successfully created a novel platform for prototyping genetic parts, contributing to Goals 2 and 13 from the Sustainable Development Goals-list. Our unique Cell-free system saves valuable time and resources, so unpredictable events due to climate change are quickly detected and counteracted. An increased fixation rate of greenhouse gases, resistances against climate change effects and production of sustainable alternatives to non-renewable substances, can be efficiently established in new crops, thus counteracting the effects of climate change. The successful development of Chloroplast Cell-Free systems of different crops and model plants contributes to the sustainable development of agriculture and improves the living conditions of people affected by climate change.
Sustainable Development Impact
Safety and Security Award
By stimulating the transplastomic approach, OpenPlast raises the bar for crop biosafety across the board. With the creation of a platform that facilitates the engineering of novel traits in the plastome of crops, we paved the way for plants that do not transmit their transgenes through pollen. This has huge implications in the prevention of risks associated with gene-flow into neighbouring fields or wild relatives; for instance: the loss of genetic diversity, contamination of non-GM crops and the spread of herbicide resistant to weeds. These are real problems with the current way we engineer plants, and mitigating them with transplastomics is a huge step forward in the struggle to promote wider public acceptance of biotechnology in agriculture. After all, we can not reap the benefits of synthetic biology if the majority of the population still sees it as a threat to the environment.
Safety and Security
A proper education is the premise for the progress we seek for green genetic engineering. Our contribution is targeted at farmers and students. We interviewed both conventional and ecological farmers and developed with their help a booklet, with around 25 pages of information material regarding genetic engineering. The booklet was later distributed among a variety of farmers.
Our second project is a long term advancement of the biology curriculum for German students. First we tested in two biology courses the value of miniPCR’s BioBits kits. These kits provide a comprehensive setup for students to experiment with transcription and translation using cell-free systems based on E.coli. With an overwhelmingly positive response from both students and teachers, we set up a financial and organisational infrastructure in collaboration with GASB, a German association for synthetic biology. This partnership will be able to cover at least 40 highschool biology courses every year.
Our Education Efforts
Best Integrated Human Practices
From the very beginning we sought the perspective of stakeholders from the academia and seed companies in the US (Corteva) and Germany (KWS) to understand what are the hurdles transplastomic plant development faces. Given their consensus around the issue of laborious transformation processes, we subsequently implemented their suggestion of utilizing OpenPlast to screen for new selection markers, paving the way for achieving chloroplast transformation of recalcitrant crops. Although we had great expression values from the T7 promoter, our exchange with partners in the academia brought to our attention the importance of having expression using endogenous transcription machinery, as it is indispensable for one of the selling points of OpenPast: prototyping metabolic pathways. From that, we optimized our extracts until we had a system capable of transcription and translation using exclusively endogenous chloroplast promoters.
Our Integrated Human Practices