In many areas of synthetic biology, like in the iGEM competition, E. coli is still the chassis most people rely on. On the contrary, it is rare to find iGEM teams working on plant synthetic biology and although the number of teams has slowly increased in the last years, most of them still focus on microalgae or cyanobacteria - organisms that are easier to handle in the time frame that iGEM provides. Apart from the slow growth of plants that makes iGEM projects with them more difficult, the lack of functional and well characterised parts for photosynthetic chassis present in the iGEM registry is another crucial factor to consider.
In our project OpenPlast, we developed Cell-Free Systems based on chloroplasts of different plants to be used as prototyping platforms for part characterization. In addition, we created a toolbox of 157 Golden Gate based parts constructed for the use in plant chloroplasts and showed that our Cell-Free Systems can effectively be used to characterize those parts in a noticeably short time. The systems we provide can serve as a basis for numerous other projects and our detailed protocols and optimisation efforts are crucial for others that want to expand this technology to even more plant chloroplasts.
Thinking of a traditional SynBio project, one of the obvious questions in terms of project design is the choice of the chassis. So, from which plant chloroplasts did we create these Cell-Free Systems?
We realised that from a biotechnological and agribusiness perspective, it might be quite important to not just choose typical plant chassis, but to look for plants in which chloroplast engineering could prove valuable. To us, this meant that if we want to show the power of the Cell-Free technology for our desired application, we want to work on several plants! In our project, we therefore worked on Cell-Free systems from chloroplasts of Tobacco, Spinach, Arabidopsis, Maize, Wheat, Rice, Soybean, Canola, Tomato and Oak. More on why we chose those plants can be found here!
We wanted to establish cell free systems from chloroplasts of various plants as prototyping platforms for metabolic networks and genetic constructs. To do this, we firstly isolate the chloroplast from the whole plant and free the cellular machinery by lysing the cell. By supplementing this extract with various chemicals, we can maintain protein expression and simulate gene expression in vitro. This has the benefit of being able to test genetic parts in a high-throughput manner without the need to introduce DNA into actual plants, a tremendously time-intensive and costly process.
We managed to get working Cell-Free systems from chloroplasts of Tobacco, Spinach, Wheat, Rice and from Oak trees (Figures 1-3). Although the expression levels in the cell-free systems from rice (Oryza sativa) and oak (Quercus robur) are lower than in our other extracts, our efforts allowed us to test the first ever biobrick for a chloroplast of a tree in our cell-free systems (Figure 3). Sadly, we did not have enough cell-free extract and time left to further optimize expression levels in those two systems, but we are confident that this can still be done in following experiments.
As written above, we started out working on even more plants than we managed to get working extracts of. Even though we were not able to create a working system from all organisms we had planned with, we are certain that this can still be achieved in future projects, as we could clearly show the power of the Cell-Free technology for rapid prototyping in the plant chloroplast.
In our extensive effort to bring this technology to the chloroplast, we went through numerous rounds of optimisation, considering a range of factors during 1) the harvest of the chloroplast, 2) the chloroplast lysis to obtain the crude extract, as well as 3) the composition of the final Cell-Free System. Learn more about our efforts here!
Previous teams have already started to work on chloroplast SynBio and built a set of parts, notably iGEM Cambridge 2016) already constructed parts for the chloroplast of Chlamydomonas reinhardtii. Nonetheless, so far teams were unable to properly characterize such parts, due to the difficult and long transformation procedures. Developing our systems for the plant chloroplast, we could show that they can indeed be employed for rapid characterisation of genetic parts, allowing us to characterise the first Modular Cloning parts for chloroplasts in the iGEM registry.
As shown in our Results and Parts section, we created a total of 157 genetic parts,83 are designed for the tobacco chloroplasts, 23 for the rice chloroplast, 11 for the chloroplast of wheat, another 10 for the spinach chloroplast and 3 parts are designed from plant viruses. In addition, our collection contains a T7 promoter, as well as 9 different reporters, 8 of which have been codon optimised for the tobacco chloroplast and one for the Chlamydomonas reinhardtii chloroplast.
We were able to show that our systems can effectively be used to characterize these parts in short time frames, as measurements can be done within one day.
Interestingly, we did not only see that our cell-free systems are indeed functioning and useable for part characterisation, but furthermore observed that parts used in chloroplast extracts of different species seem to show similar behaviour (Figure 4). In this example, various 5'UTRs were tested (the rest of the genetic construct was otherwise identical) and we could clearly observe similar behaviour in both the extracts from spinach and from tobacco.
Of course, it is nothing new that chloroplast parts from one species can be used in another, as chloroplasts of various plants are closely related. Elements from tobacco chloroplasts are commonly used in chloroplast transformations1, but it shows that our systems can flexibly be used to test a wide range of parts in chloroplasts of a desired organism.
To emphasize this point even more, we showed that our systems are capable of being used with linear DNA templates (Figure 4). This will allow to test new genetic elements without going through a whole round of cloning - instead, PCR products or ordered DNA synthesis can directly be used as a template in our CFS. This allows for a one-day cloning workflow (Figure 5) explained in more details on our Results page.
Our systems will allow researchers to drastically improve their design-build-test-learn cycle and speed up research on chloroplasts.
Linear DNA Template
One Day Cell-Free Prototyping
To show that results in our cell free systems are comparable to data gathered in vivo, we set out to transform the tobacco chloroplast with some of our construct. The goal was to measure the same set of genetic parts in vivo and in vitro to show that our system can give valuable insights into the living system.
Sadly, due to the nature of the transformation process and some hurdles we encountered, this process took quite a long time, which is why we were not yet able to perform our planned experiments in the transformed tobacco plants. Nonetheless, we were able to show successful transformation events and could even identify first signs of transgene expression in the transformed chloroplasts (Figure 6). We hope that our chloroplast toolbox will help others perform their experiments in a shorter timeframe to get chloroplast engineering done in the iGEM context! For more information on our in vivo efforts check out our in vivo diary!