Establishing cell-free systems from chloroplasts as rapid prototyping platforms for plant SynBio


Climate change presents agriculture with the biggest challenge in the history of humanity. Higher temperature, droughts and flooding will cause even greater complications and will further stress our food supply chain. To tackle all these problems we need crops, which can withstand these new conditions. But one problem we are currently facing is the speed of innovation in crop breeding and improvement, which is far too slow. In just 30 cycles of sowing and harvesting we will have reached the critical year of 2050, where humanity has to feed 10 billion people. For this reason, the goal of our project was to develop cell-free extracts from chloroplasts of various plant species, including tobacco, spinach, wheat, rice, oak and many more. On top of this we set out to characterize a vast amount of novel chloroplast parts from our toolbox collection that consists of 157 parts suitable for usage in the chloroplast. With the addition of these highly characterized and documented parts to the iGEM registry and our workflow to create chloroplast cell-free extracts, we are aiming to lay a foundation for future iGEM teams to conduct their own project on any plant chassis they could dream of.

If you want to read more about how our goals became amazing results, please visit: Results.

The Challenge

Agriculture as we know it has provided the basis for human civilization for millennia. However, the steady improvements in crop productivity we experienced since the Green Revolution are beginning to plateau [1, 2]. Considering our growing world population, rising consumption and increased biofuel use, we are in dire need of improved crop productivity. The Special Report on Climate Change and Land published by the Intergovernmental Panel on Climate Change clearly states that climate change is already negatively affecting food security around the globe, an effect that will only grow stronger as the climate crisis worsens [3]. We are clearly in need of solutions to combat the stagnation of crop productivity improvement.

The Problem

One aspect in our fight for sufficient and sustainable agricultural production is engineered crops. They bear many promises, from improved yield or better resistance against numerous biotic and abiotic stresses to increased nutritional values or sophisticated traits like more efficient carbon capture and nitrogen fixation. However, the current pace of crop engineering is slow, given that plants are inherently slow. Discovery, development, and approval of novel crop traits currently takes about 10 to 15 years, about 4 years of which are spent developing proofs of concepts and optimizing genetic constructs [4]. Something has to change for us to efficiently and quickly develop the novel crops we will need in our race against the changing climate.

Our Solution

Cell-free systems are in vitro tools capable of transcription and translation. This is achieved by creating a crude extract from an organism of choice and supplementing it with external resources, making it possible to synthesize proteins. This way, the system can be used to directly prototype genetic parts, whole metabolic pathways or characterize protein functions in an quick and easy-to-use manner. Another advantage is that cell-free systems can bypass the limitations of molecular transport across the cell wall/membrane, by directly manipulating the molecular context with e.g. the addition of non-native substrate, recombinant DNA and purified proteins or RNA [5].

In recent years, cell-free technology has gained increasing relevance in the field of synthetic biology due to its high-throughput and prototyping capabilities [6, 7, 8]. While these systems have been established for several bacterial [9, 10, 11, 12, 13, 14, 15, 16, 17] as well as a limited amount of eukaryotic systems [18, 19, 20], plants are still majorly underrepresented in this field.
With this imbalance in mind, we set our goal to expand the cell-free technology to several plant species and in our case by focusing on the chloroplasts of various plant species.

In vitro transcription or in vitro translation systems have been used in chloroplast basic research, even before chloroplast transformation was possible for any plant [21]. These systems allowed elucidating the foundational chloroplast biology, such as transcriptional [22] and translational regulation [23] and how it is affected by light [24], nuclear proteins [25] and other environmental stimuli [26].

However, nowadays such systems are not very prominent in chloroplast research and especially not in plant synbio, which is mainly due to the lack of efficient combined transcription-translation systems, which are key for the applicability in the field of synbio.

The general concept on how we obtain the chloroplast crude extracts is by first isolating the intact plastids from the plant tissue by utilizing density gradients. Subsequently, the chloroplasts are lysed and the cell debris is removed by ultracentrifugation, while retaining our desired parts of the protein biosynthesis machinery, such as the peptides, ribosomes, the endogenous transcription machinery and other important components. This way we are able to drive gene expression in vitro and can utilize our extracts for high throughput prototyping experiments. Of course many optimization and troubleshooting steps are needed to adapt this general protocol for different plant chassis, as they differ substantially due to the different chloroplast properties of the respective plant for example differences in pH, metabolic background or size of the chloroplasts.

If want to learn more about our chloroplast cell-free extract preparation in general go to our Cell-Free Page.

Project Inspiration

Very early in the project we got in contact with Lauren Clark, from the lab of Prof. Michael Jewett, who established the first cell-free system from the chloroplast of Nicotiana tabacum. Building upon her experience we wanted to expand this method to be applicable for a wider range of plant species. For the selection of species for our project we were inspired by the sixth assessment report of the Intergovernmental Panel on Climate Change (IPCC), which comes to the following conclusions: “Genetics improvement is needed in order to breed crops and livestock that can both reduce greenhouse gas emissions, increase drought and heat tolerance (e.g. rice), and enhance nutrition and food security” [3]. Based on this report, we made the creation of chloroplast cell-free systems from major crops like wheat and rice a priority in our project.

But Why Chloroplasts?

Targeting the chloroplast in plants is a promising way to generate new generations of crops that can tackle the challenges mentioned above. While genetic engineering in the chloroplast is in general more labor intensive and complex, plastid transformation offers several notable advantages relevant to plant biotechnology [27]. Due to their origin, plastids share a lot of traits with prokaryotes. The homologous recombination machinery from their cyanobacterial ancestor allows for highly efficient and precise introduction of genetic constructs into the genome [28]. Furthermore, it is possible to stack transgenes in synthetic operons, which is not possible in the nucleus. The potential for extremely high levels of expression of the gene product is also a very favorable trait unique to the chloroplast [29]. It has even been shown that gene products can exceed 10% of the total soluble protein of the plant [30]. The Prokaryotic nature of the chloroplast makes epigenetic silencing - a common problem introducing transgenes in the nucleus of plants - highly unlikely.

Improved Biocontainment

A major benefit, which is to be highlighted here, is the reduced risk of transgene escape due to the maternal inheritance of chloroplasts. Due to the widespread skepticism against genetic engineering in the general public, we believe chloroplast engineered plants can help raise acceptance of genetically modified organisms. If you want to learn more about our biocontainment effort, go to our Biocontainment Page.

Chloroplast Part Collection

The foundation of synthetic biology lies in applying engineering principles to biology and one of the key aspects for that is standardization, which is especially important for the creation of easily sharable biological parts. This mindset allows engineering projects with a higher magnitude of complexity to be achievable, like cellular logic [31, 32] or the integration of fine tuned metabolic pathways [33, 34]. The iGEM part registry is one of the cornerstones of synthetic biology. With currently over 20.000 genetic parts in standardized formats, it is one of the largest collections worldwide. From this wide range of parts only 18 are designed for the chloroplast, despite not being supported by experimental data. Even when looking beyond these Biobricks, the range of available parts in the research field is still very limited. Currently, it is only possible to express a limited amount of proteins at once in the chloroplast. Building more complex genetic circuits is impossible at this time, due to the mentioned lack of genetic tool characterization in vivo. At this point in time the repertoire of inducible parts is limited to an IPTG driven promoter [35] and a theophylline riboswitch [36], although they do not exhibit desired binarity due to the transcriptional leakiness present in the chloroplast [37]. We aim to expand the collection of plastid parts by expanding the Marburg collection [38] with chloroplast parts. Our aim was to test as many of them as we can design and build in order to get insights on which parts are suitable for chloroplast engineering projects.

Figure 1: SBOL scheme of a transcriptional unit in the chloroplast
Gene organization in the chloroplast. Symbols correspond to a 5' connector, Promoter, 5'UTR, coding sequence, 3'UTR and 3' connector respectively

A typical device for plastid expression can be divided into the following basic parts:

  1. promoter
  2. 5' untranslated region (5'UTR)
  3. coding sequence
  4. 3' untranslated region (3'UTR)

A comprehensive part collection would need all of these genetic parts with different expression strength to facilitate the construction of fine-tuned genetic devices and systems. By providing a bigger variety of available plastid parts, we aimed to minimize one of the drawbacks often associated with chloroplast engineering: unwanted homologous recombination. Due to the limited range of available chloroplast parts (also in the field), the same modules are often reused. This causes the problem that the introduced DNA might be recombined out. It has been shown that as little as 50bp can be enough for the organism to reject the provided construct [39]. That is why we strongly think it would also be desirable to develop fully Synthetic Regulatory Elements which are completely orthogonal to the endogenous promoters and UTRs and do not contain homologous sequences.

If you want to learn more about our chloroplast part collection, you can find more information on our Toolbox Page.

Human Practices

We think it is important to reach out to numerous important stakeholders of the agricultural sector, in the area of industry, politics and many more. It is also of great importance to directly talk to farmers to discuss the implication of our project in their everyday work.

If you want to know more what we achieved in our Human practice part of the project, you can read more on our Human Practice Page.

Outlook and Vision

Our vision for our chloroplast cell-free systems and our chloroplast part toolbox is that they would facilitate more complex chloroplast engineering projects in the iGEM context, in academia but also in the industrial environment. In the future, the chloroplast could be utilized as a platform to engineer the light reactions of photosynthesis, to introduce customizable microcompartments or to engineer C4 photosynthesis into C3 plants. Furthermore, plastids can be used to implement synthetic nitrogen fixation, synthetic photorespiration or synthetic carbon fixation cycles in crop plants, which would help secure global food security for an ever growing world population.

If you want to learn more about our proposed implementation of our project, you can read about it here on our Implementation Page.

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