Team:Marburg/Contribution
Contribution
Cell-free transcription and translation systems are advanced in vitro tools used as prototyping platforms for metabolic networks and gene circuits. Typically, they are based on crude cell extracts and contain the whole machinery needed for protein biosynthesis. This advantage in cell-free systems offers exciting opportunities to fundamentally transform synthetic biology. It enables new approaches for model-driven design of synthetic gene networks, rapid and portable acquisition of targeted components, as well as building synthetic cells from scratch.
Recently, cell-free technology approaches have gained more and more importance in the synthetic biology field. While the majority of cell-free systems are based on bacterial and eukaryotic cells, there are almost no cell-free systems based on plants.
Our project was focused on developing novel cell-free systems of chloroplasts from various plant species. With our Chloroplast Cell-Free system, we provide a unique test-platform, which can be easily applied to study various genetic constructs for plant engineering in a much shorter time frame.
Contribution to BBa_K1614000
The DNA concentration response of the T7 polymerase in a chloroplast cell-free system
Despite many advantages and simplicity of the system, significant differences in expression using cell-free systems can be caused by the addition of different amounts of template DNA [1]. For this reason, we decided to test the behavior of our system to different DNA concentrations.
In the following experiment we worked with the biobrick BBa_K1614000, a T7 promoter, using NanoLuc luciferase as a reporter system, within our cell-free expression measurements. The graph below indirectly shows the expression levels of this NanoLuc luciferase via the emitted luminescence (see Figure 1). To analyse the influence of DNA concentration to the target gene expression level, extracts from two different plant species (Tobacco-N. tabacum and Spinach-S. oleracea)have been used and DNA concentrations in the spectrum from 0.5 nM to 15 nM have been added to the final cell-free reaction mix.
For both N. tabacum and S. oleracea, an optimum expression level is reached at DNA concentration of 5 nM. Using higher DNA concentrations did not lead to higher expression levels.
These results show that it is essential to carefully normalize DNA concentration for the comparison of various parts in the experiments using cell-free systems. Additionally, using saturated DNA concentrations has the advantage of being less prone to variations in expression, caused using different concentrations in DNA.
Moreover, it should be more accurate not having DNA concentrations as a limiting factor in your measurements, as this could cover other effects/properties one would like to actually characterize in the experiment. It is for this reason we agreed to supplement our cell-free reactions with at least a bare minimum of 5 nM template DNA.
These data were added as a contribution to BBa_K1614000 and we hope it allows others to more carefully plan the DNA concentrations used when characterizing their parts, as there can be crucial differences depending on the concentration chosen!
Contribution to BBa_K1614000
Luminescence values are given as arbitrary units and the data is presented on a logarithmic scale. The reaction was set up with a total volume of 10µl and reaction mixtures were set up by creating a premix with all the components except the DNA template. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
In our extensive effort to bring cell-free technology to the chloroplast, we went through numerous rounds of optimisation, considering the principles of the Design–Build–Test–Learn (DBTL) cycle. We contemplated a range of factors during the harvest of the chloroplast, the chloroplast lysis to obtain the crude extract, as well as the composition of the final cell-free system. This contribution should allow others to more easily replicate our experiments and allow them to create cell-free systems on their own, from whatever plant chloroplast they want!
We succeeded in achieving working cell-free systems from chloroplasts of several agriculturally relevant plants. Moreover, we accomplished a cell-free platform with an oak extract. Even though we were not able to create a working system from all organisms we had worked with, we are certain that this can still be achieved in future projects, as we can clearly show the power of the cell-free technology for rapid prototyping in the plant chloroplast.
Establishing our cell-free system from various plant chloroplasts to diverse plant species understandably resulted in deviations from our starting point - the Tobacco chloroplast harvest and extract protocol (unpublished manuscript, patented) from the Jewett lab at the Northwestern University. Our focus was not only on optimizing the extract quality and the compositions of the measurements. We also paid great attention to the environmental impact of the production of our prototyping system as well as to its accessibility and economic competitiveness.
The following changes were made to optimize the quality of our cell-free systems and increase yield:
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Homogenization
Chloroplast isolation can be improved by optimizing medium use and blending duration. Tobacco and spinach leaves, due to their soft structure and the possibility to remove veins, were easily mechanically broken in a short blending time and with a plant mass to MCB1 buffer ratio of 1:3. We optimized this approach for fibrous plants, such as wheat and rice, whereby by adding more homogenization buffer (ratio at least 1:5) we are able to preserve the brief duration of blending and retain a high yield of intact chloroplasts. It is essential to perform the chloroplast isolation at low temperatures to ensure the integrity of the chloroplasts and inhibit enzyme activity. In our efforts to perform the experiments at as cold temperatures as possible, we tested if the extract quality could be improved by using half-frozen buffers. We observed a lower yield of chloroplasts due to difficulties with the filtration procedure of the leaf homogenate and as a result, proceeded with working with buffers at a low temperature of 4°C.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Density gradient centrifugation
The advantage of using the density gradient method for chloroplast isolation is the segregation of different organelles with little cross-contamination. We found out that the size and density of intact chloroplasts are decisive for the choice of gradient concentrations which we also used as a guideline for optimization. These vary in different plant species. In our experimental approach, intact chloroplasts from Tobacco, wheat, rice and oak isolations were successfully prepared with discontinuous Percoll gradients consisting of layers with 80%, 50% and 20% [2]. For tomato chloroplast isolations we assembled 80% and 40% Percoll gradients [3]. Chloroplasts from spinach were also obtained with sucrose density gradients of 60%, 40%, and 30% [4].
In the isolation procedure, washing of the extracted intact chloroplasts occurs after the gradient centrifugations. The washing steps are short, only 4 minutes at 1000x g. However, we found out that doubling the time of the washing steps is vital to achieving a larger volume of chloroplast extract.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Chloroplast lysis
The first method of lysing intact chloroplasts was to induce liquid homogenization by pushing the isolated intact chloroplasts through a needle 12 times. The quality of the extract was measured by the expression of luminescence. However, for specific plant species, the force of only 12 repetitions is too low and thus affects the extent of expression of the extract. We retested increasing the number of times the extract is pushed through the needles. We performed our lysis with 24 repetitions in order to achieve an ideally functioning wheat extract. For plants like rice with the particularly small size of chloroplasts, we increased the number of needle pushes to 30 times and used a smaller needle size (26 gauge instead of 25 gauge).
To see the now working wheat and rice extracts, visit our Proof of Concept page!
It was also tested whether lysis can be performed by sonication or if the increased number of pushes through a needle has a measurable effect (Figure 2). The intensity of the energy during sonication varied from a range of 100 J to 500 J. It can be assumed that sonication is also an effective method, since, despite the high expression of plasmid control, the expression of extracts sonicated with 500 J is twofold higher.
Based on these insights, we decided to continue repeating the liquid homogenization 24 times for an optimal lysis condition of wheat chloroplasts, but we propose future efforts focus on establishing a reliable lysis through sonication for larger volumes, as this could save a considerable amount of time in the lab.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Luminescence values are given as arbitrary units. The reactions were set up with a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Dialysis
To work time and cost-effectively during the extract preparation, we ceased the dialysis procedure. Dialysis has a positive effect on expression predominantly in endogenous transcription by causing changes in magnesium and potassium acetate concentrations, as well as through the removal of contaminants. In the absence of dialysis, we could not observe significant effects on the expression of the extract when using a T7 promoter (Figure 3). Those first results were not as conclusive as we would have hoped, but in consequence, dialysis was not performed anymore, in order to save resources and time and we nonetheless managed to get high expressing extracts we used throughout our project.
For a low-cost method, we advise omitting dialysis, as the cassettes are quite expensive and large buffer volumes are needed, which quickly drains chemical stocks. For troubleshooting of non-working extracts, we do advise to still try out dialysis, as this could make a difference if it is not yet working in your own hands.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Luminescence values are given as arbitrary units. The reactions were set up with a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Starch reduction
Forces acting upon the starch molecules during centrifugation cause them to eject at high velocities from the chloroplast, damaging the membrane and thus destroying chloroplasts. To reduce this issue and extract a larger fraction of intact chloroplasts, a variety of different methods were utilized.
In the absence of light, plants convert stored starch back into sugars to use in anabolic processes. All the plants we worked with were placed in the dark for 24h (wheat, rice, tomato), 48h (tobacco) or 7-12 days (oak). This approach reduced the starch levels and led to a higher yield of intact chloroplasts.
Another optimisation was the use of younger plants, as younger leaves contain less accumulated starch. A reduction in the starch of 6-week-old plants after dark incubation was clearly visible.
The Removal of stems and leaf veins was another starch-reducing factor. In plants, chloroplasts are concentrated particularly in the parenchyma cells of the leaf mesophyll. Homogenizing stems and leaf veins led to a prolonged duration of the blending step and required more buffer.
In the event of a nitrogen deficit, the starch production in wheat plants increases. To prevent this, we increased the frequency of fertilizing the plants with nitrogen-heavy fertilizer from once a week to twice a week. In the newly planted plants, a starch reduction was clearly visible during isolation.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Budget friendly extracts
We tested various ways to make our methods available worldwide, including laboratories with limited budgets. It was in our greatest interest to test cost-effective alternatives to chloroplast isolation protocols.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Percoll vs. Sucrose gradients
With Percoll being one of the most expensive reagents needed for the gradient centrifugation, our goal was to substitute this component with the cheap alternative sucrose. We marked higher extract yield and greater quality of tobacco chloroplast extracts prepared with Percoll. However, sucrose density gradient is an excellent substitute and we achieved high-quality working extracts from spinach using sucrose gradients (Figure 4).
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Figure 4.
The comparison of spinach cell-free systems created with Percoll or sucrose gradients. As reference a tobacco extract from Percoll gradients was added. The extracts were tested in varying magnesium acetate concentrations - 7, 10 and 15 mM.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Reaction volume
By downscaling the total reaction volume from 10 μl to a total of only 4 μl during measurement, we tested the extracts with selected constructs for their functionality while saving on important resources.
Despite the reduction of the total volume, the functionality of the extract can be shown (Figure 5). This optimisation allowed us to perform 2.5 times as many experiments by saving resources, with no savings in expression.
We do recommend anyone who wants to create their own systems to start with larger reaction volumes, as these are simpler to pipet together. As soon as you get these running, feel free to downscale your reactions to save resources! Of course, if you are confident enough or ideally can use a liquid handling robot, you can use smaller volumes right from the start.
Protocol Optimization to Create Chloroplast Cell-Free Extracts
Luminescence values are given as arbitrary units. The reactions were set up with a total volume of 10µl. Negative controls using only the plasmid DNA or the crude chloroplast extracts have been included respectively.
Conclusion
Through in-depth research, interaction, and communication with experts, as well as our own curiosity, we have succeeded in developing specific optimizations to adapt the work steps to produce cell-free systems from chloroplasts of different plant species. Our focus was not only on the highest possible yield of extract and maximum expression, but also on the environmental impact that the production of this system entails, as well as enabling widespread and budget-limited availability. With the help of our targeted optimizations shown here, it will be possible for future laboratories and iGEM teams to easily develop highly efficient and functional cell-free systems for chloroplasts of diverse plant species.
Sources
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- Yukawa, M., Tsudzuki, T., & Sugiura, M. (2006). The chloroplast genome of Nicotiana sylvestris and Nicotiana tomentosiformis: complete sequencing confirms that the Nicotiana sylvestris progenitor is the maternal genome donor of Nicotiana tabacum. In Molecular Genetics and Genomics (Vol. 275, Issue 4, pp. 367–373). Springer Science and Business Media LLC. https://doi.org/10.1007/s00438-005-0092-6
- Bhattacharya, O., Ortiz, I., & Walling, L. L. (2020). Methodology: an optimized, high-yield tomato leaf chloroplast isolation and stroma extraction protocol for proteomics analyses and identification of chloroplast co-localizing proteins. In Plant Methods (Vol. 16, Issue 1). Springer Science and Business Media LLC. https://doi.org/10.1186/s13007-020-00667-5
- Elias, B. A., & Givan, C. V. (1978). Density gradient and differential centrifugation methods for chloroplast purification and enzyme localization in leaf tissue. In Planta (Vol. 142, Issue 3, pp. 317–320). Springer Science and Business Media LLC. https://doi.org/10.1007/bf00385083