Team:Marburg/Cell-Free/Troubleshooting
Troubleshooting Cell-Free Extracts
Introduction
The foundation of our Openplast project was to obtain chloroplast extracts from various agriculturally relevant plants, as well as important model organisms. Lauren Clark from the Jewett Lab provided us with the Tobacco chloroplast harvest and extract protocol (unpublished manuscript, patented) which served as a basis for our optimization efforts. The principles of the protocol are summarized in the paragraph below.
The chloroplast harvest and extract protocol - the basic workflow
During the isolation procedure all the experimental steps were performed at 4 °C. First, the plant material was harvested, weighted and prepared for blender by cutting the leaves into smaller pieces. Next, the plant cells were destroyed by short blending in the homogenization buffer. After breaking the tissue, the chloroplasts were separated by a 1000x g centrifugation. The intact chloroplasts sedimented as a green pellet and were subsequently resuspended. The chloroplast suspension was distributed to 8-14 conical tubes containing Percoll or sucrose-based discontinuous density gradients. Segregation of intact chloroplasts from broken ones was achieved during 10000x g density centrifugation. The intact chloroplasts were collected with a 25 ml serological pipette and subsequently washed three times with a chloroplast wash buffer. Lysis buffer was then added and the flash-frozen isolated chloroplasts were stored at -80 °C. The next procedure was the lysis of intact chloroplasts. The chloroplasts were disrupted with a syringe by passing them through a needle 12 times. The extract was purified by differential velocity centrifugation and supplemented with GTP and amino acids which maintain protein expression and simulate gene expression in vitro. After this step dialysis was performed. Dialysis represents a major part of the preparation of intact chloroplasts. In this process, the extracts were put in dialysis cassettes, which were then placed in a specific dialysis buffer for 4 hours. The purification of the extracts was ensured by a semipermeable membrane, which separated the extracts from the dialysis buffer. Figure 1 provides an overview of the experimental workflow, firstly to obtain intact chloroplasts from plant material by shredding and centrifugation, and secondly to prepare a cell-free extract from these intact chloroplasts via needle disruption and ultracentrifugation [1].
Figure 1. (left)
In the experimental setup the plant material was collected and blended in a leaf homogenization buffer. After breaking the plant cells by blending, the chloroplasts were separated by a 1000x g centrifugation. The intact chloroplasts are then segregated from thylakoids by a 10000x g density centrifugation using Percoll gradients. Intact chloroplasts are collected and subsequently washed three times with a chloroplast buffer. The chloroplasts were disrupted with a syringe by passing them through a needle 12 times. The extract is purified by differential velocity centrifugation and supplemented by various chemicals that maintain protein expression and simulate gene expression in vitro.
Optimizing the extract quality
In our extensive effort to expand the cell-free technology that utilizes chloroplast extracts, we went through numerous iterations of the Design–Build–Test–Learn (DBTL) cycle, considering a range of the above mentioned factors during chloroplast harvesting, the isolation, the chloroplast lysis, as well as the composition of added buffers to the final cell-free system.
Optimizing medium use and blending duration
During the preliminary research on chloroplast isolation we came across the hint to work with a strongly cooled medium throughout the chloroplast separation process [2]. Therefore, we used a half-frozen leaf homogenization buffer and chloroplast wash buffer in order to use them as cold as possible and compare the isolation process with the usually used buffers that were cooled to 4 °C. The most limiting factor in this approach was the homogenate straining through the Miracloth. It was difficult to obtain all of the liquid when using the half-frozen buffer as part of it resulted in a snowball-like consistency and was, therefore, harder to press through the filtration cloth. Possibly, some chloroplast material was lost in this step. At the end of the isolation, an overall lower yield of intact chloroplasts was observed when performed with a half-frozen buffer, compared to isolation with buffers cooled to 4 °C.
In order to obtain a short blending duration, to not destroy intact chloroplasts by blending for too long, varying plant mass to buffer ratio was used for several plant species. For the model plants tobacco and spinach it was possible to use a buffer-plant mass ratio of 1:3. For plants with a higher fiber content such as wheat, rice, oak, tomato or barley, it was observed that a plant mass-buffer ratio of 1:4-5 during isolation resulted in simplified handling as well as a clear increase in extract yield.
Chloroplast washing
In the isolation procedure, washing of the extracted intact chloroplasts occurs after the gradient centrifugations. In the provided protocol, the three washing steps were relatively short, comprising of only 4 minutes each at 1000x g. In our experience, prolonging the steps is vital to achieving a higher yield of intact chloroplasts. Therefore, for all the organisms we worked with, the centrifugation times during washing were doubled. In our interview with Lauren Clark, the author of our basic protocol, we were advised to increase the duration of the washing steps in the case of an overly green supernatant.
Lysis methods
Two different methods were tested to lyse the intact chloroplasts after isolation. In the first procedure, lysis was achieved by forcing the extract through 25G needles by hand. With this liquid homogenization, organelles are lysed by forcing the suspension through a confined space, thereby shearing off the membranes [3]. The second method was to induce lysis by sonication. The number of times the chloroplasts were pushed through the needle varied, as did the intensity level during sonication. The results of the needle lysis test revealed that the number of presses in the needle method was irrelevant in spinach extract since the 12x, 24x and 42x samples had comparable values. It needs to be added that the plasmid control was abnormally high in comparison to other measurements that were performed, however, the luminescence expressions for the cell-free systems in spinach were nonetheless double the value of negative control. As for the sonication lysis test, the power of 100 J and 500 J showed similar values as the negative control of the plasmid, hence these energy levels were not proven to produce working extracts. However, the sample lysed with 300 J sonication has a comparable result to the needle pushing lysis method. Therefore, it was presumed that both approaches can be used efficiently to lyse the spinach chloroplast cells and that the number of pushes through a needle to lyse the spinach chloroplasts has no evident consequences in a range of 12-42 times. The further experiments involving tobacco and spinach proceeded with 12 times needle lysis as in the basic protocol.
Testing different lysis methods in spinach extract. The needle-pushing samples of 12x, 24x and 42x show comparable values within the standard deviation and reach around twice the value of the plasmid control. The sonication lysis method showed comparatively positive results for 300 J, nonetheless with a wide range of values.
Additionally, Lauren Clark and Dr. Michael Jewett were consulted about possible variations of the lysis method in other plant species. Lauren Clark advised us to test increasing the number of times the extract is pushed through the needle, as for specific plant species the shear force of only 12 repetitions is too low and thus affects the extent of expression of the extract. We acknowledged this remark and performed the cell lysis with 24 to 30 repetitions for chloroplast extracts that did not work previously. Through this adjustment we finally achieved a functioning rice and wheat extracts (Figures 3 & 4).
Proof of concept - working cell-free system comprising of extract from O. sativa.
Proof of concept - working cell-free system comprising of extract from T. aestivum.
Dialysis
The main goal of dialysis of extracts is the removal of contaminants but the process also causes changes in magnesium and potassium acetate concentrations. Since Lauren Clark pointed out that dialysis has a positive effect on expression predominantly in endogenous transcription and since the dialysis cassettes, as well as the components of the dialysis buffer, are expensive, it was in our great interest for the more cost-effective development of our system. Therefore, it was tested whether the absence of dialysis leads to effects on the expression of the extract when using a T7 promoter in the tobacco extract (Figure 5). The non-dialyzed extracts show a higher expression relative to the dialyzed extracts. It is unlikely that the dialysis impacts the system with T7 negatively, but rather this result might be due to the changed magnesium acetate concentration, and therefore the salt optimum for the measurement was not achieved. Next, the experiment was repeated in the cell-free system derived from spinach, however with unmeaningful results due to a wide range of values and irregularly high plasmid control (Figure 6). Additionally, the presented values are in the same orders of magnitude, which is interpreted as a comparable result in this case.
As a result, it was recognized that for cell-free systems with the T7 promoter the dialysis step in the purification procedure is not necessary. The dialysis was omitted in further chloroplast isolations and this decision immensely saved time and financial resources.
Optimizing N. Tabacum cell-free extracts: testing dialysis vs. no dialysis (linear scale). The construct with the T7 promoter was not negatively influenced by the lack of dialysis. In fact, the luminescence has a higher value in the system with a non-dialysed extract.
Optimizing S. oleracea cell-free extracts: testing dialysis vs. no dialysis (linear scale). It could not be assessed whether the cell-free system with the T7 promoter was negatively influenced by the lack of dialysis due to irregularly high plasmid control and high standard deviation. The presented values are also in the same order of magnitude, which is interpreted as a comparable result in this case.
Magnesium and potassium salt concentrations
Cell-free system performance relies on various added components including magnesium and potassium acetates. Additions of these salts have very narrow optimum windows [4] and their levels are also strongly affected by dialysis, its duration and the type of cassette used. To find the most favorable concentrations in the reaction mixture for the measurement, a range of potassium and magnesium acetate concentrations were tested. As a result, the most optimal translational buffer for non-dialysed tobacco extract consists of 30 mM KOAc and 10 mM MgOAc (Figure 7). It was also tested, what the magnesium acetate optimum in cell-free extracts is with endogenous transcription in cell-free systems of tobacco and spinach (Figure 8). This second experiment confirmed the MgOAc optimum value in N. tabacum and showed that for spinach the optimum is wider and the magnesium salt concentration can be as low as 7 mM.
The effect of varying concentrations of potassium and magnesium acetates on luminescence expression in cell-free extracts of tobacco and with the T7 construct.
Testing the effect of MgOAc concentration on luminescence expression in cell-free extracts of tobacco and spinach (that underwent dialysis) with the endogenous transcription.
Gradient concentrations depending on plant material
The advantage of using the density gradient method for chloroplast isolation is that it segregates populations of different organelles with little to none cross contamination. The size and density of intact chloroplasts is decisive for the choice of gradient concentrations. These vary in different plant species. In our experimental approach, intact chloroplasts were prepared with discontinuous Percoll gradients consisting of layers with 80%, 50% and 20% concentration [5]. Tobacco, spinach, wheat, rice and oak isolations were successfully performed with this method. Intact chloroplasts from spinach were also obtained with sucrose density gradients of 60%, 40%, and 30% [6] to test whether using a low-cost alternative to Percoll is possible. For tomato chloroplast isolations we tried a different approach and assembled 80% and 40% Percoll gradients [7].
Influence of starch content
The most frequently occurring problem was a large amount of starch in the crude plant extracts which sedimented on the side of the container during centrifugation. Starch, the energy storage molecule of plants, is produced as the product of photosynthesis inside the chloroplast. The 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:
Dark incubation
The first solution for the starch problem was the incubation of plants in darkness prior to chloroplast isolations. Under the absence of light, photosynthesis is impossible and plants instead convert stored starch back into sugars to use in anabolic (energy-requiring metabolic) processes. All the plants were placed in the dark for 24h (wheat, rice, tomato), 48h (tobacco) or 7-12 days (oak) before isolating the chloroplasts. This approach greatly reduced the starch levels and led to higher yield of intact chloroplasts.
Younger plants
Another option was the use of leaves from younger plants for the preparation of extracts, as younger leaves contain less accumulated starch. The timing of planting the greenhouse and phyto chamber was adjusted to the proposal of 6-week-old tobacco and wheat plants. A reduction in the starch of 6-week-old plants after dark incubation was clearly visible. Since the previous isolations of chloroplasts from oak trees, in which leaves of adult trees were used, showed much starch despite a dark incubation of 7-12 days, only the leaves of a young tree were harvested for the last planned isolations. They were also incubated in darkness for 7 days and as a result, did not show any starch during chloroplast harvest. However, the extracts from the young oak leaves showed no luminescence expression, while it was possible to detect expression with the chloroplasts of the older leaves (Figure 9). As there are many variables in the protocol that play into getting a working extract, we can not be sure that the younger leaves were the sole reason. More chloroplast extract preparations will have to be performed in the future to further optimize the protocol for tree leaves.
Proof of concept - Q. robur cell-free extract.
Stem removal
In plants, chloroplasts are concentrated particularly in the parenchyma cells of the leaf mesophyll. Veins and stems do not contain many chloroplasts and store a significant amount of starch and other contaminants like salts and phenols. Therefore, the removal of stems and leaf veins was another starch-reducing factor. Additionally, homogenizing these parts led to a prolonged duration of the blending step and required more buffer.
Fertilization
To further reduce starch formation, research was conducted to find other possible solutions to this problem. In the process, we came across the indication that wheat plants in particular boost their starch production in the event of a nitrogen deficit [8]. To prevent this, we began to increase the frequency of fertilizing the plants with nitrogen-heavy fertilizer to twice a week. In the newly planted plants, a starch reduction was observed during chloroplast isolation procedure. This observation could be also due to dark incubation, nonetheless we assume fertilization aided us in harvesting a higher yield of chloroplasts.
Environmental concerns
Harmful chemicals
Our project has grown with the goal of working against the symptoms of pervasive climate change. In order not to cause further damage to the environment during the production of the system, environmentally harmful substances were tried to be avoided. In the composition of the required buffers, the chronically water-polluting substance β-mercaptoethanol was added in 2 of 5 buffers. β-mercaptoethanol is reducing disulfide bonds and is thereby irreversibly denaturing the RNases being present in plant cells. Moreover, it is not only highly harmful to the environment, but also hazardous to health when inhaled or in contact with the skin. Based on this, it was decided to conduct an experiment testing the effects of the absence of β-mercaptoethanol on luminescence expression (Figure 10).
Cell-free optimization: testing whether presence or absence of β-mercaptoethanol has an effect on the luminescence measurement in the N. tabacum cell-free system.
Although the absence of β-mercaptoethanol would be of great benefit, it was clearly evident that its absence brings significant negative effects on the luminescence expression of the system. Thus, β-mercaptoethanol continued to be used in the buffer composition. Further literature research revealed that ß-mercaptoethanol has been substituted by less toxic dithiothreitol DTT in the past in RNA extractions [9]. This might also have an effect on chloroplast extracts since protein synthesis machinery relies upon RNA structures such as tRNA and ribosomes.
Plastic pollution
Plastic waste pollution resulting from laboratory work was another environmentally concerning issue we were actively working on to minimize. Obtaining the chloroplast extract is a process requiring a significant amount of plastic labware. We tested if the reuse of falcon tubes during the isolation procedure has an effect on the quality of the extract. The plasticware was thoroughly washed and rinsed with desalinated water and left to air dry. Since neither the extract yield nor the luminescence expression of the reused tubes showed any significant deviation compared to the newly used tubes, it was possible to reuse our plastic labware without any restrictions. This allowed us to reduce our overall plastic consumption by approximately 60%. For the assembly of the gradient as well as for further executions 25 ml plastic pipettes were used at the beginning. Very early on, however, we decided to switch to the more sustainable option of recyclable glass pipettes, which further minimized our plastic consumption.
Accessibility concerns
Accessibility and economic competitiveness were major concerns when exploring the experimental setup for the chloroplast isolation. We were motivated to make our methods available worldwide, including the laboratories with limited budgets. Therefore, it was in our greatest interest to test cost-effective alternatives to chloroplast isolation protocols.
Competitive alternative to Percoll-based density gradient
To achieve the goal of economic inclusivity, we explored substituting the expensive Percoll gradient with a broadly available sucrose gradient as described above [10]. Sucrose-based density gradients significantly reduce costs, as Percoll is one of the most expensive reagents needed for the chloroplast isolation protocol. Understandably, there is a trade-off when using sucrose. For instance, 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 with the endogenous promoter (Figure 11). This setup is a notable example of low-cost chloroplast isolation using plant material and sucrose available at any supermarket.
Figure 11.
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.
Reduction of the total measurement volume
By downscaling the total reaction volume during measurement, it was possible to test the extracts with selected constructs for their functionality while saving on important resources. Instead of the usual 10 μl, only a total of 4 μl of total reaction volume was used, 2 μl of which is extract (Figure 12).
Downscaling of the reaction volume.
If the 2.5-fold reduction of the total volume is included in the absolute expression of the extracts, it is recognized that despite the reduction of the total volume, the functionality of the extract can be shown. This insight allowed us to perform 2.5 times as many experiments by saving resources, with no losses in expression.
Alternative long-term storage ideas
Another concern that applies to accessibility is access to specialized equipment that allows for storage at -80 oC. We explored an idea to lyophilise the chloroplast extracts and therefore open the possibility to store them long-term at higher temperatures compatible with commonplace freezers. Lyophilisation is the removal of other frozen solvents from a solution through the process of sublimation and the removal of bound water molecules through the process of desorption. Our attempt resulted in an extract that was not fully freeze-dried due to the presence of glycerol. Glycerol is an important ingredient of the lysis buffer and improves the productivity of the extract. We were using a glycerol concentration of 10% and since the glycerol optimum is at 5-15% this experiment could be further explored with a lower concentration of the polyol compound. Due to time limitations, we were not able to continue on this matter, but future efforts should resolve this, which would not only allow for easier storage, but furthermore a much simpler exchange of cell-free extracts which could just be shipped without special cooling.
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 for the production of cell-free systems. The 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 the optimizations, it will be possible for future laboratories and iGEM teams to develop highly efficient and functional cell-free systems for chloroplasts of diverse plant species.
Sources
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- 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
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