Team:KU Leuven/Results
BLADEN
Results
On this page we highlight the results obtained from the E. coli and BY-2 experiments.
Results of E. coli Experiments
Here you will find the results of the EvolvR in Escherichia coli experiment, where we induced EvolvR in E. coli cells carrying a non-fluorescent ΔsfGFP(Y93X) cassette with a nonsense mutation. RNA-guided EvolvR can be targeted to the mutation and revert it back to wild-type, thus making sfGFP fluorescent again. More details about plasmid construction and experimental conditions can be found on the Experiments page.
First Iteration
We observed that the samples containing pEvolvR-enCas9-PolI5M or pEvolvR-enCas9-PolI3M-TBD with gRNA targeted to ΔsfGFP(Y93X) and the target pBAD-ΔsfGFP(Y93X) exhibited a drop in optical density upon induction of EvolvR expression. After being in the incubator overnight, all the bacterial cells in these samples had died. Samples carrying the same pEvolvR variant but instead with gRNA not targeted to any specific sequence (unspecific gRNA) or without a gRNA protospacer had survived. These results suggest that EvolvR may be interfering with the maintenance of the target plasmid and either destroying it or drastically reducing its copy number in the cell, resulting in the ampicillin resistance gene not providing the necessary level of resistance for survival and bacteria were left to die.
Samples containing pEvolvR-enCas9-PolI3M with targeted gRNA and target plasmid had survived and were plated on selective media (Figure 1, plates 1 and 2). Unfortunately, we did not observe any green fluorescence on those plates, suggesting that the mutation rate of the enCas9-PolI3M variant of EvolvR is not high enough to reverse the nonsense mutation in the ΔsfGFP(Y93X), which coincides with previous reports of its mutation rate [1].
Samples containing pEvolvR with sfGFP as a placeholder for a protospacer, thus inherently fluorescent, and the target plasmid were plated on media selective for both plasmids (Figure 1, plates 10 and 13) or only for the target plasmid (Figure 1, plates 11 and 12). The fraction of green colonies on plates selective only for the target plasmid is 10-fold lower than on plates selective for both plasmids, thus suggesting that EvolvR may be toxic to bacteria as they may be trying to get rid of the pEvolvR plasmid as soon as they are released from selection pressure by the antibiotic.
We also measured the forward and side scattering, and GFP fluorescence of the samples using a flow cytometer (Figure 2). Judging by forward and side scatter characteristics, the samples that have survived exhibit the normal shape of E. coli cells, confirming that an actively expressed EvolvR does not promote a change in cell shape.
We did not observe any green fluorescence in the samples containing pEvolvR-enCas9-PolI3M, targeted gRNA and target plasmid, (Figure 2, panel A) which confirms the same findings we had after inspection of the plates about enCas9-PolI3M having lower mutation rate.
We observed a fraction of green fluorescent cells in the samples carrying pEvolvR with sfGFP (Figure 2, panels B and C) that were grown overnight in the media only selective for the target plasmid. Therefore most of them had already lost the pEvolvR-sfGFP plasmid. The average fraction of green cells is the same as the fraction of green colonies that we observed on plates.
Second Iteration
Based on previous results, we decided to lower the incubation temperature after induction of EvolvR expression to reduce potential toxicity. We repeated the experiment and performed flow cytometry analysis after letting the cells grow overnight at 25°C after inducing EvolvR.
We again observed regular E. coli shape in all samples, and no green fluorescence in any of the samples containing variants of pEvolvR-targeted gRNA and the target pBAD-ΔsfGFP(Y93X) plasmid (Figure 3, panels A-C), or in any of the negative controls (Figure 3, panels D-H). These findings suggest that either the chosen protospacer sequence is not efficient, or our experimental conditions are not optimal. Further optimization of this experiment could include trying different protospacers (such as repurposing some from the "molecular ruler" experiment), different expression regulators for parts of our system, vectors with different copy number, and different experimental conditions.
Results of BY-2 Experiments
Paving the path to functional EvolvR in plant cells
Ultimately, we aimed to test the plant EvolvR constructs in Tobacco BY-2 cells. Firstly, we determined the growth rate of BY-2 cells by measuring optical density, and we confirmed that the BY-2 cell suspension culture is healthy and representative of documented BY-2 cultures. Secondly, we optimized transformation and continuous directed evolution (CDE) conditions for BY-2 EvolvR CDE experiments. We accomplished this optimization by first testing the transformation protocol both on solid and in liquid media to accelerate our future experiments. Furthermore, we assessed the effects of different antibiotics on WT BY-2 cells and Agrobacterium tumefaciens cells carrying EvolvR constructs. We also determined what combinations and concentrations were most lethal to the bacteria with minimal effect on the BY-2 cells. Next, the best sublethal (and lethal) BASTA concentrations were determined both on plates and in liquid media to optimize the selection pressure applied during later CDE experiments with BY-2 cells. Finally, we set up the EvolvR CDE experiment to validate that EvolvR is mutating the user-defined target region of the BY-2 genome. More detailed explanations and results can be found on the Experiment page and in the uploaded lab books.
Measuring BY-2 growth
The OD600 of BY-2 cell suspension culture samples were measured every 12 hours over a 7-day period in triplicate from the same BY-2 culture line. While measuring OD values, samples were diluted to ensure OD values remained below 1 to maintain accuracy. The measured OD values were multiplied with the respective dilution factor to obtain corrected values. The logarithm of the corrected OD values was plotted against time (in hours).
Figure 4(A), (B), and (C) represent the OD vs. time of cultures 1, 2, and 3, respectively. Since we only needed to calculate the growth factor and doubling time, the growth curve was not fit to a model for any further analysis. From each graph, two consecutive points were considered in the exponential phase of the growth curve, and µ was calculated, seen in Figure 4(D). In all three cases, a growth factor of 0.037 was calculated, with the corresponding td of 18 hours. This value is in good agreement with the literature which states that the doubling time of BY-2 ranges between 16-24 hours in optimal conditions [2].
Optimized transformation and CDE conditions
Before starting the BY-2 EvolvR experiments, we wanted to optimize transformation and CDE conditions. We analyzed the effect of plating versus keeping in liquid on BY-2 growth after transformation, the effect of antibiotics on A. tumefaciens and WT BY-2 cells, that of different herbicide (BASTA) concentrations and the effect of various BY-2 and A. tumefaciens volumes and concentrations.
We first evaluated the growth of BY-2 transformed cells in liquid and on solid medium. The common procedure during transformation of a BY-2 cell culture is to incubate BY-2 cells with A. tumefaciens for up to two days and then plate them on solid medium to let them grow into calli, which can take up to three weeks to become visible. This method is typically used as it allows one to isolate a single mutant and start a new transgenic culture. However, waiting for sufficient callus growth is time-consuming and negates the advantage of speed in a continuous directed evolution (CDE) experiment with EvolvR (more information can be found on the "Engineering" page). Therefore, we attempted to grow transformed BY-2 cells in liquid media immediately (48h) after transformation. Due to restrictions on the incubator space, we sought out to grow the transformed BY-2 cells shaking in deep petri dishes in 13 mL volumes for 5 days. We observed that this method did not work well for either transformed cells (with different A. tumefaciens and BY-2 concentrations) or WT BY-2 cells (Figure 5).
WT BY-2 cell growth was also tested in 50 mL conical tubes and erlenmeyers sealed with aluminum foil. Both of these methods failed as cells clustered on the bottom of the conical tubes, while cells got contaminated in erlenmeyers with aluminum foil [3]. Therefore, we decided that we cannot compromise the growing conditions of the suspension cells to save on incubator space or materials. BY-2 growth of both WT and recently transformed BY-2 cells has to be performed in erlenmeyers with a sufficiently sealing cap, taped with surgical tape to allow for oxygenation while maintaining a sterile environment.
Since we started to shift to a liquid Agrobacterium-mediated transformation (AMT) method, it became essential to determine what different antibiotic combinations and concentrations would be most effective at killing all the A. tumefaciens, while leaving the transformed BY-2 cells intact. The WT BY-2 cells would be affected by supplemented kanamycin, while the transformed BY-2 cells would contain a kanamycin resistance cassette after successful AMT. The antibiotic array we tested on our BY-2 suspension cell line consists of timentin, carbenicillin, vancomycin, kanamycin and hygromycin. In the first testing round, we supplemented the agar with an equal concentration of timentin, carbenicillin, vancomycin and kanamycin to test their effect on WT BY-2 and BY-2 cells transformed with pGGK_A-G destination vector. We did not observe large differences between the conditions (Figure 6.1A-B).
In the second round we increased the concentration of carbenicillin, vancomycin, kanamycin and added hygromycin (figure 6.2 A-D). We noticed that raising the concentration of vancomycin and carbenicillin in 2A and 2B does not affect BY-2 cell growth, while increased kanamycin concentration appears to have a marginal effect on WT BY-2 cells in 2C and 2D (Figure 6). These findings also suggest that hygromycin may make the plated cells have difficulty attaching to the solid media in 2D, making them dry up and thus not show any growth over the course of 2 weeks (Figure 6). We did not test the effects of the antibiotics again on the transformed (pGGK_A-G destination vector) BY-2 cells since these were not available anymore. Additionally, we applied these conditions to A. tumefaciens containing the pGGK_A-G destination vector and detected no growth, as expected. In the end we decided on using a combination of timentin (100 µg/mL), carbenicillin (500 µg/mL), vancomycin (200 µg/mL), kanamycin (200 µg/mL) and hygromycin (30 µg/mL) in our transformation experiments.
Next, we determined the best sublethal BASTA concentrations to be used both on plates and in liquid to optimize the selection pressure applied during CDE experiments in BY-2 cells. We determined the best BASTA concentration on plates in two rounds. In the first cycle, we tested out a large array of BASTA concentrations to rapidly narrow down the range to test in the second round. There appeared to be a clear distinction between 12 ng/mL and 1.2 ng/mL BASTA (Figure 7.1A). Therefore, we narrowed in on those concentrations and we concluded that 4.8 ng/mL of BASTA was the optimal sublethal concentration to use in CDE experiments since there is a good balance between WT BY-2 cells that survived and cells that had died and did not attach or grow on the solid medium (Figure 7.1B).
Next, we performed the same experiments in liquid BY-2 cell suspension cultures over multiple rounds. We first tested the same BASTA concentration array as before to narrow down the concentration range to test in the second round. These cells exhibited the same pattern as the BY-2 calli on solid medium (figure 7.2A). Due to incubator space constraints, we tested only one extra concentration between 12 ng/mL and 1.2 ng/mL BASTA. From this experiment we concluded that a concentration between 6 ng/mL and 1.2 ng/mL is the optimal sublethal concentration to use in liquid media CDE experiments (figure 7.2B). A more specific concentration has not been determined yet.
Additionally, during the transformation experiments, we tested different transformation conditions involving BY-2 and Agrobacterium concentrations and volumes to determine the best transformation environment specific to our project. We decided on 4 mL of a 3/40 BY-2 dilution since this seemed to have the best density compared to the transformation procedure we witnessed in our training. The Agrobacterium concentration or volume did not seem to matter much in the end.
EvolvR transformed into the BY-2 genome
After optimizing the transformation and CDE conditions, we transformed BY-2 cells with a plant destination vector carrying EvolvR. This is an enCas9-PolI5M-unspecific construct (pGGK_35SP_NLS-N7_enCas9-PolI5M_P2A-mCherry-NLS_35ST_unspecific-gRNA) which would be used as a control in CDE experiments and does not target the glutamine synthetase gene. This vector includes an mCherry reporter, which localizes to the nucleus as it is fused to a nuclear localization signal (NLS). We visualized mCherry using a fluorescence confocal microscope and were able to determine that we have successfully transformed BY-2 cells with this EvolvR construct and that we have created a transgenic BY-2 cell line containing EvolvR. Figure 8 reveals this pattern clearly while being absent in the WT negative control. Based on this we can conclude that we can transform EvolvR into BY-2 cells. Therefore, we have built and designed all the necessary parts and protocols for the application of EvolvR in plant cells to optimize target genes in CDE experiments.
Performing EvolvR and CDE in BY-2 plant cells
Finally, we wanted to validate whether EvolvR is mutating the user-defined target region of the BY-2 genome, which we chose to be the glutamine synthetase gene encoding for the target of BASTA herbicide. As the BY-2 genome is not fully characterized, we sequenced the target native BY-2 gene so as to choose the best protospacers for the EvolvR experiments in BY-2. These protospacers were then inserted into the gRNA scaffold in our plant EvolvR constructs, as described on the Experiments page. Unfortunately, due to time constraints, this is where we had to stop our experiments.
As mentioned on the proof-of-concept page, our plan was to transform a WT BY-2 cell suspension culture with an EvolvR construct carrying the BASTA target protospacer. After 48 hours, we would add an array of antibiotics at specific concentrations determined in the optimization experiments described above. These antibiotics would kill off the Agrobacterium without harming the transformed BY-2 cells. The WT BY-2 cells will be affected by supplemented kanamycin, while the transformed BY-2 cells will contain a kanamycin resistance cassette after successful AMT. The selection of transformed BY-2 cells out of a liquid transformation mixture with WT BY-2 cells and Agrobacterium would be continued throughout the whole experiment to ensure only transformed BY-2 cells survive. Initial conclusions regarding EvolvR activity in plant cells can be drawn by sequencing the BY-2 genes targeted by EvolvR.
If we can demonstrate that the targeted mutation rate in the target gene is higher than the global mutation rate, we can infer that EvolvR is active in BY-2 plant cells. Additionally, at 3 days post-transformation, we would add a sublethal concentration of BASTA as described in the transformation optimization experiments above to start the CDE experiment. This selection pressure would cause the desired mutations generated by EvolvR to make more abundant as they would multiply faster due to higher fitness compared to the less BASTA-resistant variants. By harvesting the BY-2 cells again and sequencing the target gene, we can showcase that EvolvR is indeed functional in plant cells and together with CDE this method can be applied to a BY-2 cell suspension culture to accelerate plant evolution.
EvolvR is a very promising tool for continuous directed evolution of plants, which has never been described before. We have shown that a large construct containing EvolvR can effectively be transformed into BY-2 cells and our next steps are to validate the activity of EvolvR in these cells to confirm that it is able to mutate a target genomic region in BY-2 cells. The toolkit, protocols, and hardware designs we have built in our project will certainly advance the field of plant biotechnology towards a future in which plants can rapidly be evolved towards novel phenotypes which can help us battle the effects of climate change and meet the growing food demand.
References
[1] Halperin, S. O., Tou, C. J., Wong, E. B., Modavi, C., Schaffer, D. V., & Dueber, J. E. (2018). CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature, 560(7717), 248–252. https://doi.org/10.1038/s41586-018-0384-8
[2] Santos, Rita B, et al. “Putting the Spotlight Back on Plant Suspension Cultures.” Frontiers in Plant Science, vol. 7, no. 2016, 2016, p. 297.
[3] Experimental Plant Division, R. B. R. C. (2021, September 7). RPC00001: Nicotiana tabacum by-2 cell suspension culture. rpc00001: Nicotiana tabacum BY-2 cell suspension culture - BRC plant cell line documentation. Retrieved from
https://plant.rtc.riken.jp/resource/cell_line/web_documents/cell_lines/rpc00001.html