Overview
In this study, we aimed to construct a cell-free transcription-based biosensing platform, 'MITSUNARI,' by extending the ROSALIND system and applying it to the detection of nitrate and ammonium ions. Initially, we constructed all plasmids and attempted to purify the target proteins. Upon the completion of these steps, we proceeded sequentially with proof-of-concept and system functionality tests.
Although we were unable to demonstrate clear sensing capabilities for nitrate and ammonium ions in all six Input circuits, we achieved progress that suggests the potential applicability of the system for sensing, warranting further validation. A significant achievement was demonstrating that the T7 Two-hybrid system can be applied to MITSUNARI.
Regarding the Transcription circuits, we successfully performed in vitro transcription using purified T7 RNA polymerase, allowing us to optimize the reaction conditions and characterize the system for further experiments.
For the five Output circuits, we were able to fully or partially demonstrate functionality in some cases, while in others, we made progress that could lead to additional verification.
Detailed results are described in the following sections.
NH4+ Input circuits
(1)GlnA/GlnR - T7 Two hybrid system
Design
The system contains GlnA, GlnR-Leucinezipper-AN3.5 fusion protein (hereafter referred to as GlnR-LzA), T7 RNA Polymerase-Leucinezipper-BN3.5 (hereafter referred to as T7RNAP-LzB), a transcription template DNA of a GlnR binding sequence-T7 Promoter d1-Output sequence (hereafter referred to as template), Glu, and ATP.
In the absence of ammonium, GlnR-LzA does not bind to the template, so transcription is not activated. As the ammonium concentration increases, GlnR-LzA binds to template in proportion to the concentration and T7RNAP-LzB is recruited to Template through the interaction between LzA and LzB, activating transcription.
Consequently, transcription is activated in an Ammonium-dependent manner, and the Ammonium concentration is converted into the degree of transcription activation (transcription rate or transcription amount).
For GlnR-LzA, three candidate designs were created through Modeling: two variants with different fusion positions of GlnR-LzA and GlnA-LzA (which, theoretically, can recruit T7RNAP-LzB just like GlnR-LzA). The most optimal one is planned to be tested through an assay.
Main Achievements
- Mass culture and purification were performed for GlnA, GlnR, GlnR-Leucine zipper AN3.5, GlnR mutant-Leucine zipper AN3.5, GlnA-Leucine Zipper AN3.5, Leucine Zipper AN3.5-GlnR, T7-Leucine Zipper, and Zinc finger-Leucine Zipper.
- Using a positive control with the Zinc finger-Leucine Zipper, it was demonstrated that the two-hybrid system utilizing the Leucine Zipper can be applied in vitro.
- It was also proven that the two-hybrid system can be applied to the GlnR system using a GlnR mutant with high DNA binding activity regardless of glutamine concentration.
View details
Plasmid Construction
The plasmids used in this experiment (GlnA, GlnR, GlnR-Leucine zipper AN3.5, GlnR mutant-Leucine zipper AN3.5, GlnA-Leucine Zipper AN3.5, Leucine Zipper AN3.5-GlnR, T7-Leucine Zipper, and Zinc finger-Leucine Zipper) were constructed through XE cocktail assembly, and their successful construction was confirmed by Sanger sequencing.
Protein Purification
As shown in Figure1, SDS-PAGE bands were observed after mass culture and purification.
SDS-PAGE of protein purification
Proof of concept with Zinc Finger
To demonstrate that transcription via the two-hybrid system can be applied in vitro, we conducted a demonstration experiment using a Zinc finger. After optimizing the concentrations of T7 RNA Polymerase-Leucine Zipper BN3.5 and Zinc finger-Leucine Zipper AN3.5, as shown on the Engineering Page, we performed an assay while varying the concentration of GlnR-Leucine zipper AN3.5. The results are shown in Figure 2. A more than twofold difference in fluorescence values was observed between the negative control and the assay with 7 μM Zinc finger-Leucine Zipper AN3.5. Although the high background remains an issue, this experiment demonstrated that the two-hybrid system can be utilized in vitro.
The figure shows how the increase in fluorescence from 0 to 60 minutes changes with varying concentrations of Zinc Finger-Leucine Zipper, while the concentration of T7 RNA Polymerase-Leucine Zipper was fixed at 0.7 μM. The negative control represents the condition where the concentration of Zinc Finger-Leucine Zipper is 0, and the error bars indicate the range of measurement errors. The DNA Template concentration was set at 0.265 μM.
Proof of concept with GlnR
Next, we proceeded with a proof of concept using a GlnR mutant that exhibits high DNA binding activity regardless of glutamine concentration. When GlnR-Leucine Zipper was introduced, a fluorescence increase of up to approximately 10 times was observed compared to the negative control. This suggests that the binding activity of GlnR(mutant)-Leucine Zipper may be higher than that of Zinc Finger-Leucine Zipper. The reason why fluorescence increased when the concentration of GlnR-Leucine Zipper decreased is discussed in Modeling 6. In any case, it was demonstrated that GlnR(mutant)-Leucine Zipper can initiate transcription by binding to DNA. Although a complete proof of concept for glutamine concentration-dependent transcription activation was not achieved within the project period, the results suggest that it could be achieved through further optimization of conditions and protein design.
The change in fluorescence from 0 to 60 minutes when varying the concentration of GlnR-Leucine Zipper AN3.5. The negative control represents the condition where GlnR-Leucine Zipper AN3.5 was not included. The error bars indicate the range of measurement errors. The DNA Template concentration was set at 0.26 μM, and the T7 RNA Polymerase-Leucine Zipper concentration was set at 0.7 μM.
(2)GlnA/GlnR - Repressor system
Design
The system contains GlnA, GlnR, T7 RNA Polymerase (hereafter referred to as T7 RNA Polymerase), a transcription template DNA of the T7 Promoter-GlnR binding sequence-Output sequence (hereafter referred to as template), Glu, and ATP.
In the absence of ammonium, GlnR does not bind to the template, so transcription occurs constitutively. As the ammonium concentration increases, GlnR binds to the template in proportion to the concentration, and transcription is suppressed by its repressor activity.
As a result, transcription is inhibited in an ammonium-dependent manner, and the ammonium concentration is converted into the degree of transcriptional repression (transcription rate or transcription amount).
Main Achievements
- Successfully constructed all three plasmids used in the system.
- Successfully purified all three proteins required for the system.
- Demonstrated that the GlnR mutant (GlnRN95), which constitutively binds to DNA, can efficiently inhibit in vitro transcription. This suggests the potential for the system to function after further optimization of reaction conditions and protein modifications.
View details
Plasmid Construction
The plasmids used in this experiment (GlnA, GlnR, and GlnRN95) were constructed using XE cocktail assembly, and the accuracy of their sequences was confirmed by Sanger sequencing.
Protein Purification
As shown in Figure 4, SDS-PAGE bands were observed following mass culture and purification.
SDS-PAGE band of GlnA, GlnR, and GlnRN95
System Test
To investigate the system, we measured the repressor activity of GlnR using in vitro transcription and fluorescence measurement of the aptamer, which is the transcription product, under conditions without glutamine and with an excess of glutamine. The transcription template encoded the T7 Promoter-GlnR binding sequence (R2)-Broccoli aptamer. Purified and stocked GlnA and GlnR were added to the system, and a sample using GlnRN95 instead of GlnR was also tested as a positive control.
The change in fluorescence from 0 to 60 minutes at Gln concentrations of 0 mM (-) and 20 mM (+) is shown. "GlnRN95" refers to the sample where the constitutive DNA-binding mutant GlnRN95 was added instead of GlnR, while "T7" represents the condition where neither GlnA nor GlnR was added. The error bars indicate the range of measurement errors. The template concentration was set at 0.125 μM, GlnA at 5 μM, and GlnR/GlnRN95 at 1 μM.
Although GlnR slightly inhibited transcription, its repressor activity did not show Gln dependency. On the other hand, GlnRN95 was able to efficiently suppress transcription.
While this result did not prove the system's functionality, it demonstrated that the repressor activity of GlnRN95 was sufficient to inhibit T7 RNA Polymerase transcription. This suggests that the system has the potential to function after further optimization of reaction conditions and protein modifications. Additionally, it indicated that repressor-type sensor proteins could be applied as an input format in the MITSUNARI platform.
(3)GlnA/TnrA - Activator system
Design
In the system, GlnA, TnrA, T7 RNA Polymerase (hereafter referred to as T7 RNA Polymerase), the transcription template DNA (hereafter referred to as Template) containing the T7 Promoter-TnrA binding sequence-Output, Glu, and ATP are present. In the absence of ammonia, TnrA binds to the Template, constitutively repressing transcription. As the ammonia concentration increases, TnrA dissociates from the DNA in proportion to the concentration, lifting the repression and activating transcription. This allows ammonia-dependent transcription activation, converting the ammonia concentration into the degree of transcription activation (transcription rate or amount).
Main Achievements
- Plasmids were constructed.
- Protein purification was successfully achieved.
View details
Plasmid Construction
The plasmid used in this experiment (TnrA) was constructed using XE cocktail assembly, and its generation was confirmed by Sanger sequencing.
Protein Purification
As shown in Figure 6, SDS-PAGE bands were observed following mass culture and purification.F
SDS-PAGE gel of purified TnrA
Pull down
We conducted a pull-down assay to determine whether TnrA binds to its specific DNA recognition sequence.
As a result of the pull-down, after quantification using qPCR, the input and sample were converted to equivalent amounts, and a bar graph was created showing the percentage of the sample relative to the input on the vertical axis. The TnrA control indicates the sample that was amplified by qPCR using a plasmid that does not contain the TnrA binding sequence.The data was collected three times, and standard error was used as the error bars.
Transcription
We investigated how TnrA affects the transcription of Broccoli transcription DNA, which contains the TnrA-specific recognition sequence downstream of the promoter, in the presence and absence of glutamine. As a result, when examining the fluorescence intensity at the start of transcription and the change in fluorescence after 25 minutes, TnrA did not exhibit the expected activity.
The change in fluorescence from 0 to 25 minutes in the presence and absence of TnrA and glutamine. The error bars indicate the range of measurement errors. The DNA Template concentration was set at 1.25 μM, and the T7RNAP concentration was set at 0.6 μM.
Based on the results of the pull-down assay and transcription assay, it is considered that the reason TnrA did not exhibit the expected activity could be due to an issue with the design of the Broccoli transcription DNA, which includes the TnrA-specific recognition sequence downstream of the promoter.
NO3- Input circuits
(4)NasR - Split T7RNAP system
Design
The system contains NasR-Split NT7 fusion protein (hereafter referred to as NasR-NT7), MCP-Split CT7 fusion protein (hereafter referred to as MCP-CT7), RNA encoding NasR binding sequence-MCP binding sequence (hereafter referred to as RNA scaffold), and transcription template DNA encoding T7 Promoter-Output.
In the absence of nitrate, NasR-NT7 remains unbound, so it does not cause Split T7 reassociation, keeping transcription off. As the nitrate concentration increases, NasR-NT7 binds to RNA scaffold in proportion to the concentration. Since MCP-CT7 is always bound to RNA scaffold, NasR-NT7 and MCP-CT7 bind adjacently to scaffold RNA, causing Split T7 to physically come into close proximity and reassociate, thereby turning transcription on.
As a result, transcription is activated in a nitrate-dependent manner, and the nitrate concentration is converted into the degree of transcriptional activation (transcription rate or transcription amount).
For the assays conducted in the process of constructing this system, we adopted the aforementioned interlocking peptide sequences, Leucine Zipper-AN3.5 and Leucine Zipper-BN3.5, as a positive control for in vitro activity testing of Split T7 system using NT7/CT7 pair used in this study. We prepared fused NT7-LzA, CT7-LzB, and NT7 and CT7 alone as a negative control.
For testing the entire system, we used constitutive RNA-binding mutant NasR (R193A)-NT7 as a positive control.
Main Achievements
- Successfully constructed all seven plasmids for the system.
- Successfully purified two of the seven proteins to be used: NasR-NT7 and NasR(R193A)-NT7.
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Plasmid Construction
The plasmids used in this experiment (NasR-NT7, NasR(R193A)-NT7, MCP-CT7, NT7-LzA, CT7-LzB, NT7, and CT7) were constructed using XE cocktail assembly, and the accuracy of their sequences was confirmed by Sanger sequencing.
Protein Purification
For NasR-NT7 and NasR(R193A)-NT7, as shown in Figure9, SDS-PAGE bands were observed following mass culture and purification.
For MCP-CT7, NT7-LzA, CT7-LzB, NT7, and CT7, despite multiple purification attempts while adjusting mass culture and purification conditions as detailed in the notes, clear SDS-PAGE bands were not observed at the expected positions. This is likely due to decreased solubility and folding issues caused by the split design. Given that NasR-NT7 and the NT7-NLP7-CT7 construct were successfully purified in small quantities, it suggests that purification might be achievable through fusion with a solubility tag or by using an expression system other than the pET system.
This represents a milestone that could enable more efficient research in our future studies and in other efforts to apply the Split T7 system in vitro.
SDS-PAGE gel of N-T7RNAP-NasR
SDS-PAGE gel of Nas(R193A)-NT7RNAP
(5)NLP7 - Split T7RNAP system
Design
The system contains NT7-NLP7-CT7 fusion protein (hereafter referred to as NT7-NLP7-CT7) and transcription template DNA encoding T7 Promoter-Output.
In the absence of nitrate, the conformation of NT7-NLP7-CT7 keeps Split T7 fragments at both ends separated, preventing their reassociation, and thus transcription remains off. As the nitrate concentration increases, NT7-NLP7-CT7 undergoes a conformational change that brings Split T7 fragments into close proximity, allowing them to reassociate, which turns transcription on.
As a result, transcription is activated in a nitrate-dependent manner, and the nitrate concentration is converted into the degree of transcriptional activation (transcription rate or transcription amount).
Main Achievements
- Successfully constructed the plasmids used in the system.
- Successfully purified the proteins used in the system.
View details
Plasmid Construction
The plasmid used in this experiment (NT7-NLP7-CT7) was constructed using XE cocktail assembly, and the accuracy of its sequence was confirmed by Sanger sequencing.
Protein Purification
After attempting purification with varying culturing and purification conditions, a faint band was observed on the SDS-PAGE during the third trial. The low yield may be due to the large molecular size of the protein (206.96 kDa) and the instability of the Split T7RNAP, as previously suggested. The sample was concentrated using an Amicon filter and used for assays.
SDS-PAGE gel of purified proteins
System Test
Using NT7-NLP7-CT7 as the polymerase, we conducted a transcription assay under both nitrate-free and nitrate-excess conditions. The template used was the T7 promoter-Broccoli aptamer sequence, and the fluorescence of the transcription product was observed using a qPCR machine.
Transcription reactions were performed under four conditions: total protein concentration of 3.5 µM (+) and 0 µM (-) of NT7-NLP7-CT7-containing protein solution, and NaNO3 concentration of 0 mM (-) and 100 mM (+). The difference in fluorescence intensity between 0 minutes and 60 minutes after the start of the reaction was measured using a qPCR device and displayed in a bar graph. Each condition was tested with three samples, and the error range was indicated.
In subsequent experiments, using the same template, buffer, and temperature conditions, transcription with regular T7 RNA Polymerase yielded fluorescence intensities ranging from 600,000 to 1,800,000. Therefore, we concluded that transcription by NT7-NLP7-CT7 did not occur regardless of the presence or absence of nitrate. Possible causes include low protein purity, abnormal folding, decreased in vitro activity of NLP7, the conformation of NLP7 being unsuitable for Split T7 RNA Polymerase reconstitution at any stage, or experimental errors.
The results of this system provided insights for improving the purification conditions of NT7-NLP7-CT7. The application of this system as an in vitro nitrate sensor or in the MITSUNARI platform will be part of future plans.
(6)NLP7 - Directory Reporting system
Design
This system utilizes the nitrate-dependent conformational change of NLP7.
In response to nitrate, NLP7 changes to a more folded conformation, bringing the fused Split-mCitrine or NanoBiT (Split luciferase) into close proximity, resulting in their reassociation and emitting fluorescence or luminescence.
The nitrate concentration is reported as fluorescence intensity or luminescence intensity, allowing nitrate sensing.
In this project, this system is an exception, as it reports directly without involving transcription. Although it is not included in the MITSUNARI platform, it was tested due to its potential as the simplest and most robust nitrate-sensing system.
Main Achievements
- Successfully constructed the plasmids used in the system.
- Successfully purified the proteins used in the system.
View details
Plasmid Construction
The plasmids used in this experiment (NLP7-mCitrine and NLP7-NanoBit) were constructed using XE cocktail assembly, and their generation was confirmed by Sanger sequencing.
Protein Purification
As shown in Figure12, SDS-PAGE bands were observed following mass culture and purification.
SDS-PAGE gel of LgBiT-NLP7-SmBiT
NLP7-mCitrine
We investigated whether NLP7-mCitrine exhibits the desired activity in response to changes in nitrate ion concentration. The bacterial lysate expressing NLP7-mCitrine was incubated at room temperature for 15 minutes while varying the nitrate concentration, and the fluorescence intensity was measured.
Fluorescence intensity after incubating the bacterial lysate expressing NLP7-mCitrine with varying concentrations of nitrate ions for 15 minutes. The error bars indicate the range of measurement errors.
NLP7-NanoBit
We measured how the luminescence intensity changes by varying the concentration of NLP7-NanoBit in the presence and absence of nitrate ions.
The change in luminescence intensity of NLP7-NanoBit at different concentrations, with nitrate ion concentrations of 1 mM and 0 mM in the solution. The error bars indicate the range of measurement errors.
In both cases, no significant differences in fluorescence intensity or luminescence intensity were observed in the presence and absence of nitrate. Possible reasons for not exhibiting the expected activity include changes in the conformation of NLP7 due to substances present in the medium or high concentrations leading to the reconstitution of reporters between proteins.
Transcription circuit
(7)T7RNAP transcription
Design
The system contains T7 RNA Polymerase and transcription template DNA for T7 promoter-Output.
A continuous transcription reaction occurs, producing RNA from the output sequence.
Main Achievements
- Successfully purified the proteins to be used.
- Optimized the enzyme concentration and template concentration suitable for the sensing system using a method to measure transcription activity through the green fluorescence of the Broccoli Aptamer.
- Established a correlation between the observed fluorescence intensity and the actual absolute amount of transcribed RNA when using the measurement method based on Broccoli Aptamer.
View details
The plasmids used in this experiment were purchased from Addgene.
Protein Purification
As shown in Figure15, SDS-PAGE bands were observed following mass culture and purification.
SDS-PAGE gel of purified T7 RNA Polymerase
Enzyme Concentration Optimization
The need to optimize the concentration of T7 RNA Polymerase for more efficient measurements and cost-effective sensing was raised. Additionally, our preliminary experiments (using PAGE and qPCR devices, as referenced in Engineering) observed a phenomenon where fluorescence peaked immediately after transcription and then declined, despite no significant impact from RNases or other factors, indicating that the concentration of enzymes or contaminants might be the cause.
Therefore, we sequentially reduced the enzyme concentration from a "slightly high" level described in the literature to determine the optimal concentration that demonstrated the desired fluorescence intensity enhancement for sensing.
Using a transcription template that transcribes the Broccoli fluorescent aptamer, we conducted the transcription reaction in the presence of the fluorescent dye DFHBI-1t. Fluorescence intensity was measured using the SYBR GREEN filter on the ABI Step One Plus. Our purified T7 RNA polymerase (at 1x, with a final concentration of 4.8 μM) was diluted twofold, and the resulting solution was reacted as described in the Experiments section. The blue wavelength values of the raw data from the Step One Plus were measured every minute for one hour and compared.
The transcription activity was confirmed by varying the concentration of the purified T7 RNA polymerase and measuring the fluorescence. The template concentration was set at 1.25 μM, and each condition was executed with one sample.
As a result, it was found that the unstable fluorescence enhancement waveform at excessively high T7 RNA Polymerase concentrations became more stable and approached a linear relationship with appropriate dilution. Possible causes include the transcription inhibition effect reported in the literature due to high T7 RNA Polymerase concentrations and the reduction of effects caused by high contaminant concentrations. This indicates that under the reaction and reagent conditions with our purified T7RNAP, a final concentration of 0.6 μM is optimal.
Template Concentration Optimization
To improve the practicality and functionality of the system, there is a need for more economical experiments and to measure the activity enhancement of proteins such as GlnR and NanoBiT, which exhibit negative effects at high concentrations. In this context, the necessity to optimize the DNA Template concentration in a lower range has been raised. Therefore, the Template concentration will be decreased sequentially from the "saturation concentration for transcription activity" indicated in the literature to determine a lower concentration that allows for sufficient fluorescence measurement.
Except for the Template concentration, the reaction conditions and measurement methods were the same as described above, with a final concentration of T7 RNA Polymerase set at 0.6 μM and the measurement time set to 90 minutes. The Template concentrations were sequentially reduced to 1.25 μM, 0.5 μM, 0.25 μM, 0.125 μM, 0.05 μM, 0.025 μM, 0.0125 μM, and 0 μM. Three samples were taken for each concentration, and the average values of the measured fluorescence raw data (Blue) for each measurement point were plotted as a line graph.
The concentration of the template was varied, and the fluorescence intensity of the transcribed Broccoli Aptamer was measured every minute. Three samples were measured for each condition, and their average values were plotted on the graph. The horizontal axis represents time (minutes), and the vertical axis represents the raw data fluorescence intensity.
As the concentration of the template was sequentially decreased, it was observed that transcription activity initially increased up to a certain concentration, after which the rate of increase in transcription activity gradually plateaued.
This indicates that the minimum final concentration of T7 RNA Polymerase that allows for a sufficient increase in fluorescence from the transcription reaction is estimated to be 0.125 μM, providing a guideline for the design of systems and experiments where low template concentrations should be applied.
Broccoli fluorescence intensity - RNA absolute concentration mapping
In constructing all systems, we decided to use the same method of measuring fluorescence with Broccoli for the transcription activity assays of the Input system. On the other hand, in many Output system assays, quantitated RNA that has already been transcribed will be added to measure the reporting. To enable the discussion of sensing functionality by linking the assay results of the Input system with those of the Output system in the data, we established a correlation between the fluorescence intensity measured using the aforementioned method and the actual absolute amount of RNA produced at that point in time.
Measurement of Fluorescence Intensity: Data from the samples with a final concentration of T7 RNA Polymerase at 0.6 μM from the aforementioned Enzyme Concentration Optimization experiment were used.
Measurement of Absolute RNA Amount: The T7 RNA Polymerase concentration was set at 0.6 μM, and the transcription reaction was conducted under the same reagent and temperature conditions as those used in the qPCR device. Samples were collected from the reaction system every 10 minutes after the start of transcription, with a specified volume (1 µL was taken from a 100-fold diluted solution.) taken, and the RNA concentration was measured using the Qubit RNA HS Assay Kit on the Thermo Fisher Qubit 4.
Table concentration of quantified RNA
| time | Sample 1 | Sample 2 | Sample 3 |
|---|---|---|---|
| 10 min | 10.41 μM | 13.22 μM | 12.04 μM |
| 20 min | 21.96 μM | 25.26 μM | 23.02 μM |
| 30 min | 22.67 μM | 37.78 μM | 29.75 μM |
| 40 min | 29.99 μM | 46.28 μM | 36.36 μM |
| 50 min | 37.78 μM | 47.22 μM | 37.78 μM |
| 60 min | 54.54 μM | 62.57 μM | 45.33 μM |
This established a correlation between Broccoli fluorescence intensity and absolute RNA concentration. Strictly speaking, it would be desirable to conduct additional experiments considering the differences in reaction conditions and template concentrations; however, this provided a basis for linking the Input and Output systems, offering a reference for discussions regarding the overall sensor system.
Output circuit
(8)Fluorescent RNA Aptamer
Design
When the Broccoli sequence RNA is transcribed by the Input, the RNA folds constitutively and binds to DFHBI-1t, emitting green fluorescence. This converts the transcription amount into fluorescence intensity, which is reported.
Main Achievements
- Complete functionality and high fluorescence intensity were demonstrated.
View details
The experimental content overlaps with the previously mentioned section (7) on T7RNAP transcription.
It was demonstrated that T7RNAP-mediated transcription resulted in sufficiently high fluorescence, fully functioning as a reporter. Additionally, it was used as a reporter for assays in Input systems (1) to (5). It was also utilized in separate hardware experiments, where a clear increase in fluorescence intensity was observed with our prototype device.
The Broccoli Aptamer is a reporter that our sensing platform, ROSALIND, had already used for sensing field samples, so its functionality is not a novel achievement in itself. However, being able to incorporate it as the first reporter with complete and sufficient functionality for sensing nitrate and ammonium is a significant accomplishment for achieving the project's objectives.
(9)Cas7-11 - Chromoprotein system
Design
The system contains the Cas7-11-csx29-guide RNA complex (hereafter referred to as the Cas complex), the CBM-truncated csx30-Spycatcher003 complex, the Spytag003-chromoprotein complex, and cellulose crystals.
Before the reaction, the CBM-truncated csx30-Spycatcher003 complex and the Spytag003-chromoprotein complex are automatically covalently linked by Spy system to form the CBM-truncated csx30-Spycatcher003-Spytag003-chromoprotein complex (hereafter referred to as the chromoprotein module). The chromoprotein module then automatically binds to cellulose crystals via CBM9.2, causing the cellulose crystals to precipitate as a solid phase along with it.
Thus, it typically exists as a colored precipitate and a colorless supernatant.
When the target RNA transcribed by Input system is present, csx29 in the Cas complex is activated and cleaves the truncated csx30 in the chromoprotein module. After cleavage, the fragment containing the chromoprotein is released from the cellulose crystals, resulting in the coloring of the supernatant.
This allows the transcription quantity to be converted into the amount of chromoprotein cleaved, which is reported as the intensity of the color in the supernatant.
Chromoproteins can be freely selected from multiple colors.
Additionally, because the chromoprotein module is split into two parts via the Spycatcher-Spytag system, the chromoprotein side can be easily generated by simply adding the 16-amino-acid Spytag003. This makes it a versatile module that allows for the easy creation of many variations. It is also easy to extend if one wishes to use other colored chromoproteins or GFP as reporters.
Main Achievements
- Successfully purified the proteins.
- Confirmed the cleavage of CBM9.2-truncated csx30-Spycatcher003r by Cas7-11.
- Verified the binding of CBM to RAC.
View details
Plasmid Construction
The plasmids used in this experiment (CBM9.2-truncated csx30-Spycatcher003 and Spytag003-Chromoprotein) were constructed using XE cocktail assembly, and their generation was confirmed by Sanger sequencing.
Protein Purification
As shown in Figure18, SDS-PAGE bands were observed following mass culture and purification.
SDS-PAGE of purified proteins
SDS-PAGE of purified Cas7-11
Proof of concept with Cas7-11
We confirmed that Cas7-11-Csx29 recognizes and cleaves truncated csx30. We varied the concentrations of Cas7-11-Csx29 and target RNA and confirmed the cleavage activity using SDS-PAGE.
SDS-PAGE gel of csx30 cutting assay Cleavage of CBM9.2-truncated csx30-Spycatcher003r by Cas7-11-Csx29. The concentration of CBM9.2-truncated csx30-Spycatcher003r was kept constant at 2.5 μM, while the concentrations of Cas7-11-Csx29 were varied at 1 μM, 0.5 μM, and 0.25 μM, and the concentrations of target RNA were varied at 2.5 μM, 0.25 μM, and 0 μM.
Proof of concept with Spytag-Spycatcher
We confirmed whether CBM9.2-truncated csx30-Spycatcher003 and Spytag003-Chromoprotein bind together through the Spy system using SDS-PAGE.
We mixed the purified eluted fractions of CBM9.2-truncated csx30-Spycatcher003 and Spytag003-asPink and incubated them at room temperature for 65 minutes, after which the results were confirmed using SDS-PAGE.
SDS-PAGE gel of the result of spytag reaction
In the mixed samples, the band for Spytag003-asPink, which was at a lower concentration, disappeared, and the appearance of a new band with a higher molecular weight than these two proteins was confirmed. (The appearance of multiple bands may be due to translation termination products or cleaved products containing either Spytag or Spycatcher.)
The results align with the expectation that Spycatcher and Spytag form a common bond with nearly 100% efficiency. Therefore, it is believed that the Spy system is functioning completely.
Proof of concept with CBM
We used SDS-PAGE to verify whether CBM9.2-truncated csx30-Spycatcher003-Spytag003-Chromoprotein binds to cellulose. A solution containing cellulose and CBM9.2-truncated csx30-Spycatcher003-Spytag003-Chromoprotein was mixed and centrifuged, and then the proteins present in the supernatant and pellet were examined using SDS-PAGE.
On the left is a mixture of Spytag003-Chromoprotein and RAC after centrifugation. On the right is a mixture of CBM9.2-truncated csx30-Spycatcher003-Spytag003-Chromoprotein and RAC after centrifugation.
A mixed solution of Spytag003-gfasPurple and CBM9.2-truncated csx30-Spycatcher003r, which was incubated at room temperature for 30 minutes, and a solution containing an equal amount of only Spytag003-gfasPurple were both mixed with cellulose and incubated at room temperature for another 30 minutes. After incubation, the mixture was centrifuged to precipitate the cellulose. The proteins present in the supernatant were then analyzed using SDS-PAGE.
This experiment confirmed that Spytag003-gfasPurple alone was unable to bind to cellulose, while CBM9.2-truncated csx30-Spycatcher003r-Spytag003-gfasPurple successfully bound to cellulose.
(10)Split APEX system
Design
The system utilizes the peroxidase activity of APEX as a chromogenic reporter system. When there is RNA (RNA scaffold) transcribed from the Input system, MCP-AP and PCP-EX bind adjacently, leading to the reconstitution of Split APEX, which triggers a chromogenic reaction through its peroxidase activity. The transcription amount is reported as chromogenic intensity.
Main Achievements
- Successfully constructed the plasmids.
- Successfully purified the proteins.
View details
Plasmid Construction
The plasmids used in this experiment (MCP-AP and PCP-EX) were constructed using XE cocktail assembly, and their generation was confirmed by Sanger sequencing.
Protein Purification
As shown in Figure24, SDS-PAGE bands were observed following mass culture and purification.
SDS-PAGE of purified proteins (MS2 coat protein -split APEX2)
SDS-PAGE of purified proteins (PP7 coat protein-split APEX2)
(11)MS2/PP7-NanoBiT
Design
The system is a luminescent reporter system utilizing split luciferase. The MCP-SmBiT and PCP-LgBiT bind to the RNA scaffold transcribed by the Input system, leading to the reconstitution of NanoBiT, which emits light due to luciferase activity. The transcription amount is reported as luminescence intensity.
Main Achievements
- Successfully constructed the plasmids.
- Successfully purified the proteins.
- Confirmed that luminescence intensity varies with RNA concentration.
View details
Plasmid Construction
The plasmids used in this experiment (MCP-SmBiT and PCP-LgBiT) were constructed using XE cocktail assembly, and their generation was confirmed by Sanger sequencing.
Protein Purification
As shown in Figure 25, SDS-PAGE bands were observed following mass culture and purification.
SDS-PAGE of purified PP7 coat protein - LgBiT.
SDS-PAGE of purified MS2 coat protein - SmBIT
Proof of concept with MS2/PP7-NanoBit
We confirmed whether the recombination of MCP-SmBiT and PCP-LgBiT changes the luminescence intensity based on RNA concentration.
The change in luminescence intensity when varying the concentration of the scaffold RNA in a solution containing 1 nM of MCP-SmBiT and 1 nM of PCP-LgBiT, respectively.
The decrease in luminescence intensity when the RNA concentration was higher compared to the protein concentration is believed to be due to the binding of proteins to different RNA scaffolds, which inhibited the reconstitution of NanoBiT.
(12)Cas-Nanolock
Design
The system is a luminescent reporter system utilizing split luciferase. HiBiTm is fused to the NanoBiT in a configuration that allows for constitutive reconstitution, creating a barrier to reconstitution via truncated csx30. The Cas7-11-csx29 activated by the RNA (target RNA) transcribed from the Input system cleaves the truncated csx30, removing HiBiTm, which allows NanoBiT to reconstitute and emit light due to luciferase activity. The transcription amount is reported as luminescence intensity.
Main Achievements
- Successfully constructed the plasmids.
- Successfully purified the proteins.
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Plasmid Construction
The plasmid used in this experiment (Cas-Nanolock) was constructed using XE cocktail assembly, and its generation was confirmed by Sanger sequencing.
Protein Purification
As shown in Figure27, SDS-PAGE bands were observed following mass culture and purification.
SDS-PAGE of purified Nanolock.
Summary
In this experiment, we obtained the aforementioned results. (For more detailed experimental conditions and certain omitted milestones in progress, please refer to the Engineering and Note sections.) The results indicate that we successfully reproduced elements of the original ROSALIND platform in the MITSUNARI platform. Additionally, we either demonstrated the feasibility of implementing some extended features or made progress towards their implementation, achieving significant milestones that validate the feasibility and functionality of the platform.
For future experiments, we plan to conduct repeated experiments, optimization, and characterization of the components of the MITSUNARI system, which are currently under implementation. This will enable us to verify its functionality and prepare it for practical application as a biosensing platform. Moreover, to realize the project's goal of sensing nitrate and ammonium ions, we will address the aforementioned missing validations and conduct additional experiments and optimizations to demonstrate the system's functionality.
Furthermore, several additional steps are required for the system's practical application in the field. These steps include encapsulating the system into a cartridge for long-term storage while maintaining activity, measuring and mitigating the effects of components present in field samples (extraction solutions from various soils) on the system, constructing a calibration system to correct for measurement variations caused by different environmental factors in the field, and integrating the system with hardware devices used in agricultural settings. Although we had planned to address these aspects during this project, they were deferred due to the incomplete construction of MITSUNARI. Completing these validations and countermeasures through additional experiments to ultimately develop a system applicable to field conditions is part of our future plan.