Overview

We designed a genetic circuit system with the aim of sensing NO₃⁻ and NH₄⁺. Furthermore, with a view to future applications for sensing other substances, enhancing agricultural management, and contributing to various other synthetic biology projects, our genetic circuit system has been constructed as a versatile system, with everything except for the initial Input stage serving as a sensing platform named MITSUNARI.

This system is designed to be practical, rapid, cost-effective, and simple, and is composed of a transcription-only cell-free system that significantly expands upon the ROSALIND sensing system[1]. While whole-cell sensors can be equipped with precise functions, their societal implementation is challenging due to regulations such as the Cartagena Protocol on Biosafety. Therefore, we chose to use a cell-free sensor. The reason for using a sensor based solely on transcription, without involving the translation system, is that cell-free translation systems are costly and complex, making them potentially difficult to control. In this platform, all reactions of several types of sensor proteins that receive the sensing targets (in this case, NO₃⁻ and NH₄⁺) are converted into T7RNAP transcription and output to several selectable reporters. The concentration of the sensing targets is indicated by the speed and intensity of the reporting.

Overview

Input consists of sensor genes that, in response to NH₄⁺ and NO₃⁻, respectively, undergo reactions such as binding to DNA, dissociating from DNA, RNA binding, and structural changes. Through several mechanisms, all of these reactions are converted into T7RNAP transcription. While a single Input circuit is practically sufficient for both NH₄⁺ and NO₃⁻, we tested three circuits for each to select the most high-performing one from among the candidate genes.

Transcription is the core of this platform. The responses from Input system are all integrated at this stage and connected to multiple selectable reporters. This is made possible by a well-characterized and robust transcription reaction.

Output consists of multiple groups of reporters that vary intensity depending on the amount of transcribed RNA and the transcription speed. These include fluorescence, luminescence, and colorimetric. Each reporter has its own advantages, such as robust reactions, not requiring excitation light, high sensitivity, being observable with the naked eye, and the ability to simultaneously halt the transcription reaction. This platform allows of the free selection of Output format that best suits the intended purpose.

Additionally, as an option, extra efforts have been made to expand the applicability of MITSUNARI.

The designs of each system are summarized below.

Circuit overview provides a summary of the general design and function of each system. Background covers the origins and principles of the genes and chemical reactions used in each system.

Circuit design describes the behavior of the actual designed systems. Finally, Initial Plan outlines how we plan to construct, characterize, and utilize each system in this project.

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NH4+ Input circuits

(1)GlnA/GlnR - T7 Two hybrid system

Circuit overview
This is an extension of E. coli two-hybrid system. GlnR binds to DNA in an NH₄⁺-dependent manner and activates transcription by guiding T7RNAP to the T7 promoter d1 via the leucine zipper. The NH₄⁺ concentration is converted into an increase in transcription rate, allowing the sensing of NH₄⁺. Background

The GlnA/GlnR system is an NH₄⁺-dependent transcription repression mechanism in Bacillus subtilis[2]. GlnA is a glutamine synthetase that synthesizes glutamine from NH₄⁺, glutamate, and ATP. It also binds with the product glutamine to form the GlnA-FBI complex. GlnR is a repressor but cannot bind to DNA or function on its own. Its binding to DNA is stabilized by the GlnA-FBI complex, and it gains repressor activity by binding as a dimer to specific DNA sequences. Additionally, a constitutive DNA-binding mutant (GlnRN95) has been reported[3].

Leucinezipper-AN3.5 and Leucinezipper-BN3.5 are de novo designed short peptide sequences that bind to each other with a dissociation constant of 10^-9 nM[4]. They are primarily expressed in a form fused to arbitrary proteins A and B, and are used to facilitate a weak interaction between A and B.

T7 Two-hybrid System is a system that mimics the well-known transcription ON/OFF switching system, the two-hybrid system used in yeast, by employing T7RNA polymerase/T7Promoter commonly used in E. coli[5]. The T7 promoter is a constitutive expression promoter, but its variant with five nucleotides removed from the 5' end (T7 Promoter d1) has reduced affinity for T7RNAP, resulting in significantly decreased expression. However, by linking T7RNAP with any DNA-binding protein via a leucine zipper and guiding T7RNAP to the vicinity of the T7 Promoter d1 through the activity of the DNA-binding protein, transcriptional activity is greatly enhanced. This system is used to measure the activity of arbitrary DNA-binding proteins.

In this case, we designed a circuit that activates transcription in an NH₄⁺-dependent manner using Two-hybrid system by applying GlnA-FBI and GlnR to arbitrary DNA-binding proteins.

In previous studies, purification and in vitro testing of the DNA-binding activity of GlnA and GlnR were conducted. As for T7 Two-hybrid System, there is an example where Zincfinger268 was used as a DNA-binding protein in vivo.

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 NH₄⁺, GlnR-LzA does not bind to template, so transcription is not activated. As the NH₄⁺ 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 NH₄⁺-dependent manner, and the NH₄⁺ 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 LzA and GlnA-LzA (which, theoretically, can recruit T7RNAP-LzB just like GlnR-LzA). The most optimal one will be tested through an assay.

• All proteins to be used will be expressed and purified for the in vitro assay.
• Test the DNA binding of GlnR. The binding of the constitutive DNA-binding mutant of GlnR to DNA, the glutamine-dependent DNA binding of GlnA/GlnR, and the NH₄⁺-dependent DNA binding of GlnA/GlnR will be tested, in that order. The reaction conditions and optimization of GlnR binding sequence will also be performed.
• Test T7 Two-hybrid system in vitro.T7 As a positive control, Zincfinger268-LzA, which has been demonstrated to function in previous literature, will be used in place of GlnR-LzA for reproducibility experiments and optimization of reaction conditions in vitro. We will also attempt to optimize T7 Promoter d1 sequence in vitro.
• Test the system. The entire system will be tested to check transcriptional activation using GlnR positive control, glutamine-dependent transcriptional activation, and NH₄⁺-dependent transcriptional activation. Optimization of reaction conditions and selection of GlnR-LzA variants will be conducted. Ultimately, a system that most dramatically activates transcription in an NH₄⁺-dependent manner will be established.

(2)GlnA/GlnR - Repressor system

Circuit overview
This system utilizes the original repressor activity of GlnR. GlnR binds to DNA in an NH₄⁺-dependent manner and suppresses T7 transcription. The NH₄⁺ concentration is converted into a decrease in transcription rate, then NH₄⁺ is sensed. Background For GlnA/GlnR system, please refer to Background of the previous item (1) GlnA/GlnR - T7 Two-hybrid system. In this case, we will directly utilize the original NH₄⁺ concentration-dependent repressor activity of GlnA/GlnR system. In previous studies, purification and in vitro testing of the DNA-binding activity of GlnA and GlnR were conducted.

The system contains GlnA, GlnR, T7 RNA Polymerase (hereafter referred to as T7RNAP), a transcription template DNA of the T7Promoter-GlnR binding sequence-Output sequence (hereafter referred to as template), Glu, and ATP. In the absence of NH₄⁺, GlnR does not bind to template, so transcription occurs constitutively. As the NH₄⁺ concentration increases, GlnR binds to template in proportion to the concentration, and transcription is suppressed by its repressor activity. As a result, transcription is inhibited in an NH₄⁺-dependent manner, and the NH₄⁺ concentration is converted into the degree of transcriptional repression (transcription rate or transcription amount).

• All proteins to be used will be expressed and purified for the in vitro assay.
• Measure the DNA-binding/repressor activity of GlnR. (1)The activity assay, characterization, and optimization in the GlnA/GlnR - T7 Two-hybrid system will be conducted in parallel. Additionally, changes in transcription amount and transcription rate in response to varying ammonium concentrations will be recorded to establish a single sensing system. Ultimately, we aim to establish a system that most dramatically suppresses transcription in an ammonium-dependent manner.

(3)GlnA/TnrA - Activator system

Circuit overview
This system utilizes the original activator activity of TnrA. TnrA binds to DNA in an NH₄⁺-dependent manner and activates T7 transcription. The NH₄⁺ concentration is converted into an increase in transcription rate, then NH₄⁺ is sensed. Background

GlnA/TnrA system is an NH₄⁺-dependent transcription activation mechanism in B. subtilis[6]. GlnA is the same as the one used in the GlnA/GlnR system. Please refer to Background of (1) GlnA/GlnR - T7 Two-hybrid system. TnrA naturally forms a dimer and binds to specific DNA sequences, acting as a repressor. In the presence of NH₄⁺, it binds to the GlnA-FBI complex, dissociates from DNA, and exhibits activator activity that turns transcription on. In this case, we will directly utilize the original NH₄⁺ concentration-dependent activator activity of GlnA/TnrA system. In previous studies, purification and in vitro testing of the DNA-binding activity of GlnA and TnrA were conducted.

The system contains GlnA, TnrA, T7 RNA Polymerase (hereafter referred to as T7RNAP), a transcription template DNA of the T7 Promoter-TnrA binding sequence-Output sequence (hereafter referred to as template), Glu, and ATP. In the absence of NH₄⁺, TnrA binds to template, which constitutively suppresses transcription. As the NH₄⁺ concentration increases, TnrA dissociates from DNA in proportion to the concentration, lifting the repression and activating transcription. As a result, transcription is activated in an NH₄⁺-dependent manner, and the NH₄⁺ concentration is converted into the degree of transcriptional activation (transcription rate or transcription amount).

• All proteins to be used will be expressed and purified for the in vitro assay.
• Measure the DNA dissociation/activator activity of TnrA. The tests will be conducted in the following order: glutamine-dependent dissociation of GlnA/TnrA from DNA, and NH₄⁺-dependent dissociation of GlnA/TnrA from DNA. This will be performed using a pull-down assay or activator activity measurement. Optimization of reaction conditions will also be conducted. Additionally, changes in transcription amount and transcription rate in response to varying NH₄⁺ concentrations will be recorded to establish a single sensing system. Ultimately, we aim to establish a system that most dramatically suppresses transcription in an NH₄⁺-dependent manner.

NO3- Input circuits

(4)NasR - Split T7RNAP system

Circuit overview
This system utilizes the NO₃⁻-dependent RNA binding activity of NasR. In the presence of NO₃⁻, NasR and MS2 bind adjacently to RNA, and the fused Split T7 RNAPs reassociate, resulting in transcription activation. The NO₃⁻ concentration is converted into an increase in transcription rate or amount, then NO₃⁻ is sensed. Background

NasR is an RNA-binding protein derived from Klebsiella oxytoca. It has a characteristic of binding to a specific tandem hairpin sequence in RNA in a NO₃⁻-dependent manner and functions as an anti-transcription terminator in vivo. Several mutants that constitutively bind to RNA have also been reported[7][8]. Split T7 RNA Polymerase System consists of two fragments of artificially split T7 RNA Polymerase (Split NT7 and Split CT7). Each fragment is inactive on its own,but when the two fragments are brought into close proximity through external factors such as protein-protein interactions, they reassociate and regain transcriptional activity, thus activating transcription. Split NT7 used in this study was modified through directed evolution in previous research to make spontaneous reassociation more difficult[9]. MS2 coat protein (hereafter referred to as MCP) is a capsid protein from MS2 bacteriophage and constitutively binds to a specific hairpin structure in RNA[10]. In this system, a mechanism using the RNA-binding activities of NasR and MCP to induce the reassociation of Split T7 was designed. In previous studies, NasR and MCP have been successfully purified, and MCP has been used in an in vitro reconstitution system of a different split protein (NanoBiT). There is also an example of Split T7 RNA Polymerase System using Leucinezipper as the external factor for binding in vivo.

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 NO₃⁻, NasR-NT7 remains unbound, so it does not cause Split T7 reassociation, keeping transcription off. As the NO₃⁻ 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 NO₃⁻-dependent manner, and the NO₃⁻ 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, Leucinezipper-AN3.5 and Leucinezipper-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.

• All proteins to be used will be expressed and purified for the in vitro assay.
• NasR activity test Test the NO₃⁻-dependent RNA-binding activity of NasR-NT7 (and NasR(R193A)-NT7).
• In vitro activity test of Split T7 system Perform an in vitro activity test of Split T7 system. Use NT7-LzA/CT7-LzB as the positive control and NT7/CT7 as the negative control, and confirm that only the positive control exhibits transcriptional activity.
• Testing the entire system Test the entire system and confirm that transcriptional activity increases in a NO₃⁻ concentration-dependent manner. Optimize reaction conditions, protein concentrations, and RNA scaffold sequences to establish a well-functioning sensing system. Ultimately, we aim to establish a system that most dramatically suppresses transcription in an NO₃⁻-dependent manner.

(5)NLP7 - Split T7RNAP system

Circuit overview
This system utilizes the NO₃⁻-dependent conformational change of NLP7. In response to NO₃⁻, NLP7 shifts to a more folded conformation, bringing Split T7 fragments fused at both ends into close proximity, resulting in their reassociation and transcription being turned on. The NO₃⁻ concentration is thus converted into an increase in transcription rate or amount, allowing NO₃⁻ sensing. Background

NLP7 is a transcriptional regulatory protein from Arabidopsis thaliana. In a previous study, when a gene with split mCitrine, a fluorescent protein, fused to both the N-terminus and C-terminus of NLP7 was expressed in plant cells, it was reported to function as a NO₃⁻-responsive biosensor[11]. This is because, as the NO₃⁻ concentration increases, NO₃⁻ binds to NLP7, causing a significant conformational change that brings the N-terminus and C-terminus into a conformation suitable for the reconstitution of the split protein.

Details on Split T7 system can be found in Background of item (4) NasR - Split T7RNAP system.

Inspired by previous studies, we hypothesized that split T7RNAP could also be reconstituted in response to NO₃⁻ concentration, and we designed the system accordingly. In previous studies, there have been examples of purification of NLP7 and NanoBiT in isolation. NLP7-mCitrine has been shown to function as a NO₃⁻ sensor within living plant systems.

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 NO₃⁻, the conformation of NT7-NLP7-CT7 keeps Split T7 fragments at both ends separated, preventing their reassociation, and thus transcription remains off. As the NO₃⁻ 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 NO₃⁻-dependent manner, and the NO₃⁻ concentration is converted into the degree of transcriptional activation (transcription rate or transcription amount).

• All proteins to be used will be expressed and purified for the in vitro assay.
• Testing the entire system Test the entire system and confirm that transcriptional activity increases in a NO₃⁻ concentration-dependent manner. Optimize reaction conditions and protein concentrations to establish a well-functioning sensing system. Ultimately, we aim to establish a system that most dramatically suppresses transcription in an NO₃⁻-dependent manner.

(6)NLP7 - Directory Reporting system

Circuit overview
This system utilizes the NO₃⁻-dependent conformational change of NLP7. In response to NO₃⁻, 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 NO₃⁻ concentration is reported as fluorescence intensity or luminescence intensity, allowing NO₃⁻ sensing. In this project, this system is an exception, as it reports directly without involving transcription. Although it is not included in MITSUNARI platform, it was tested due to its potential as the simplest and most robust NO₃⁻-sensing system. Background

For information about NLP7, refer to Background of item (5) NLP7 - Split T7RNAP system mentioned earlier. mCitrine is a yellow-green fluorescent protein, which is a mutant variant of GFP derived from Aequorea victoria. Split-mCitrine consists of two fragments (Split NmCitrine and Split CmCitrine) that have been artificially split. Each fragment is non-functional on its own, but when brought into close proximity by external factors such as protein-protein interactions, they reassociate and emit fluorescence[12]. NanoBiT is a split luciferase developed by Promega for measuring protein-protein interactions and consists of two fragments, LgBiT and SmBiT[13]. Each fragment is inactive on its own, but when brought into close proximity by external factors such as protein-protein interactions, they reassociate and emit luminescence through luciferase activity. The dissociation constant between LgBiT and SmBiT is relatively high at 190 μM, which reduces spontaneous reconstitution in solution[13]. Inspired by previous studies, we hypothesized that split T7RNAP could also be reconstituted in response to NO₃⁻ concentration and designed the system accordingly. In previous studies, there have been examples of purification conducted individually for NLP7, mCitrine, and NanoBiT. Moreover, as mentioned earlier, Split NmCitrine-NLP7-Split CmCitrine fusion protein has been shown to function as a NO₃⁻ reporting system within plant cells.

Fluorescent Reporting: The system contains a Split NmCitrine-NLP7-Split CmCitrine fusion protein (hereinafter referred to as NmCitrine-NLP7-CmCitrine). In the absence of NO₃⁻, the conformation of NmCitrine-NLP7-CmCitrine keeps the two ends of the Split mCitrine apart, preventing their reassembly and resulting in no fluorescence. As the NO₃⁻ concentration increases, NmCitrine-NLP7-CmCitrine undergoes a conformational change that brings the two ends of the Split mCitrine into proximity, allowing them to reassemble and emit fluorescence. This results in a NO₃⁻-dependent enhancement of fluorescence, where the NO₃⁻ concentration is converted into fluorescence intensity.

Luminescence Reporting: The system contains SmBiT-NLP7-LgBiT fusion protein (hereinafter referred to as SmBiT-NLP7-LgBiT). In the absence of NO₃⁻, the conformation of SmBiT-NLP7-LgBiT keeps the NanoBiT fragments at either end apart, preventing reassembly and resulting in no luciferase activity. As the NO₃⁻ concentration increases, the SmBiT-NLP7-LgBiT undergoes a conformational change, bringing the NanoBiT fragments closer together for reassembly, which restores luciferase activity and produces luminescence. This process enhances luminescence in a NO₃⁻-dependent manner, converting the NO₃⁻ concentration into luminescence intensity.

• Express and purify all proteins to use for in vitro assays.
• Test the entire system The entire system will be tested to confirm that fluorescence and luminescence increase in a NO₃⁻ concentration-dependent manner. Optimizations of reaction conditions and protein concentrations will be performed to refine the sensing system. Ultimately, the goal is to establish a system that produces the most dramatic fluorescence and luminescence in response to NO₃⁻.

Transcription circuit

(7)T7RNAP transcription

Circuit overview
This is an in vitro transcription system that utilizes T7 RNA Polymerase (including T7 Two-hybrid System and Split T7 System). It is a core component of our sensing platform, MITSUNARI, where the concentration of the target substance is converted into transcription speed and quantity, which is then reported through selectable outputs. While the actual transcription step is included on the input side, this section focuses on characterizing the transcription by T7 RNA Polymerase in advance. Background T7 RNA Polymerase is an RNA Polymerase derived from the T7 phage. It possesses RNA polymerase activity on its own, recognizes the T7 Promoter with high specificity, and performs transcription constitutively. It is produced in high quantities. Due to these characteristics, it is one of the representative RNA polymerases used for in vitro transcription[14]. In prior research, T7 RNA Polymerase has been purified for use in in vitro transcription systems such as Pure-system, with various characterization and optimization of reaction conditions reported[15]. Additionally, several methods have been reported that utilize qPCR equipment and fluorescent RNA aptamers to measure transcription activity in real-time, quantifying transcription levels as fluorescence from the aptamers[15]. Furthermore, commercial kits are available for performing transcription reactions using T7 RNA Polymerase in vitro.

Circuit 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.

• Express and purify all proteins to use for in vitro assays.
• Conduct activity tests, necessary optimizations, and characterization assessments. Test for normal transcription activity through PAGE analysis of the transcription products.

The assay for measuring transcription activity will involve transcribing a fluorescent RNA aptamer as the transcription product and measuring its fluorescence intensity in real-time using a qPCR machine. This method will measure baseline transcription activity under normal conditions, and optimizations and calibrations of reaction conditions and protein/template concentrations will be conducted with reference to previous literature. Additionally, since it is challenging to perform a complete end-to-end assay for all combinations of input and output within this project, the RNA absolute quantity will be obtained from the input assay after a set period post-reaction initiation. This will be correlated with the reporting intensity from the output assay based on the initial RNA amount added. To achieve this, the observed fluorescence intensity of the aptamer will be matched with the actual absolute quantity of transcribed RNA using a Qubit RNA quantification kit. Ultimately, the necessary characterization for the overall system assay will be completed, establishing the most efficient conditions for transcription initiation.

Output circuit

(8)Fluorescent RNA Aptamer

Circuit overview
The fluorescent RNA aptamer is used in a fluorescent reporter system. The transcribed RNA autonomously adopts a higher-order structure based on its sequence, forming an aptamer that binds to a specific fluorescent dye, resulting in enhanced fluorescence. The transcriptional quantity is reported as fluorescent intensity. Background

The Broccoli RNA aptamer is an artificially designed RNA aptamer that autonomously adopts a hairpin structure and binds to the fluorescent dye DFHBI-1t, significantly enhancing its fluorescence[16]. While there are many types of fluorescent RNA aptamers, the combination of DFHBI-1t and Broccoli is known for its high fluorescence intensity and low background, making it a high-performance fluorescent RNA aptamer. In this study, we adopt the RNA that emits fluorescence directly as a reporter, anticipating that it will be inexpensive, robust, and provide sufficient intensityPrevious research has introduced a system called "ROSALIND," which employs fluorescent aptamers as reporters in an in vitro transcription system where transcriptional activity varies with the concentration of the sensing target. This platform is considered a prototype of MITSUNARI. Additionally, there have been prior studies using this Broccoli aptamer in ROSALIND for sensing substances in environmental waters, such as rivers. There are also papers that utilize the fluorescence of the Broccoli aptamer for in vitro transcription activity tests measured with qPCR machines.

Circuit design
When the RNA of the Broccoli sequence is transcribed in response to the input, it undergoes structural folding and binds to DFHBI-1t, emitting green fluorescence. This allows the transcription quantity to be converted and reported as fluorescent intensity.

Initial Plan
No specific proteins are present in this system. Characterization and optimization of reaction conditions were performed in the previous section (7) with T7RNAP transcription, and it is used as a reporter during the assay of Input system. It can be directly employed as a NO₃⁻ and NH₄⁺ sensor. Ultimately, it is intended to be completed as one of the options for Output system that reports using the simplest and highest-intensity fluorescence.

(9)Cas7-11 - Chromoprotein system

Circuit overview
The system utilizes the RNA-dependent protease activity of Cas7-11-csx29 and a colorimetric reporter system using chromoproteins. The CBM9.2-truncated csx30-Spycatcher003r-Spytag003-chromoprotein module exists as a solid-phase precipitate bound to cellulose aggregates. In response to Input, Cas7-11-csx29 cleaves the truncated csx30 within the module, releasing the fragment containing the chromoprotein from the precipitate, which colors the supernatant. The transcription quantity is reported as the color intensity of the supernatant. Background

Cas7-11, csx29, and csx30 are all proteins that constitute the Cas7-11 system from Desulfonema ishimotonii. Cas7-11 and csx29 exist in a complex with guide RNA, and when target RNA binds, Cas7-11 cleaves the target RNA while the activated csx29 cleaves csx30 at the 429th amino acid. The truncated csx30 consists of only the sequence from amino acids 396 to 565, and like the full-length csx30, it is subject to cleavage by csx29[17]. The chromoproteins tsPurple, gfasPurple, asPink, aeBlue, amajLime, and cjBlue are six colors from a 14-color GFP homolog family chromoprotein color palette designed for expression in E. coli[18]. CBM9.2 is a cellulose-binding module from xylanase 10A of Thermotoga maritima. It binds to cellulose crystals and precipitates as a solid phase along with the cellulose crystals[19].

Spy system is a technique that uses Spytag and Spycatcher to form covalent bonds between proteins. When one protein contains the Spytag sequence and the other contains the Spycatcher sequence, mixing the two proteins allows for the formation of a covalent bond between Spytag and Spycatcher, linking the proteins together. Spytag003 and Spycatcher003 are improved variants of the Spytag-Spycatcher system developed in previous research[20]. In this study, we designed a reporting system that combines these components to "reveal" the chromoprotein, relying on its clear visual reporting capability. Previous research has reported Cas7-11 system, CBM9.2, and the Spy system function in vitro after purification. There are also reports of the purification of chromoproteins. Additionally, there are examples of constructing similar systems that do not use Cas system or Spy system and are not RNA-dependent, utilizing TEV protease instead[21].

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.

• Express and purify all proteins to use for in vitro assays.
• Confirm the activity of Cas7-11 system, Spy system, and CBM9.2 in reproducibility experiments.
• Conduct tests on the entire system. Optimize reaction conditions and refine it as a reporter system. Ultimately, the goal is to complete it as a highly versatile reporting system that enables clear reporting observable by eye.

(10)MCP/PCP-Split APEX2 system

Circuit overview
This is a colorimetric reporter system that utilizes the peroxidase activity of APEX. When the RNA scaffold transcribed from Input system is present, MCP-AP and PCP-EX bind together, reconstituting the split APEX and triggering a color reaction through its peroxidase activity. The transcription quantity is reported as color intensity.

Background

APEX2 is a mutant of soybean ascorbate peroxidase that oxidizes various substrates. APEX2 can be used as a visual reporter by oxidizing molecules that produce fluorescence or pigments. In previous research, a Split-APEX2 that is reconstituted only in the presence of protein-protein interactions was developed using directed evolution methods[22]. MS2 coat protein (MCP) and PP7 coat protein (PCP) are the coat proteins of the MS2 bacteriophage and PP7 bacteriophage, respectively, and they constitutively bind to specific hairpin structures of RNA[10][23]. The current system utilizes the RNA-binding activity of MCP and PCP to reassemble Split APEX for colorimetric reporting. The addition of hydrogen peroxide during the reaction allows for precise control of the reaction time by stopping the transcription reaction. Previous research has demonstrated the functionality of a nearly identical system in vivo. There have also been instances where MCP and PP7 were purified and used in an in vitro system for reconstituting similar split proteins (NanoBiT).

The system contains MCP-Split N-APEX2 fusion protein (hereafter referred to as MCP-AP) and PCP-Split C-APEX2 fusion protein (hereafter referred to as PCP-EX). When the RNA scaffold transcribed by Input system is present, MCP-AP and PCP-EX bind together and come into close proximity, allowing Split APEX2 to reconstitute. After a set period of transcription, hydrogen peroxide and substrate are added (the transcription reaction is halted by the hydrogen peroxide). The already reconstituted Split APEX2 then oxidizes the substrate into a colorimetric product, resulting in a color change. This allows the transcription quantity to be converted into color intensity, which is reported.

• Express and purify all proteins to use for in vitro assays.
• Test the entire system. Optimize reaction conditions and other parameters. Ultimately, the goal is to complete it as a colorimetric reporting system that allows for precise control of reaction time.

(11)MCP/PCP-NanoBiT

Circuit overview
This is a luminescent reporter system that utilizes split luciferase. When MCP-SmBiT and PCP-LgBiT bind to RNA scaffold transcribed by the Input system, NanoBiT is reconstituted, resulting in luminescence due to luciferase activity. The transcription quantity is reported as luminescent intensity. Background

NanoBiT is Split luciferase mentioned in section (6) NLP7 - Directory Reporting system. MCP and PCP are RNA-binding proteins described in section (10) Split APEX2 system. In this system, the RNA-binding activities of MCP and PP7 are utilized to reconstitute NanoBiT. This provides an advantage as it allows for luminescent reporting without the need for light irradiation or observation at specific wavelengths. Previous research has demonstrated the functionality of the same system in vitro. This system serves as a reproducibility experiment for that work[24].

The system contains MCP-SmBiT fusion protein (hereinafter referred to as MCP-SmBiT) and the PCP-LgBiT fusion protein (hereinafter referred to as PCP-LgBiT). When the RNA scaffold transcribed by Input system is present, MCP-SmBiT and PCP-LgBiT bind together and come into close proximity, reconstituting NanoBiT. After a set period of transcription, the addition of luciferin (flucoumarin) triggers luminescence from the already reconstituted NanoBiT. This allows the transcription quantity to be converted into luminescent intensity, which is reported.

• Express and purify all proteins to use for in vitro assays.
• Test the entire system. Optimize reaction conditions and other parameters. Ultimately, the goal is to complete the system so that it reports luminescence in the clearest manner according to the transcription quantity.

(12)Cas-Nanolock system

Circuit overview
This is a luminescent reporter system that utilizes Split luciferase. HiBiTm is fused to NanoBiT through truncated csx30 in a configuration that allows for constitutive reassembly. Cas7-11-csx29, activated by RNA (targetRNA) transcribed from Input system, cleaves truncated csx30, removing HiBiTm and allowing NanoBiT to reconstitute, resulting in luminescence due to luciferase activity. The transcription quantity is reported as luminescent intensity.

Background

Cas7-11, csx29, and truncated csx30 are part of target RNA-dependent protein cleavage system mentioned in section (9) Cas7-11 - Chromoprotein system. NanoBiT is a Split luciferase composed of two fragments, SmBiT and LgBiT, as described in section (6) NLP7 - Directory Reporting system. HiBiT system is a Split luciferase consisting of HiBiT and LgBiT, developed by Promega[25]. HiBiT system has the same origin and the LgBiT module as NanoBiT, but unlike NanoBiT, it has the property of constitutively reconstituting itself. Therefore, when SmBiT and HiBiT are present simultaneously, HiBiT preferentially binds to LgBiT. HiBiTm is a variant of HiBiT that constitutively binds to LgBiT, but the reconstituted HiBiTm-LgBiT does not exhibit luciferase activity. Consequently, it functions as a competitive inhibitor of HiBiT and SmBiT.

In this system, the principle of the (9) Cas7-11 - Chromoprotein system is extended to luminescent reporting by utilizing the release of inhibition by HiBiTm, allowing for the reconstitution of luciferase. This provides an advantage as it enables luminescent reporting without the need for light irradiation or observation at specific wavelengths. Previous research has constructed a similar system called "Nanolock" in vitro, which uses TEV protease instead of truncated csx30 and is not RNA-dependent[26].

Circuit design
The system contains Cas7-11-csx29-guideRNA complex (hereafter referred to as the Cas complex) and SmBiT-LgBiT-truncated csx30-HiBiTm complex (hereafter referred to as Cas-Nanolock). Normally, SmBiT and HiBiTm are in close proximity to LgBiT due to their fusion, which allows HiBiTm to preferentially bind to LgBiT, resulting in a lack of luciferase activity. When the RNA (targetRNA) transcribed from Input system is present, the activated csx29 from the Cas complex cleaves truncated csx30, separating HiBiTm from LgBiT. With the linker that kept HiBiTm and LgBiT close together cleaved, the fused SmBiT can now preferentially bind to LgBiT, reconstituting NanoBiT. After a set period of transcription, the addition of luciferin (flucoumarin) triggers luminescence from the already reconstituted NanoBiT. This allows the transcription quantity to be converted into luminescent intensity, which is reported.

• Express and purify all proteins to use for in vitro assays.
• The in vitro activity tests of Cas system will refer to the results from the (9) Cas7-11 - Chromoprotein system.
• Test the entire system, and optimization of the reaction conditions will be performed. Ultimately, the goal is to complete the system so that it reports luminescence in the clearest manner according to the transcription quantity.

Optional

(13)閾値の系

Circuit overview
The threshold system is designed to enable the observation of target concentration, which is represented analogously by the intensity of the reporter, as a digital signal. This allows for results to be read visually or with simple optical sensors. It operates by being added between transcription and output and can be applied simplyby modifying the sequence of the RNA output from the existing Input system. Background There are precedents in prior research where a similar system was constructed in vitro, and this system represents a replication of that work[27]. It is introduced to expand the applications that can utilize the MITSUNARI platform.

The system contains two types of incomplete transcription templates, as well as short oligo DNA sequences known as Blocker and Activator. The Blocker is present in excess relative to both the templates and the Activators, allowing the threshold value to be adjusted based on the amount of Blocker. Each transcription template encodes a sequence consisting of a Blocker complementary sequence T7 promoter-CoActivator sequence, as well as a Blocker complementary sequence T7 promoter-Output sequence. The region from the 5' end to the middle part of the T7 promoter is single-stranded. Due to the incompleteness of the T7 promoter, T7 RNA polymerase cannot transcribe this transcription template. The Blocker has a complementary sequence to the Blocker part of the template and includes a blocking arm of a certain length at the 5' end. The Activator is complementary to the single-stranded portion of the T7 promoter in the template. When the Activator binds to the missing part of the T7 promoter, it completes the T7 promoter, allowing T7 RNA polymerase to transcribe the template. However, under normal conditions, the longer homologous region of the Blocker binds strongly to the template while the blocking arm covers the missing part of the T7 promoter, inhibiting the annealing of the Activator, thus preventing transcription from occurring. CoActivator is an RNA that is not initially present in the system. It is fully complementary with the Blocker and the blocking arm portion. The Output RNA is an RNA sequence required by any Output system and is typically transcribed by Input system. When applying the threshold system, Input system transcribes the CoActivator instead of the Output RNA. Once the transcribed RNA (CoActivator) enters the system by Input system, the free Blocker begins to preferentially bind to it. At this stage, Output system does not emit any reports. The amount of free Blocker functions as the "threshold" in this system. When the amount of CoActivator transcribed by Input exceeds this threshold (the amount of free Blocker), the Blocker bound to the template starts to release and bind to the longer complementary sequence of CoActivator. This release lifts the blocking effect, allowing the Activator to bind to the template, enabling T7 RNA Polymerase to begin transcribing the template. One of the two templates transcribes the CoActivator, creating a positive feedback loop where the transcribed CoActivator alleviates the blocking of another template, leading to an explosive increase in CoActivator transcription that releases all template blockages. Simultaneously, the other template also experiences a complete release from blocking, starting to transcribe Output RNA at maximum efficiency. This results in a rapid influx of RNA into Output system, generating a swift and intense reporting signal. This mechanism allows for digital signal-like reporting.

The threshold can be adjusted by varying the amount of Blocker, allowing for the preparation of multiple sensors with different Blocker quantities. By assaying them simultaneously, it is possible to measure the target concentration across a certain range.

Initial Plan
There are no specific proteins inherent to this system. Test the entire system, and optimize reaction conditions. Ultimately, it is planned to be developed as an optional module that can be easily applied to MITSUNARI.

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