Design

Throughout the project, T7 RNA Polymerase is purified to obtain sufficient yield and purity for use in in vitro transcription experiments and will be stored in a form suitable for preservation over several months. (The activity is verified in the cycle mentioned in the T7 RNA Polymerase section later.) The T7 RNA Polymerase are sourced from Addgene. Based on the expression and purification protocols from the IDT-contributed paper and the protocols used by iGEM Kyoto 2021, creating a standardized protocol applicable to other proteins are planned for expression and purification. Yield assessments are conducted using A280 measurements with a Nanodrop and the Lambert-Beer law. Purity assessments are planned to be performed using SDS-PAGE. If successful, it is expected that, based on the PI's and team members' experience, a yield of about 10 mg and a purity of approximately 90% can be achieved. The stocking method is planned to be reconsidered once the yield is confirmed.

Build

The plasmid Addgene124138 (pQE30-His-T7RNAP) [1] was used and transformed into BL21(DE3) E. coli. Expression and purification were carried out under the conditions described in the Experiment and Notebook, as per the design.

Test

According to the conditions described in the Experiment and Notebook, A280 measurements using a Nanodrop and total protein concentration measurements based on the Lambert-Beer law were conducted for each purification fraction. Subsequently, SDS-PAGE was performed on Fractions 1 and 2, which had relatively high concentrations, under the conditions described in the Experiment and Notebook, to assess purity.

Figure1

Picture of SDS gel for first purification. The band presumed to be T7 RNA Polymerase is enclosed by a red line.

As a result, the concentration obtained was approximately 2.6% of the expected yield, which was low. Regarding purity, although a band corresponding to the molecular weight of T7 RNA Polymerase was observed in the SDS-PAGE, the purity was estimated to be around 50% by visual inspection. All fractions were considered purification failures and were discarded.

Learn

The yield and purity were significantly below the target. Since the plasmid was a commercial product and the acquisition of the intended T7 RNA Polymerase itself was confirmed, it is believed that modifying some conditions for expression and purification could improve this purity and yield. Although the exact cause could not be directly determined from the results, potential causes included, as unchangeable factors, differences in the expression vector compared to the reference experiments, differences in the reagents and equipment used for purification, and inherent differences in the protein's properties, which might have contributed to the inevitable reduction in yield. Modifiable factors included insufficient culture size, incomplete sonication, insufficient bead quantity, and inadequate technique.

Design

Cycle 1 is repeated under improved conditions. Throughout the project, it was planned to purify T7 RNA Polymerase with sufficient yield and purity for use in in vitro transcription experiments, and to store it in a form suitable for preservation over several months. (The activity is verified in the cycle mentioned in the T7 RNA Polymerase section later.) It was decided to use T7 RNA Polymerase purchased from IDT. Based on the expression and purification protocols from the IDT-contributed paper, the protocols used by iGEM Kyoto 2021, and the learnings from Cycle 1, an improved standardized protocol applicable to other proteins was created for expression and purification. Compared to Cycle 1, the culture size was increased, sonication time was extended, the bead quantity relative to culture size was increased, and more skilled techniques were applied. Yield assessment was conducted using A280 measurements with a Nanodrop and the Lambert-Beer law, while purity assessment was performed using SDS-PAGE. If successful, it was expected to achieve a yield commonly obtained through protein purification using the AKTA Start purification system, which is typically around 10 mg, with a purity of approximately 90%. The stocking method was to be reconsidered once the yield was confirmed.

Build

The plasmid Addgene124138 (pQE30-His-T7RNAP) was used and transformed into BL21(DE3) E. coli. Expression and purification were carried out under the improved conditions from Cycle 1, as described in the Experiment and Notebook, according to the design.

Test

A280 measurements using a Nanodrop and total protein concentration measurements based on the Lambert-Beer law were conducted for each purification fraction under the conditions described in the Experiment and Notebook.

As a result, the concentration obtained was about 44% of the expected yield. It was determined that Fractions 2 and 3 needed to be concentrated based on their concentration. The Amicon ultrafiltration method, which members had experience with, was selected for concentration. Concentration and desalting were performed using Amicon-0.5 30K, and concentration measurements were conducted using the same procedure. Based on the concentration, 80% glycerol was added to achieve a final concentration of 30%, and the samples were aliquoted into 10 μl portions and stored at -80°C. Subsequently, an SDS-PAGE purity assessment was conducted under the conditions described in the Experiment and Notebook.

Figure2

Picture of SDS gel for purification on cycle 2. The band presumed to be T7 RNA Polymerase is enclosed by a red line.

Although a yield loss of about 50% was observed with this process, it was considered an acceptable value. Regarding purity, a band corresponding to the molecular weight of T7 RNA Polymerase was observed, and the purity was estimated to be around 50–60% by visual inspection.

Learn

Although the yield and purity were somewhat below expectations, the purification was considered successful since there was deemed to be a sufficient quantity for future assays. The causes for not meeting the initial expectations remained consistent with the assumptions stated in Cycle 1, and no specific cause was identified. However, considering the possibility of unchangeable factors and the fact that the purification was ultimately successful, this was deemed acceptable. Based on this procedure and results, a standardized protocol applicable to other proteins will be created. Regarding stocking, since it was confirmed that relatively low-concentration fractions could be handled without issues through Amicon concentration and desalting, this will continue to be adopted as a standard procedure. For storage at -80°C, because proteins are completely frozen, aliquoting is necessary to avoid refreezing, which makes it difficult to flexibly adjust the required amount for future assays and adds extra effort. Therefore, it is necessary to consider better methods.

Design

In the planned experiments, it was expected that the purification of over 30 different proteins would be necessary. It was considered undesirable to individually set or optimize expression and purification conditions for each protein based on respective references, given the limited number of available incubators and the effort involved. Therefore, we decided to use Cycle 2 as a reference and purify other proteins using standardized conditions with as much commonality as possible in culture conditions and buffers. Based on the conditions from Cycle 2 and relevant references, we attempted to standardize the culture conditions, composition of purification buffers, and reagents used. Additionally, we aimed to standardize the bead conditions and the imidazole concentration in the fractions.This approach was tested on proteins such as His-GlnA, His-GlnR, His-GlnRN79-(GGGGS)-LeucinzipperA3.5-(GGGGS)5-GlnRC40, His-GlnRN95-(GGGGS)-LeucinzipperA3.5, His-TnrA, His-GlnRN95, His-MCP-SmBiT, and His-PCP-LgBiT. If the purification is successful, it is expected that all proteins will achieve purity and concentration levels comparable to Cycle 2. While some failures were anticipated, it was expected that applying individual conditions only to the unsuccessful cases would save overall effort and time.

Build

The plasmids to be used were constructed. Inserts were designed to have pseudo Prefix-suffixes for XE-cocktail assembly mentioned in the Overview. These were either directly synthesized as Gblocks by IDT or constructed via PCR using sequences obtained from Gblocks and the Distribution kit, along with standard oligos from Eurofinsgenomics. The vector used was the pET11a vector purchased from Addgene, which was digested with restriction enzymes at various sites according to the affinity tags used. These components were assembled using XE-cocktail, transformed into DH5α, and plasmids were obtained through colony direct PCR, liquid culture, and Miniprep. The insert sequences were confirmed by Sanger sequencing through Azenta. Only plasmids with confirmed correct sequences were used in subsequent experiments. In later cycles, plasmids were constructed using the same procedure (in some cases, Gene Fragments synthesized by Twist were also used). Correctly constructed plasmids were transformed into BL21(DE3), and purification was attempted under the standard conditions described in the Experiment and Notebook. Standardization efforts included unified expression conditions (IPTG 0.1 mM, chilling on ice upon reaching OD 0.6, 16°C overnight), using Tris Buffer instead of phosphate buffer, unifying the pH of Tris-HCl used (pH 7.9), standardizing bead quantity (1 ml), and standardizing fraction collection volume (500 μl).

Test

For each purified fraction, concentration measurements were conducted using A280 and the Lambert-Beer law (when the extinction coefficient exceeded 10,000) or the Pierce 660 method (when proteins with an extinction coefficient of 10,000 or below were included in the measurements for that day). Additionally, SDS-PAGE was performed on fractions with relatively high concentrations to assess purity.

Figure3

SDS gels of proteins which are purified by standardized protocol.

For details on concentration, please refer to the Notebook. Refer to the above for SDS-PAGE results. Except for GlnA, no proteins reached sufficient concentrations, and in terms of purity, some target bands were faint or even absent, indicating a high overall failure rate in purification(Figure3). For proteins deemed to have been successfully purified, concentration was performed using Amicon-0.5 3K, and glycerol was added to reach a final concentration of 50%. These were stored at -20°C as stock solutions.

Learn

Possible causes for the purification failures included reduced cell yield due to cold shock, cultivation temperatures being too low, insufficient IPTG concentration, inappropriate salt concentration or pH, unsuitable buffer type, and excessive bead quantity relative to expression levels. For proteins that failed purification with this standard protocol, it was indicated that conditions tailored to each individual protein would be necessary. It was found that the standard protocol resulted in far lower yields and purity (i.e., purification failures) than anticipated, suggesting that applying conditions tailored to each protein from the start would ultimately lead to more efficient experimentation. Regarding stock preparation, there were no issues in the preparation process or storage, and unless negative impacts on activity are identified in subsequent assays, it is considered best to proceed with this method. It was decided that this stock preparation method will be adopted going forward.

Design

In Cycle 3, some proteins could not be recovered efficiently. Therefore, to recover the target proteins in higher quantities and purity, large-scale cultivation of E. coli and protein purification were conducted by referring to papers that described the purification methods for each protein. Additionally, based on previous cycles, the amount of Ni-NTA beads was adjusted. The purity and concentration of the purified proteins were measured using SDS-PAGE, Pierce 660 nm, and the Lambert–Beer law. If the purification proceeded as intended, it was expected that protein purity would increase, and higher-concentration protein stocks would be obtained.

Build

Plasmids confirmed to match the target sequence by Sanger sequencing were used and transformed into BL21(DE3) E. coli. According to the design, cultivation and purification were carried out using the protocol described in the Experiment and Notebook, which was based on the referenced papers.

Test

According to the conditions described in the Experiment and Notebook, A280 measurements using a Nanodrop and total protein concentration measurements based on the Lambert-Beer law were conducted for each purification fraction. Subsequently, SDS-PAGE was performed on fractions with relatively high concentrations under the conditions described in the Experiment and Notebook to assess purity. As a result, the purity and concentration exceeded those of the proteins purified in Cycle 3.

Learn

Through this cycle, it was found that the culture conditions of E. coli and the buffer conditions for purification have a significant impact on protein production and purification. It was learned that following the optimized conditions indicated in previous studies is the best strategy to reduce failures in protein purification.

Design

To use in future assays, it was necessary to confirm that T7 RNA Polymerase (T7 RNA Polymerase) is active and that transcription can be observed as the fluorescence of the RNA aptamer (Broccoli). Therefore, as a preliminary experiment, a plan was made to conduct a transcription reaction with the stock T7 RNA Polymerase and confirm clear fluorescence using Qubit. If the experiment is successful, a clear increase in green fluorescence is expected to be observed before the transcription reaction begins.

Build

The purchased Addgene124138 (pQE30-His-T7RNAP) was used as the plasmid, expressed in BL21(DE3), purified, and stored at -80°C. It was thawed for use. Details are described in the Protein Purification section. The fluorescent dye DFHBI-1T was purchased from Cayman Chemical Company, and the transcription template was prepared by annealing standard oligos from Eurofinsgenomics in TE Buffer.

Test

The reagents were prepared, and the transcription reaction was conducted under the conditions described in the Experiment and Notebook. Measurements were taken in green fluorescence mode on Qubit for the control (no enzyme) and at 0 minutes, 1 hour, and 19 hours after the transcription started. In the Notebook, each sample was measured three times to account for instrument measurement errors, but only the first measurement result is shown.

Result

Figure4

Fluorescence of Broccoli captured by the iPhone 14 camera on a transilluminator. The left side shows the blank, and the right side shows the transcribed sample.

A clear increase in green fluorescence was observed. Additionally, when photographed on the transilluminator, a sufficient increase in fluorescence was visibly confirmed, as shown in Figure 4.

Learn

It was confirmed that the transcription reaction could be clearly observed with the Broccoli aptamer using the prepared T7 RNA Polymerase stock, indicating that the T7 RNA Polymerase has transcriptional activity and that the Broccoli aptamer will be useful for confirming transcription reactions in future assays. The transcription reaction was clearly detectable within about one hour, which is consistent with the data from the reference paper[2] and can serve as a guide for determining transcription times in future transcription assays.

Design

To use in future assays, it is necessary to confirm that the transcription products from the T7 RNA Polymerase stock are not adversely affected by contamination such as RNase and that the transcription products are of sufficient quality. Additionally, it should be verified that the transcription reaction occurs at a sufficiently high level. Therefore, both the commercial T7 RNA Polymerase transcription kit (Megascript, Thermo) and the T7 RNA Polymerase stock will be used to transcribe the Broccoli Aptamer Template (69 bp in total length, 52 bp downstream from the T7 promoter, referred to as the 'Broccoli Template') and a template suitable for in vitro transcription (834 bp in total length, 603 bp between the T7 promoter and T7 terminator, a part of the piK006 His-GlnR plasmid, referred to as the '600 bp Template'). These will be analyzed together with a DNA oligo ladder using urea-denatured PAGE. If the T7 RNA Polymerase is unaffected by contamination and has sufficient activity, it is expected that a slightly fainter single band of the transcription product compared to the commercial product should be observed.

Build

The T7 RNA Polymerase used in the experiment was expressed by introducing the plasmid purchased from Addgene into E. coli. DFHBI-1T was purchased from Cayman Chemical Company, and the MEGAscript™ T7 Transcription Kit from Thermo Fisher was used as a positive control.

Test

A 2-hour transcription reaction was performed under the conditions described in the Experiment and Notebook, and the frozen samples were subjected to urea-denatured PAGE, followed by EtBr staining and imaging. Since an RNA ladder was not available, a 100 bp ladder from TOYOBO and a standard oligo from Eurofinsgenomics were used instead. In the first run, the gel quality was poor, resulting in smeared bands, so the gel was remade, and the sample amount and ladder combination were adjusted before rerunning the electrophoresis. Photos of both attempts are provided(Figure5 and Figure6). The electrophoresis and gel preparation were conducted under the conditions described in the Experiment and Notebook.

Figure5

Urea-PAGE for quality check of transcription product 1

Figure6

Urea-PAGE for quality check of transcription product 2

Bands were observed at positions that are likely correct. Although they were not perfect single bands, the purity was comparable to the commercial kit, so it is considered acceptable. The concentration also appeared to be at a sufficiently high level, with bands slightly fainter than the commercial kit but still adequate.

Learn

The results were roughly as expected. Although the electrophoresis was somewhat unclear and the transcription products, including those from the commercial kit, were not perfect single bands, these differences are not considered problematic. It appears that the homemade T7 RNA Polymerase stock was not significantly inhibited by RNase, and the transcription activity itself is deemed sufficient. This indicates that the T7 RNA Polymerase stock is of adequate quality for use in future assays.

Design

The experiment conducted in Cycle 1 was scaled down to a 10 µl system, and fluorescence was measured over time. According to previous studies, there are examples where fluorescence was measured over time using a qPCR machine [2]. As described in the Experiments, fluorescence was measured using the ABI Step One Plus. If the system functioned as intended, it was expected that fluorescence would increase proportionally with time over one hour.

Build

The T7 RNA Polymerase used in the experiment was expressed by introducing the plasmid purchased from Addgene into E. coli. DFHBI-1T was purchased from Cayman Chemical Company, and the MEGAscript™ T7 Transcription Kit from Thermo Fisher was used as a positive control.

Test

Following previous research, fluorescence intensity was measured using the SYBR Green filter on the ABI Step One Plus. The T7 RNA Polymerase purified by us and the T7 RNA Polymerase from the Thermo Fisher MEGAscript™ T7 Transcription Kit were reacted as described in the Experiments, and the blue wavelength raw data values from the Step One Plus were measured every minute for one hour and compared. As a result, the expected change in fluorescence intensity over time was not observed. Compared to the T7 RNA Polymerase used as a positive control, our purified T7 RNA Polymerase showed a rapid increase in fluorescence intensity at the initial stage, followed by a rapid decrease in fluorescence intensity.

Figure7

We compared the activity of our purified T7 RNA polymerase with that of ThermoFisher's MEGAscript™ T7 Transcription Kit using fluorescence intensity. Additionally, we compared the changes in activity with and without the DNA transcription template.

Learn

The reasons for the lack of the expected change in fluorescence intensity observed during this cycle were considered as follows: the presence of contaminants in the purified protein stock affected the fluorescence intensity, and the high concentration of the purified protein led to the depletion of rNTPs in the reaction solution.

Design

The results from Cycle 3 confirmed differences in activity between our purified T7 RNA Polymerase and the T7 RNA Polymerase from the Thermo Fisher MEGAscript™ T7 Transcription Kit. It was considered that either the contaminants present in the purified protein, the high concentration of T7 RNA Polymerase, or both might be the contributing factors. Therefore, to mitigate the impact of both factors and examine the activity of T7 RNA Polymerase, the stock of our purified T7 RNA Polymerase was diluted, and its activity was investigated using the ABI Step One Plus, as in Cycle 3.

Build

As in Cycle 3, the T7 RNA Polymerase used in the experiment was expressed by introducing the plasmid purchased from Addgene into E. coli. DFHBI-1T purchased from Cayman Chemical Company was used.

Test

As in Cycle 3, fluorescence intensity was measured using the SYBR Green filter on the ABI Step One Plus, following previous research. Our purified T7 RNA Polymerase was diluted twofold successively, and the resulting solutions were reacted as described in the Experiments. The raw data values at the blue wavelength from the Step One Plus were measured every minute for one hour and compared. As a result, as the stock was further diluted, fluorescence intensity increased proportionally with time, while the maximum fluorescence intensity after a one-hour reaction decreased.

Figure8

The transcription activity of the purified T7 RNA polymerase was confirmed by fluorescence while varying its concentration. The template concentration was set at 1.25 μM.

Learn

Through Cycle 4, the optimal protein concentration for the T7 RNA Polymerase transcription experiment in the Experiments was determined.

Design

In Cycle 4, the optimal concentration of T7 RNA Polymerase for in vitro transcription reactions using our purified T7 RNA Polymerase was determined. To establish a correspondence between the measured fluorescence intensity and RNA concentration for modeling purposes, the change in RNA concentration over time following the start of transcription was measured.

Build

As in Cycle 4, the T7 RNA Polymerase used in the experiment was expressed by introducing the plasmid purchased from Addgene into E. coli. Samples were collected from the reaction system every 10 minutes from the start of the reaction, and RNA concentration was measured using the Qubit RNA HS Assay Kit with Thermo Fisher's Qubit .

Test

Incubation was performed under the same reaction conditions as in Cycle 4, and samples were collected from the reaction system every 10 minutes from the start of the reaction. RNA concentration was measured using the Qubit RNA HS Assay Kit with Thermo Fisher's Qubit 4. The changes in RNA concentration over time obtained from this experiment showed a correlation with the fluorescence intensity obtained in Cycle 4. The results obtained from this cycle were used for modeling and applied in subsequent experiments.

Measured RNA concentration time course on cycle5

Learn

This experiment confirmed the correlation between fluorescence intensity and RNA concentration. Through this experiment, fluorescence intensity values were used to determine RNA concentration in subsequent experiments.

Design

In assays, for measuring the repressor or activator activity of GlnR and TnrA, reaction conditions with a lower template concentration compared to the protein might be preferable. However, based on the concentration of protein stocks available at this point and the descriptions in reference papers, it was found that the protein concentration should not or could not be excessively higher relative to the template concentration, which was set at a final concentration of 1.25 μM. Therefore, it was preferable to perform assays with as low a template concentration as possible. In this cycle, the template concentration was gradually lowered while measuring transcription to identify the lowest template concentration at which sufficient transcription could still be measured. It was anticipated that, at a certain dilution level, a stage would be reached where the increase in fluorescence due to transcription would become linear and clearly observable. This stage was considered the lower limit for the template concentration.

Build

As in Cycle 4, the T7 RNA Polymerase used in the experiment was expressed by introducing the plasmid purchased from Addgene into E. coli.

Test

The reaction conditions, except for the template concentration, were kept the same as in Cycle 4, and the measurement time was set to 90 minutes. The template concentration was decreased in the following order: 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 fluorescence raw data (Blue) at each measurement point were plotted on a line graph (Figure9).

Figure9

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 the average values were plotted on the graph. The horizontal axis represents time (minutes), and the vertical axis represents the raw fluorescence intensity data.

Learn

The minimum final concentration at which sufficient fluorescence increase was observed through a normal T7RNAP transcription reaction was estimated to be 0.125 μM. This provided a guideline that, in subsequent assays, the template concentration should be set to 0.125 μM when using a lower template concentration.

Design

The proteins used in this project, GlnR and TnrA, are designed to interfere with transcription by binding to DNA. In subsequent experiments, to rule out the possibility that GlnR and TnrA are not binding to DNA and thus not interfering with T7 RNA Polymerase transcription, as well as to investigate the optimal DNA binding sequence for GlnR, a pull-down assay was planned. Considering the possibility that pull-down might not be performed correctly due to competition between Ni-NTA beads and DNA or failure in protein recovery by Ni-NTA, it was also decided to perform SDS-PAGE on the pull-down samples.

Build

For the experiment, plasmids were created to produce GlnR mutants that always bind to the DNA sequence (GlnRN95 and GlnRN95-(GGGGS)-LeucinezipperA3.5) and TnrA. The sequences were designed based on information from references [3][4][5]. The vector used was a PET11a vector with the sequence between Xba1 and BamH1 at the 5' end, to which a modified sequence from Pet15b containing the His-Thrombin site replaced with a 3C site at the insert side of Xba1 was added. For the insert, gBlocks ordered from IDT were used. After confirming that the completed plasmids matched the target sequences via Sanger sequencing by AZENTA, they were transformed into BL21 and expressed and purified under the conditions described in the Experiment and Notebook. Additionally, the template used for the pull-down assay was ligated into the Sma1 site of the pUC118 vector under the conditions described in the Experiments and Notebook after being PCR-amplified from oligos ordered from Eurofins. These sequences were confirmed to match the target sequences by Sanger sequencing at AZENTA.

Test

Pull-down and qPCR were conducted under the conditions described in the Experiments and Notebook. SDS-PAGE was also performed under the same conditions.

Figure10

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. L2, L3, and L4 represent the types of transcription templates, respectively. The control indicates the sample that was amplified by qPCR using a plasmid that does not contain the binding sequence. The data was collected three times, and standard error was used as the error bars.

Figure11

SDS-PAGE gel after pull-down assay.

The pull-down results suggested a tendency for GlnRN95 and TnrA to bind to DNA, but due to high background and errors, the results were not sufficiently significant. Additionally, GlnRN95-(GGGGS)-LeucinezipperA3.5 did not show a clear binding tendency. SDS-PAGE analysis revealed that GlnRN95 and TnrA were sufficiently pulled down regardless of the presence of DNA. Although GlnRN95-(GGGGS)-LeucinezipperA3.5 initially had a low protein amount (possibly due to an experimental design error), it was confirmed that pull-down was still achieved.

Learn

Contrary to expectations, the results indicated a rather low (and likely not significant) DNA-binding activity. Possible reasons for this include potential inhibition of activity by the His-tag, the possibility that the conformation that binds DNA as a dimer cannot coexist with binding to Ni-NTA, and the possibility that the strength of DNA binding is insufficient for the pull-down assay. Although the results were insufficient to demonstrate DNA binding, they were not entirely negative. Given that repeating the pull-down assay is unlikely to yield improvements based on the identified causes, it was concluded that it would be better to proceed with measuring the repressor and activator activity of GlnR and TnrA as they are.

Design

We aimed to confirm whether the repressor activity of GlnR and the activator activity of TnrA are exhibited in T7RNAP in vitro transcription. Both proteins, along with GlnA, were added to the transcription system, and T7RNAP transcription activity was measured under two conditions: one with no Gln present in the solution and the other with an excess of Gln. It was hypothesized that with GlnR, normal transcription would occur under the condition with no Gln, while significantly suppressed transcription would occur under the excess Gln condition. With the constitutive DNA-binding mutant of GlnR (GlnRN95), significantly suppressed transcription was expected under both conditions. For TnrA, it was anticipated that transcription would be significantly suppressed under the no-Gln condition and proceed normally under the excess Gln condition.

Build

Plasmids coding for GlnR, GlnRN95, TnrA, and GlnA were created. Inserts were synthesized as Gblocks by IDT, and the vector was pET11a digested with XbaⅠ and BamHⅠ, assembled using XE-cocktail assembly. The proteins were designed to have a His-tag and Thrombin cleavage site fused from the XbaⅠ site of the pET15b plasmid. The sequences were confirmed by Sanger sequencing, and expression, purification, and stocking were conducted in BL21(DE3) as described in the Experiment and Notebook. Considering the potential impact of the His-tag on activity, fractions of all four proteins with the His-tag cleaved by GST-Thrombin from GE Healthcare were prepared. The GST-Thrombin was not removed after the treatment. Three types of transcription templates with the GlnR binding sites R1 and R2 and the TnrA binding site placed between the T7 promoter and the Broccoli aptamer sequence were created by annealing standard oligos from Eurofins Genomics. For GlnR and TnrA, reaction buffers with the composition deemed optimal were prepared by comparing the reference literature with the buffer composition used for transcription. Refer to the Notebook for details.

Test

In the reaction buffers for GlnR and TnrA, various reagents for transcription were mixed, and the fluorescence of the transcribed Broccoli was measured using a qPCR machine to assess transcription activity. The final concentrations were as follows: GlnR and piK029: 1 µM, TnrA: 2 µM, GlnA: 5 µM, and Template: 0.125 µM. For the combinations of protein type, template type, and the presence or absence of Thrombin treatment, the following 10 combinations were tested:

• GlnA/GlnR-Template (R1 or R2)-Thrombin (digested or undigested)
• GlnA/GlnRN95-Template (R1 or R2)-Thrombin (digested or undigested)
• GlnA/TnrA--Thrombin (digested or undigested) Transcription activity was measured under two conditions: without Gln and with an excess of Gln (20 mM), with three samples for each combination. The average value of the difference in fluorescence between 0 minutes and 60 minutes after the reaction start was plotted on a bar graph.

Figure12

GlnR TnrA First Assay. The vertical axis represents the increase in fluorescence values (a.u.) from 0 to 60 minutes. The error bars indicate the standard error. The experiment was conducted in three wells, and the average is shown in the bar graph.

There was no clear difference for the combination of GlnR and Template R1 with or without Gln or with or without Thrombin treatment. Similarly, the combination of GlnRN95 and Template R1 showed little difference from GlnR, and there was no difference due to Thrombin treatment. For the combination of GlnR and Template R2, there was a tendency for transcription to increase in the presence of Gln, while Thrombin treatment seemed to slightly suppress transcription regardless of the presence or absence of Gln. The combination of GlnRN95 and Template R2 appeared to suppress transcription more clearly than the other GlnR and GlnRN95 samples. For TnrA, there was no noticeable difference due to the presence or absence of Gln or Thrombin treatment.

Learn

In the combination of GlnRN95 and R2, the expected transcriptional repression effect appeared to be exerted. However, in the combination of GlnR and R2, the observed activity was the opposite of what was originally anticipated, which could be due to a possible mix-up of reagents. Therefore, a cautious repetition of the experiment will be conducted to confirm this. For the other combinations, the expected activities were not observed. Since no clear impact of Thrombin treatment was observed overall, it was suggested that Thrombin treatment is unnecessary. It was also implied that the binding sequence included in the GlnR template R2 is superior to R1 in terms of the level of repression by GlnRN95 and the differentiation in assays involving GlnR. As there was no distinct activity observed overall, it became apparent that the reaction conditions need to be fundamentally improved. Although there is some doubt about the activity of the proteins themselves, given the effort required to purify the proteins again, it was concluded that it would be better to improve the reaction conditions to enhance activity or to obtain additional data for further analysis.

Design

The experiment was fundamentally the same as in Cycle 2, but with improved conditions. Since there was no clear impact of Thrombin treatment, only untreated Thrombin samples were used. The T7 RNA Polymerase concentration was doubled to make differences in transcription levels more evident. Considering the potential negative effects of glycerol, sodium acetate, and NaCl on transcription, the regular transcription buffer previously used for transcription was employed for the reaction and protein preparation instead of the reaction buffer. In Cycle 2, the R2 template showed a clearer difference for GlnR, so only the R2 template was used. To observe the effects of the presence of glycerol, Gln, and differences in transcription templates on the transcription reaction, a negative control containing only T7 RNA Polymerase, without enzymes such as GlnA, was prepared for the experiment.

Build

As in Cycle 2, the same enzymes and templates were used. Thrombin treatment and reaction buffer preparation were not performed.

Test

In the reaction buffers for GlnR and TnrA, various reagents for transcription were mixed, and the fluorescence of the transcribed Broccoli was measured using a qPCR machine to assess transcription activity. The final concentrations were as follows: GlnR and piK029: 1 µM, TnrA: 2 µM, GlnA: 5 µM, and Template: 0.125 µM. For the combinations of protein type, template type, and the presence or absence of Thrombin treatment, the following five combinations were tested:

• GlnA/GlnR-Template R2-Thrombin undigested
• GlnA/GlnRN95-Template R2-Thrombin undigested
• GlnA/TnrA-Thrombin undigested
• T7 only-Template (R2 or TT04 (TnrA Template)) Transcription activity was measured under two conditions: without Gln and with an excess of Gln (20 mM), with three samples for each combination. The average value of the difference in fluorescence between 0 minutes and 60 minutes after the reaction start was plotted on a bar graph (figure13).

Figure13

GlnRTnrA 2nd Assay The vertical axis represents the increase in fluorescence values (a.u.) from 0 to 60 minutes. The error bars indicate the standard error. The experiment was conducted in three wells, and the average is shown in the bar graph.

For the combination of GlnR and R2, there was no clear difference in the presence or absence of Gln, but considering a 10% significance level, a significant difference was observed, suggesting a possible effect. For the combination of T7 and R2, there was no significant difference with or without Gln. The combination of GlnRN95 and R2 showed a clear transcriptional repression effect, consistent with Cycle 2. Regarding TnrA, transcription activity remained at a very low level regardless of the presence of GlnA, and it was also low in the absence of TnrA.

Learn

For GlnR, although there was no reversal of activity with the change in conditions, its activity was barely detectable. On the other hand, the transcriptional repression activity of GlnRN95 was shown to be functional. This suggests that it might be possible to make the system work through improvements in protein purification, reaction conditions, or design. Regarding TnrA, the fact that transcription activity was very low even without TnrA suggests a significant issue with the design or quality of the template. It is likely necessary to investigate either a design flaw in the template or an unexpected incident during its preparation or storage.

Design

We decided to use the T7 two-hybrid system as a mechanism for inducing transcription only when a DNA-binding protein binds to DNA. This system employs the concept from a previous in vivo study [6] and applies it in vitro. Specifically, it utilizes a set of hetero-interacting leucine zipper structures, Leucine Zipper AN3.5 and Leucine Zipper BN3.5 [7]. The fusion proteins of DNA-binding protein-Leucine Zipper AN3.5 and T7RNAP-Leucine Zipper BN3.5 interact within the system, leading to transcription activation only when the DNA-binding protein binds to DNA. To demonstrate that this system can work in vitro, we decided to conduct experiments using a zinc finger protein fused with the leucine zipper structure, which always binds to DNA. For the assay, we used DNA with a sequence containing the zinc finger binding site, T7 promoter, and Broccoli aptamer. When the DNA-binding protein binds to DNA, transcription is expected to occur, resulting in the production of the fluorescent aptamer and subsequent fluorescence derived from Broccoli.

Build

For the experiment, plasmids were created to produce Zinc Finger-Leucine Zipper AN3.5 and T7 RNA Polymerase - Leucine Zipper BN3.5. The sequences were designed based on information from paper [7]. The vector used was a PET11a vector with the sequence between Xba1 and BamH1 at the 5' end, to which a modified sequence from Pet15b containing the His-Thrombin site replaced with a 3C site at the insert side of Xba1 was added. For the insert, gBlocks ordered from IDT were used. The completed plasmids were transformed into BL21, and expression and purification were conducted under the conditions described in the Experiment and Notebook. Additionally, the transcription template was prepared by PCR amplification of oligos ordered from Eurofins, followed by purification.

Test

The concentration of T7 RNA Polymerase-Leucine Zipper BN3.5 was varied, and the experiment was conducted to compare fluorescence values with and without the presence of Zinc Finger-Leucine Zipper AN3.5. The experiment was performed under the conditions described in the Notebook and Experiment. The concentration of Zinc Finger-Leucine Zipper AN3.5 in the reaction mixture was fixed at 1.76 μM, while the concentration of T7 RNA Polymerase-Leucine Zipper BN3.5 was changed in two-fold increments. The results are shown in Figure 14.

Figure14

A graph showing the difference in fluorescence values between 0 and 60 minutes in the Broccoli Assay with and without the presence of Zinc Finger-Leucine Zipper. The concentration of T7RNAP-Leucine Zipper was varied, and the DNA concentration was set at 0.265 μM.

Learn

As the concentration of T7RNAP-Leucine Zipper increased, the fluorescence values rose both in the presence and absence of the zinc finger. When comparing the conditions with and without the zinc finger, an optimal concentration was observed, which in this case was around 0.7 μM, showing approximately a 2.5-fold difference, and was considered the optimal condition.

Design

In Cycle 1, the optimization of the T7 RNA Polymerase-Leucine Zipper BN3.5 concentration was conducted. It was determined that the system worked best with T7 RNA Polymerase-Leucine Zipper BN3.5 at a concentration of 0.7 μM. Based on this, the concentration of T7 RNA Polymerase-Leucine Zipper BN3.5 was fixed at 0.7 μM, and the concentration of Zinc Finger-Leucine Zipper AN3.5 was varied.

Build

The concentration of T7 RNA Polymerase-Leucine Zipper BN3.5 was fixed at 0.7 μM, and the concentration of Zinc Finger-Leucine Zipper AN3.5 was varied.

Test

The experiment was conducted under the conditions described in the Notebook and Experiment. The concentration of Zinc Finger-Leucine Zipper AN3.5 was set at 7 μM, 3.5 μM, and 1.75 μM for the experiments. The results are shown in Figure 15.

Figure15

A figure showing how the fluorescence increase from 0 to 60 minutes changes when the concentration of Zinc Finger-Leucine Zipper is varied, with the concentration of T7 RNA Polymerase-Leucine Zipper 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 error. The DNA template concentration was set at 0.265 μM.

Learn

It was found that transcription was activated as the concentration of zinc finger increased. Additionally, displaying the error range demonstrated the usefulness of the two-hybrid system. However, it also revealed the issue of high background noise.

Design

In Cycle 2, it was demonstrated that the two-hybrid system is useful, but it also highlighted the issue of a high value in the negative control. Therefore, it was considered that optimizing the T7 promoter d1 in the transcription template could achieve a lower background.

Build

T7 promoter d1 is a modified version of the T7 promoter with the first 5 bases removed. It was considered important to alter the remaining 12 bases to lower the affinity. Therefore, to maximize the changes in the bases, the transcription template was altered one base at a time from the 5' end, using the following substitutions: A→C, T→G, G→T, and C→A.

Test

A transcription assay was conducted using templates with altered bases. Initially, eight prepared promoters were screened, but only the one with a single base substitution(TT18) at the first position showed effective transcription (Figure 16). Subsequently, a transcription assay was performed using only the template with the single base substitution at the first position, but no difference was observed between the presence and absence of the zinc finger. Therefore, it was concluded that the original promoter was optimal (Figure 17).

Figure16

The results of the sequence screening. TT18-TT25 represents the type of template, and the vertical axis shows the difference in fluorescence values from 0 to 60 minutes. The plus sign indicates the presence of Zinc Finger-Leucine Zipper AN3.5, while the minus sign indicates its absence.

Figure17

A graph comparing the fluorescence values from 0 to 60 minutes with and without the Zinc Finger-Leucine Zipper using the promoter with the first base of the original T7 promoter d1 altered. The DNA concentration was 0.17 μM, and the concentration of Zinc Finger-Leucine Zipper was 7 μM.

Learn

It was concluded that the original promoter was optimal, and based on this, an assay for GlnR was conducted.

Design

Since the usefulness of the two-hybrid system was confirmed, a transcription experiment was conducted using a positive control with GlnR connected to the leucine zipper to verify whether the two-hybrid system could also be applied to GlnR.

Build

A mutant of GlnR that binds to DNA even at low glutamine concentrations was obtained from reference [8], and a plasmid with Leucine Zipper AN3.5 connected to its C-terminus was created using synthetic DNA ordered from IDT. The vector used was a PET11a vector with the sequence between Xba1 and BamH1 at the 5' end, to which the insert-side sequence from Pet15b Xba1 was added. The completed plasmid was transformed into BL21, and expression and purification were conducted under the conditions described in the Experiment and Notebook. The transcription template was designed by replacing the Zinc finger region used in Cycle 1 with a DNA sequence called GlnRAo2, and it was created by performing PCR on oligos ordered from Eurofins, followed by purification.

Test

Figure18 shows a comparison of the fluorescence value changes in the transcription assay while varying the concentration of GlnR-Leucine Zipper AN3.5, compared with the negative control (without GlnR-Leucine Zipper AN3.5). The DNA template concentration was 0.26 μM, and the T7RNAP-Leucine Zipper concentration was 0.7 μM. At all concentrations of GlnR-Leucine Zipper AN3.5, a higher change in fluorescence value was observed compared to the negative control. Additionally, as the concentration of GlnR-Leucine Zipper AN3.5 increased, a trend of decreasing fluorescence values was observed.

Figure18

A graph showing the change in fluorescence values from 0 to 60 minutes when the concentration of GlnR-Leucine Zipper AN3.5 was varied. The negative control represents the case without GlnR-Leucine Zipper AN3.5. The DNA template concentration was 0.26 μM, and the T7RNAP-Leucine Zipper concentration was 0.7 μM. The experiment was conducted in three wells, and the standard error was used as the error bars.

Learn

It was demonstrated that the two-hybrid system can be applied to biosensors using GlnR.

Design

Next, mutants were designed with the leucine zipper structure inserted into the linker region of GlnR, as well as mutants with the leucine zipper structure inserted into GlnA. The design details are described in Part 4 of the Modeling page, where AlphaFold was used to select regions that would likely have minimal impact on protein function for the design.

Build

For the protein with the leucine zipper AN3.5 incorporated within GlnR, a vector with the sequence between Xba1 and BamH1 of PET11a at the 5' end, to which the insert-side sequence from Pet15b Xba1 was added, was used. For proteins with the leucine zipper AN3.5 incorporated at the N-terminus of GlnR or GlnA, a vector with the sequence between Xba1 and BamH1 of PET11a at the 5' end, to which the insert-side sequence from Pet15b Xba1 with the His-Thrombin site replaced with a 3C site was used. The completed plasmids were transformed into BL21(DE3), and expression and purification were conducted under the conditions described in the Experiment and Notebook.

Test

After preliminary experiments, it was determined that using the GlnR with the Leucine Zipper AN3.5 inserted internally and Leucine Zipper AN3.5-GlnA was the best approach. The results of the experiment conducted under four conditions (0 mM and 20 mM glutamine, with and without GlnR) are shown in Figure 19. Due to large errors, no significant difference was observed between the presence and absence of glutamine.

Figure19

In the GlnR+ condition, the experiment was conducted with GlnR at 1.8 μM and GlnA at 5 μM (GlnR- serves as the negative control). The experiment was performed in three wells, and the difference between 0 and 60 minutes was shown in a bar graph. The DNA template concentration was 0.26 μM, and the T7-Leucine Zipper concentration was 0.35 μM. The error bars indicate the standard error.

Additionally, for the protein with a leucine zipper attached to GlnA, experiments were conducted under four conditions: with and without glutamine, and with and without the protein. Based on preliminary experiments, 0.88 μM of Leucine Zipper-GlnA was determined to be optimal, and the experiment was conducted in three wells, with the results shown in Figure 20. Again, no significant difference was observed between the presence and absence of glutamine.

Figure20

The results of the experiment conducted under two conditions of glutamine concentration (20 mM and 0 mM) are shown, divided into GlnA+, GlnR+ (with GlnA and GlnR present) and GlnA-, GlnR- (without GlnA and GlnR). The concentration of GlnA was 0.88 μM when present, and the concentration of GlnR was 1.1 μM when present. The DNA template concentration was 0.26 μM, and the concentration of T7 RNA Polymerase-Leucine Zipper BN3.5 was 1 μM. The fluorescence values represent the difference between 0 minutes and 60 minutes. The error bars indicate the standard error.

Learn

Even with the addition of the leucine zipper to GlnR and GlnA, we were not able to clearly distinguish the glutamine concentration trend within the project's timeframe. Considering that the positive control in Cycle 4 worked well, it is possible that issues with protein storage conditions or the concentrations of GlnA and GlnR were the cause. We aim to optimize these aspects in the future to enable effective glutamine sensing.

Design

In the reaction involving Cas7-11, two particularly important processes are the RNA-dependent cleavage of csx30 by Cas7-11-csx29 and the formation of a covalent bond between Spycatcher and Spytag. Both of these can be confirmed through SDS-PAGE, so the first step is to verify this activity. The cleaved bands and the bands after the binding should be observable using SDS-PAGE.

Build

For the Cas7-11-csx29 activity test, plasmids for co-expressing Cas7-11 and guide RNA, as well as the csx29 plasmid, were purchased from Addgene (Addgene 197612[9] SUMO_TwinStrep_Cas7-11_crRNA / Addgene 197613 pCDF_6xHIS_Csx29[10]). These plasmids were transformed into the same BL21(DE3) strain to co-express the Cas7-11-csx29-guide RNA complex, which was then purified. Due to limitations on available reagents, the purification procedures mentioned in the reference, including Streptactin pulldown, SUMO tag cleavage, Heparin HiTrap, and Superose, were omitted, and only Ni-NTA pulldown was performed. The sample was purified and concentrated. Additionally, the insert of CBM9.2-truncated csx30-Spycatcher was synthesized by IDT and introduced into the pET11a vector using XE-cocktail assembly. A pseudo prefix-suffix and Spytag were introduced into the sequence obtained from Distribution via PCR. By introducing this into the pET11a vector using XE-cocktail assembly, a Chromoprotein-Spytag was created for the activity test of the Spy system. Both constructs were completed as described in the Experiment and Notebook sections and were expressed and purified using BL21(DE3). These proteins were not stocked, and the fractions obtained after purification or concentration were used directly for the assay. The target RNA for Cas7-11 was generated by PCR using Standard Oligos from Eurofins Genomics to create a transcription template, which was then transcribed in vitro.

Test

For the Cas7-11 cleavage activity test, a solution of Cas7-11-csx29-guide RNA was mixed with a solution of CBM9.2-truncated csx30-Spycatcher, and both were incubated at 37°C for 65 minutes, with one set including the target RNA and the other set without it, before being frozen at -80°C. For the Spy system activity test, a solution of CBM9.2-truncated csx30-Spycatcher was mixed with a solution of asPink-Spytag and incubated at room temperature (24°C) for 65 minutes, then frozen at -80°C.

The following day, the samples were thawed, and SDS-PAGE was performed to ensure that the protein amounts were uniform across all lanes for each assay, along with the protein solutions prior to mixing, and bands were observed.

Figure21

Picture of SDS-PAGE for cycle1 of Cas 7-11.

In the Spy system activity test, some bands present in the lower concentration asPink-Spytag solution disappeared in the mixed solution, and new bands likely corresponding to the binding products were observed. In the Cas7-11 cleavage activity test, no bands indicating the cleavage of CBM9.2-truncated csx30-Spycatcher were observed.

Learn

It was found that the Spy system was almost completely bound and functional. Regarding Cas7-11-csx29-guide RNA, the low purity during purification made it unclear which bands were correct, suggesting that the concentration may have been insufficient or the purification may have failed. A likely reason for this could be that the large SUMO tag was not cleaved, which may have negatively affected the activity. It was considered necessary to incorporate some of the omitted purification steps to obtain a fraction with higher purity and to perform SUMO tag cleavage for Cas7-11-csx29-guide RNA.

Design

In Cycle 1, no cleavage of the spycatcher-Csx30-CBM complex by Cas7-11 was observed. One possible factor for the lack of expected activity from Cas7-11 in Cycle 1 was the presence of the uncut Twinstreptag from the Cas7-11 protein, which may have interfered with its activity. Therefore, it was anticipated that using a protein with the tag cleaved would allow for the cleavage of the spycatcher-Csx30-CBM complex. Cleavage was assessed and confirmed using SDS-PAGE.

Build

Cas7-11 and Csx29 proteins used in the experiment were derived from plasmids purchased from Addgene. The spycatcher-Csx30-CBM complex was created using a plasmid containing Csx30 obtained from Addgene and a sequence amplified by PCR from sequences purchased from IDT, which was then introduced into the pET11a vector. Ulp1 was purchased as an insert from Twist and introduced into the pET11a vector. Additionally, the desired sequence was confirmed to match by Sanger sequencing from AZENTA. These constructs were introduced into BL21 and proteins were purified under the conditions described in the Experiments and Notebook sections.

Test

Following the conditions outlined in the Notebook, a certain amount of Ulp1 was added to the elution of the Cas7-11 and Csx29 complex, and after incubation to cleave the tag, the spycatcher-Csx30-CBM complex was cleaved according to the conditions specified in the Notebook. As a result, SDS-PAGE was performed to confirm activity, but the cleavage of the spycatcher-Csx30-CBM complex could not be observed, as shown in the Figure22.

Figure22

Results of the Cas7-11 cleavage assay. The experiment examined whether the SpyCatcher-Csx30-CBM complex was cleaved in the presence and absence of the Cas7-11-Csx29 complex and target RNA.

Learn

From these results, it was determined that the protein concentration was not optimal. Additionally, it was considered that the cleavage of the Twinstreptag by Ulp1 had not occurred.

Design

In Cycle 2, no cleavage of the spycatcher-Csx30-CBM complex by Cas7-11 was observed. One possible reason for the lack of expected activity from Cas7-11 in Cycle 2 was the presence of the uncut Twinstreptag from the Cas7-11 protein, which may have interfered with its activity. Therefore, the concentration of Ulp1 was gradually increased to identify the appropriate concentration for effective tag cleavage during incubation. Cleavage was assessed and confirmed using SDS-PAGE.

Build

The Cas7-11 and Csx29 proteins used in the experiment were derived from plasmids purchased from Addgene. Ulp1 was obtained as an insert from Twist and introduced into the pET11a vector. Additionally, Sanger sequencing from AZENTA confirmed that the desired sequence matched. These constructs were introduced into BL21, and the proteins were purified under the conditions described in the Experiments and Notebook sections.

Test

Following the conditions outlined in the Notebook, the concentration of Ulp1 was gradually increased for the Cas7-11 and Csx29 complex bound to Strep-Tactin beads. The proteins in the supernatant were then confirmed using SDS-PAGE to check for cleavage. As a result, it was confirmed that cleavage of the Twinstreptag by Ulp1 occurred at a certain concentration, as shown in the Figure23.

Figure23

SDS-PAGE gel of SUMO cutting. Results of the experiment in which the Twin-Strep-tag fused to Cas7-11 was cleaved using SUMO protease. The concentration of SUMO protease was gradually increased, and the proteins present in the Strep-Tactin beads and the supernatant were analyzed using SDS-PAGE.

Learn

Comparing the results of Cycle 2 and Cycle 3, it was evident that the cleavage of the Twinstreptag was not sufficiently achieved in Cycle 2.

Design

In Cycle 2, one reason for the lack of cleavage of the spycatcher-Csx30-CBM complex was not only the insufficient cleavage of the tag but also the inadequate concentration of the protein. Therefore, using the Cas7-11 and Csx29 complex, which was confirmed to have sufficient cleavage of the Twinstreptag throughout Cycle 3, the protein concentrations were adjusted to discover the optimal concentration for cleavage. The results were confirmed by SDS-PAGE.

Build

Cas7-11 and Csx29 proteins used in the experiment were derived from plasmids purchased from Addgene. The spycatcher-Csx30-CBM complex was created using a plasmid containing Csx30 obtained from Addgene and a sequence amplified by PCR from sequences purchased from IDT, which was then introduced into the pET11a vector. Ulp1 was purchased as an insert from Twist and introduced into the pET11a vector. Additionally, the desired sequence was confirmed to match by Sanger sequencing from AZENTA. These constructs were introduced into BL21 and proteins were purified under the conditions described in the Experiments and Notebook sections. Furthermore, the Cas7-11 and Csx29 complex, which was confirmed to have the Twinstreptag cleaved in Cycle 3, was used.

Test

Following the conditions outlined in the Notebook, the concentrations of the Cas7-11 and Csx29 complex and the target RNA were adjusted, and the cleavage activity of the spycatcher-Csx30-CBM complex was confirmed by SDS-PAGE. As a result, under the conditions of Cycle 2, it was determined that while the concentration of the target RNA was sufficient, the concentration of the Cas7-11 and Csx29 complex was inadequate, as shown in the Figure24.

Figure24

SDS-PAGE gel of third assay. Experiment confirming the cleavage of the SpyCatcher-Csx30-CBM complex using the Cas7-11-Csx29 complex, which was verified to have its Twin-Strep-tag cleaved by SUMO protease. The cleavage activity was assessed by varying the concentrations of the Cas7-11-Csx29 complex and target RNA, followed by analysis using SDS-PAGE.

Learn

Throughout Cycle 4, sufficient concentrations of the Cas7-11 and Csx29 complex, as well as the target RNA, were identified for cleaving the spycatcher-Csx30-CBM complex using the Cas7-11 and Csx29 complex.

Design

The Cas7-11 and Csx29 proteins used in the experiment were derived from plasmids purchased from Addgene. The spycatcher-Csx30-CBM complex was constructed using a plasmid containing Csx30 obtained from Addgene and sequences purchased from IDT, which were amplified by PCR and introduced into the pET11a vector. The Ulp1 insert was purchased from Twist and introduced into the pET11a vector as well. Furthermore, verification of the desired sequences was confirmed by Sanger sequencing performed by AZENTA. These constructs were then introduced into BL21 cells, and the proteins were purified under the conditions described in the Experiments and Notebook sections. Additionally, the Cas7-11 and Csx29 complex, which was confirmed to have its Twin-strep-tag cleaved in Cycle 3, was used in this experiment.

Build

The Cas7-11 and Csx29 proteins used in the experiment were derived from plasmids purchased from Addgene. The spycatcher-Csx30-CBM complex was constructed using a plasmid containing Csx30 obtained from Addgene and sequences purchased from IDT, which were amplified by PCR and introduced into the pET11a vector. The Ulp1 insert was purchased from Twist and introduced into the pET11a vector as well. Furthermore, verification of the desired sequences was confirmed by Sanger sequencing performed by AZENTA. These constructs were then introduced into BL21 cells, and the proteins were purified under the conditions described in the Experiments and Notebook sections. Additionally, the Cas7-11 and Csx29 complex, which was confirmed to have its Twin-strep-tag cleaved in Cycle 3, was used in this experiment.

Test

Following the conditions described in the Notebook, the concentrations of the Cas7-11, Csx29 complex and target RNA were adjusted, and the cleavage activity of the CBM-Csx30-spycatcher003-spytag003-gfasPurple complex bound to cellulose was assessed using SDS-PAGE. As a result, no cleavage of the CBM-Csx30-spycatcher003-spytag003-gfasPurple complex bound to cellulose by the Cas7-11, Csx29 complex was observed.

Figure25

Cleavage experiment of the CBM-Csx30-spycatcher003-spytag003-gfasPurple complex bound to cellulose in the presence and absence of Cas7-11.

Learn

Potential factors for the lack of cleavage by Cas7-11 include the possibility that the cellulose interfered with Cas7-11's ability to cleave Csx30, or that gfasPurple similarly hindered the cleavage activity. Additionally, the high concentration of the CBM-Csx30-spycatcher003-spytag003-gfasPurple complex might have contributed to the cleavage not proceeding as expected.