iGEM 2025 — Team china-xjtu

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# Notebook

December 2024 - January 2025

### Team Assembly & Project Launch

Project officially launched with core team recruitment completed within one week, forming a "2+3+2" tiered structure:

* 2 dry lab members (responsible for bioinformatics analysis and AI structure prediction)
* 3 wet lab members (responsible for plasmid construction for protein characterization and detection)

First discussion meeting held on December 13 to clarify the technical approach of "AI-assisted protein design + experimental validation closed-loop."

Team Building
Project Planning
Technical Approach

February - March 2025

### Topic Focus & Resource Preparation

During a collective discussion on February 26, team members shared diverse research directions. After faculty guidance, the team finalized the project topic three days later: allulose production, focusing on directed enzyme evolution and modification.

Completed iterations of recruitment posters on March 10, attracting over 60 applicants university-wide. Ultimately selected 10 students to join the team, expanding to 17 members with defined roles and group leaders.

Laboratory application at Xingqing Campus approved on March 15.

Topic Determination
Team Expansion
Resource Preparation

April 2025

### AI Model Development & Data Production

During April 15-17, the wet lab team evaluated RFdiffusion, Protein MPNN, and Alpha Protein2 before selecting David Baker Lab's Protein MPNN as the backbone sequence generation engine and rapidly completing local deployment.

April 27-29: Wet lab team completed laboratory space planning over the weekend. Following the "three zones, two channels" standard (reagent zone, operation zone, instrument zone; separate personnel and material flow), the lab passed the school's safety inspection on the first attempt.

AI Models
Lab Planning
Technical Evaluation

May - June 2025

### Project Iteration & Optimization

May 22: Group discussion reassessed sequence design, synthesis, and characterization phases, identifying sequence improvement opportunities.

June 4: Task reassignments following personnel changes; team leader formally confirmed.

June 8: Introduced IMODS to predict DTE binding poses with target proteins, providing high-confidence templates for subsequent mutation site screening.

June 20: Submitted two predicted DTE mutant sequences for company synthesis. Designed corresponding primers based on sequence design to prepare for experimental work.

Project Iteration
Task Assignment
Sequence Design

June - July 2025

### Plasmid Construction & Experimental Validation

June 27: Group discussion finalized plasmid construction strategy, designing DTE enzyme expression systems and RNA-pepper-based allulose biosensors.

July: The wet lab group completed the construction of three plasmids, establishing the expected system through PCR, sequencing, and other steps.

Plasmid Construction
Experimental Validation
Biosensors

August 2025

### Team Exchange & Competition Preparation

August 6-8: Team traveled to Shenzhen for CCiC, received judge feedback, and refined subsequent experimental plans.

August 10: Project exchange with teams from Yan'an University and Northwestern Polytechnical University to share experiences.

Team Exchange
Competition Preparation
Experience Sharing

August - September 2025

### System Characterization & Data Closure

Late August - Mid-September: The wet lab group completed characterization of all constructed plasmids and performed quantitative system characterization with closed-loop validation.

System Characterization
Data Closure
Quantitative Analysis

Mid-September - Present

### Experimental Data Integration & Analysis

Integrated and analyzed experimental data, summarized project outcomes, and prepared project reports and paper writing.

Data Integration
Outcome Summary
Report Writing

# Protocols

![进度指示图片](https://static.igem.wiki/teams/5890/alo/12c905f248b198b39bf1495fd371412f.webp)

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Document Navigation

* [1.Overview](#introduction)
* [2. Design](#design)
  + [2.1 Design of a novel DTE enzyme](#enzyme)
  + [2.2 Design of the DTE Enzyme Expression System](#system)
  + [2.3 Design of an RNA-Pepper-Based Allulose Biosensor](#biosensor)
  + [2.4 RNA-pepper-based DTE enzyme activity characterization system](#pepper)
* [3. Build](#build)
  + [3.1 Construction of the DTE enzyme expression system](#constructiona)
  + [3.2 Construction of an Allulose Biosensor Based on RNA-pepper](#constructionb)
  + [3.3 Construction of the RNA-pepper-based DTE enzyme activity characterization system](#constructionc)
* [4. Test](#test)
  + [4.1 DTE Enzyme Expression System Verification and Catalytic Activity Assessment](#assessment)
  + [4.2 Performance testing of the RNA-pepper-based allulose biosensor](#performance)
  + [4.3 Detection Using an RNA-Pepper-Based DTE Enzyme Activity Characterization System](#detection)
* [5. Learn](#learn)

# Engineering

## Content Overview  To further confirm whether the enzymes designed by the dry lab group using AI tools possess genuine catalytic activity, we characterized the catalytic performance of DTE enzymes through wet lab experiments. During the formal experimental process, we primarily designed: * A fructose biosensor based on pepper fluorescent RNA; * A plasmid for expressing the DTE enzyme; * Integration of both systems to measure DTE enzyme catalytic capacity.  The entire process followed the iGEM-recommended DBTL (design, build, test, learn) cycle, where we continuously refined our working system through feedback. Below are the completed experimental steps and their underlying logic. 2. Design2.1 Design of a novel DTE enzyme  We aimed to redesign the DTE enzyme sequence while preserving catalytic activity.  We first analyzed the structure of the native DTE enzyme (using PDB: 2OU4 as a template) to identify key regions that must be preserved as a conservative design strategy (Table 1). D-Tagatose-3-Epimerase (DTEase) is a key enzyme catalyzing C3-position epimerization of various ketosides, holding significant value in rare sugar biosynthesis. This study uses DTEase from Pseudomonas cichorii (PDB: 2OU4) as the design template. This enzyme exhibits a typical metal-dependent (β/α)₈ barrel structure. Its active site comprises a Mn²⁺ ion coordinated by four highly conserved residues (Glu152, Asp185, His211, Glu246), responsible for substrate deprotonation and protonation. Furthermore, 2OU4 forms a stable homodimer in its native state. Its active pocket is located on one side of the dimer interface, exhibiting an open, loose substrate-binding characteristic that enables it to efficiently catalyze the C3 epimerization reactions of both D-tagatose and D-fructose simultaneously.  D-tagatose-3-epimerase (DTEase): 2ou4 sequence:  MNKVGMFYTYWSTEWMVDFPATAKRIAGLGFDLMEISLGEFHNLSDAKKRELKAVADDLGLTVMCCIGLKSEYDFASPDKSVRDAGTEYVKRLLDDCHLLGAPVFAGL TFCAWPQSPPLDMKDKRPYVDRAIESVRRVIKVAEDYGIIYALEVVNRFEQWLCNDAKEAIAFADAVDSPACKVQLDTFHMNIEETSFRDAILACKGKMGHFHLGEAN RLPPGEGRLPWDEIFGALKEIGYDGTIVMEPFMRKGGSVSRAVGVWRDMSNGATDEEMDERARRSLQFVRDKLA  DOI：10.1016/j.jmb.2007.09.033;10.1002/cbic.201402620 Table 1. Key Functional Regions and Design Strategy for DTE | Region Category | Residue ID (A) | Mutability | | --- | --- | --- | | Catalytic Core | A152/A185/A211/A246 | Fixed | | Dimer Interface | A103-263 (key residues) | Fixed | | Hydrophobic Core | A30/50/100/120/150/180/200/230/250/280 | Mutable | | Surface Charge | A10/20/60/90/130/170/220/260/290 | Mutable | | Remaining 90% | All remaining residues | Mutable |  Using 2OU4 as a template, we performed structure-based sequence prediction with LigandMPNN: 1. Designated Mn²⁺-coordinating residues A152/A185/A211/A246 and critical sites like the dimer interface as fixed regions (non-mutable), while classifying the remaining ~90% of residues as redesignable regions; 2. Enhanced Val/Ile/Leu in the hydrophobic core and Asp/Glu/Lys/Arg in surface charge regions via residue-specific preference optimization, while globally shielding free Cys to mitigate oxidation risks; 3. Ensured synchronous optimization of both chains using symmetry constraints, incorporating ligand side-chain environment sensing and packing algorithms to prevent collisions between new sequences and metal ions/substrates; 4. Through iterative refinement, we optimized surrounding residues within the overall framework of the new enzyme's renew series. From 32×2 samples, we rapidly screened relatively stable variants DTE-1 and DTE-2 using the Expasy ProtParam tool.  We performed molecular docking of D-fructose into DTE-1 and DTE-2 using CB-Dock2. Both enzymes exhibited well-defined binding pockets with favorable Vina scores (–5.1 and –5.0 kcal·mol⁻¹, respectively). Template-based refinement using 2qum further confirmed substrate-binding capability, with contact scores of 12.4 (DTE-1) and 54.7 (DTE-2). Although DTE-1 binds in a shifted cavity and DTE-2 shows moderate pocket similarity to the reference enzyme 2OU4, both retain key interactions necessary for substrate recognition. Complementary docking via DiffDockfurther revealed that DTE-2 displays a clear preference for D-tagatose over D-fructose,[combined with our findings from atomic-interaction analysis and molecular dynamics with binding free energy calculations,](https://2025.igem.wiki/china-xjtu/model) suggesting enhanced catalytic pairing propensity toward the target substrate. Collectively, these results imply that both DTE-1 and DTE-2 possess detectable catalytic activity. 2.2 Design of the DTE Enzyme Expression System  To enable sustained DTE enzyme expression, we employed the high-copy pSEVA341 plasmid with a porin promoter, ensuring stable expression and functionality of the DTE enzyme in bacteria. ![Figure 1A](https://static.igem.wiki/teams/5890/dry-lab-modelling-pictures/figure-4.webp) DTE enzyme expression system（[BBa\_251J7RL3](https://registry.igem.org/parts/bba-251j7rl3)、[BBa\_25S11TZB](https://registry.igem.org/parts/bba-25s11tzb)、[BBa\_258V0N5W](https://registry.igem.org/parts/bba-258v0n5w)） 2.3 Design of an RNA-Pepper-Based Allulose Biosensor ![Figure 1B](https://static.igem.wiki/teams/5890/wetlab-picture/wet.avif) Allulose sensor based on RNA-pepper([BBa\_25N1O2HD](https://registry.igem.org/parts/bba-25n1o2hd))   Validating DTE enzyme catalytic activity hinges on detecting allulose concentrations in the environment by coupling fluorescent signals to allulose levels. A 2019 study reported designing a GFP-fluorescent synthetic dye—(4-((2-hydroxyethyl)(methyl)amino)-subbenzylidene)cyanophenylacetonitrile (HBC), featuring a structurally rigid electron acceptor and a strong electron donor. Paired with the HBC-binding aptamer Pepper, it enhances HBC fluorescence by over 3000-fold in vitro and exhibits bright fluorescence when expressed in *E. coli* cells. Compared to other currently available fluorescent reporter groups, it offers an order-of-magnitude improvement in fluorescence intensity and activation factor, a one- to two-fold increase in affinity, enhanced thermal stability by approximately 20°C, superior pH tolerance, broader spectral range, and higher signal-to-noise ratio, providing an effective solution for characterizing allulose synthase activity. Therefore, based on the fluorescent RNA pepper-HBC system, we constructed a biosensor for DTE enzyme activity. By utilizing allulose to release the PsiRA repression effect on RNA pepper expression, we achieved biological detection of allulose concentration. 2.4 RNA-pepper-based DTE enzyme activity characterization system  By integrating the two aforementioned systems, we constructed a dual-plasmid system to enable comparative analysis of different DTE enzyme activities.  Using electroporation, p341-porin-DTE-WT, p341-porin-DTE-1, and p341-porin-DTE-2 were separately transformed into a strain containing pET28-porin-psiRA-pepper to establish the dual-plasmid system for enzyme activity characterization. Through the allulose-mediated PsiR repression release system, the allulose concentration was coupled with pepper expression levels and final fluorescence intensity to indirectly characterize the catalytic efficiency of DTE enzymes. ![Figure 1C](https://static.igem.wiki/teams/5890/dry-lab-modelling-pictures/figure-1.webp) RNA-pepper-based DTE enzyme activity characterization system（[BBa\_25SX79U8](https://registry.igem.org/parts/bba-25sx79u8)、[BBa\_254CVCZ7](https://registry.igem.org/parts/bba-254cvcz7)、[BBa\_25M55R6E](https://registry.igem.org/parts/bba-25m55r6e)） 3. Build3.1 Construction of the DTE enzyme expression system  Utilizing the laboratory's existing DTE-WT sequence and PSEVA341 plasmid, we added restriction sites to both ends of the sequence via PCR. The resulting p341-porin-DTE-WT plasmid for DTE enzyme expression was constructed through Golden Gate assembly. We ordered the designed synthetic sequences for DTE1 and DTE2 from BGI-Hexa and obtained the p341-porin-DTE-1 and p341-porin-DTE-2 plasmids using the same method. ![Figure 2A](https://static.igem.wiki/teams/5890/wetlab-picture/figure-2a.webp) Schematic diagrams of DTE enzyme expression system plasmids(1:DTE-WT plasmid map; 2: DTE-1 plasmid map; 3: DTE-2 plasmid map) 3.2 Construction of an Allulose Biosensor Based on RNA-pepper  Constructing the pET28-porin-psiRA-papper plasmid as a biosensor for DTE enzyme activity based on the fluorescent RNA pepper-HBC system. The pET28-porin-psiRA-papper plasmid was transformed into Escherichia coli BL21. ![Figure 2B](https://static.igem.wiki/teams/5890/wetlab-picture/figure-2b.webp) Plasmid map of the Allulose sensor based on RNA-pepper 3.3 Construction of the RNA-pepper-based DTE enzyme activity characterization system  Using electroporation, p341-porin-DTE-WT, p341-porin-DTE-1, and p341-porin-DTE-2 were separately transformed into strains containing pET28-porin-psiRA-papper to establish a dual-plasmid system for enzyme activity characterization.  The successful construction of the above-mentioned systems was verified by colony PCR and DNA sequencing. 4. Test4.1 DTE Enzyme Expression System Verification and Catalytic Activity Assessment  To further confirm the catalytic activity of the DTE enzyme, we employed an Agilent 1260 liquid chromatograph for precise detection of allulose in the culture system. We first prepared monosaccharide and mixed sugar solutions of D-fructose and D-allulose, determining their retention times under experimental conditions. Subsequently, we tested E. coli DH5α not transformed with the DTE enzyme expression plasmid, confirming that conventional E. coli cannot catalyze the conversion of D-fructose to D-allulose.  To confirm the system's proper expression function, RT-qPCR was employed to detect the expression levels of the three DTE enzymes, using E. coli DH5α without the plasmid as a control. Results indicated that all three systems functioned well, exhibiting significant DTE enzyme expression levels. ![Figure 2B](https://static.igem.wiki/teams/5890/wetlab-picture/figure-4b.webp) Relative expression levels of three DTE genes  Subsequently, we analyzed the post-reaction culture supernatants from single-plasmid strains harboring p341-porin-DTE-WT, p341-porin-DTE-1, and p341-porin-DTE-2. However, the results showed that the allulose content produced by all three strains was extremely low, despite previous fluorescence level measurements indicating that all three DTE enzymes possessed a certain degree of catalytic activity. This suggests potential issues with the reaction conditions. 4.2 Performance testing of the RNA-pepper-based allulose biosensor  The successfully constructed pET28-porin-PsiRA-Pepper plasmid was efficiently transformed into E. coli BL21. The recombinant E. coli was cultured in LB medium at 37°C for 10-12 hours. Substrate D-allulose was added at various concentrations: 0.001, 0.01, 0.1, 0.1, 10, 100, 1000 mM/L. After 2 h of continued incubation, HBC dye was added. Changes in fluorescence intensity following the addition of different D-allulose concentrations were measured. Results demonstrated the system functions effectively, capable of detecting allulose concentration changes. ![Figure 2B](https://static.igem.wiki/teams/5890/wetlab-picture/figure-5a-new.webp) Test results of the RNA-pepper-based allulose biosensor  Through induction with varying concentrations of allulose, we demonstrated that this biosensor exhibits a significant response to allulose within the concentration range of 10 to 1000 mM. The relationship between the output signal intensity and the input concentration approximately follows a sigmoidal curve, which is consistent with theoretical predictions and confirms the proper functioning of the system. 4.3 Detection Using an RNA-Pepper-Based DTE Enzyme Activity Characterization System  Next, the activity of different DTE enzymes was characterized using a dual-plasmid system. First, the optimal fructose induction concentration was determined. The p341-porin-DTE-WT and pET28-porin-psiRA-pepper dual-plasmid system was selected. Catalytic reactions were conducted at 100, 200, 300, 400, and 500 mM/L. HBC buffer was added to the supernatant, and fluorescence levels were measured using a microplate reader. The optimal catalytic concentration was confirmed to be 300 mM/L. To determine reaction duration, we employed the dual-plasmid system of pET28-porin-psiRA-pepper and pSEVA341-porin-DTE-WT under 300 mM/L catalytic conditions, measuring fluorescence intensity at 2, 4, and 6 hours. Considering the degradation curve of pepper mRNA obtained in the dry experiment, we concluded that fluorescence intensity beyond 6 hours no longer accurately reflects actual allulose concentrations. Therefore, subsequent measurements will focus on monitoring fluorescence intensity changes during the 6-hour catalytic process.  In a 300 mM/L fructose environment, monitoring the fluorescence intensity changes over time for different dual-plasmid systems revealed no significant differences in DTE enzyme activity during the 2-hour reaction. However, at 4 hours, the fluorescence intensity corresponding to the newly designed DTE enzyme showed a certain degree of reduction. At 6 hours, the fluorescence intensity of DTE1 exhibited a relatively significant increase compared to both WT and DTE2. It can be reasonably inferred that both the newly designed DTE1 and DTE2 enzymes possess catalytic activity, with DTE1 potentially exhibiting a slightly higher conversion rate than the WT (wild-type) DTE enzyme. ![Figure 5e](https://static.igem.wiki/teams/5890/wetlab-picture/figure-5e.webp) Comparison of catalytic results for different DTE enzymes Through our analysis, we have successfully constructed the system for DTE enzyme expression and indirectly measured the activity of different DTE enzymes. According to the fluorescence data, the novel DTE enzyme DTE1, which was designed with the assistance of AI tools, showed a higher conversion rate after 6 hours of catalysis. However, due to the inherent instability of pepper RNA, definitive conclusions cannot be drawn based solely on fluorescence levels. It is necessary to further refine the liquid chromatography experimental protocol to obtain precise quantitative data, which is essential for accurately calculating key indicators such as the enzyme's conversion rate. 5. Learn  Subsequent analysis revealed: although fluorescence data indicated catalytic activity across different DTE enzymes, alonose sensor measurements suggested all three enzymes produced alonose levels around 10 mM—far below the 30% conversion rate reported in prior literature. Therefore, we conclude that insufficient bacterial biomass and overall DTE enzyme quantity prevent the production of adequate allulose. Consequently, for industrial allulose production, considerations must extend beyond the catalytic activity of DTE enzymes to include fundamental factors such as enzyme expression levels and cell density.

![进度指示图片](https://static.igem.wiki/teams/5890/alo/12c905f248b198b39bf1495fd371412f.webp)

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![进度指示图片](https://static.igem.wiki/teams/5890/alo/12c905f248b198b39bf1495fd371412f.webp)

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Document Navigation

* [1.Construction Results](#construction)
  + [1.1 Construction of DTE Enzyme Expression Systems](#constructiona)
  + [1.2 Construction of an RNA-pepper-based DTE enzyme activity characterization system](#constructionb)
* [2. Test Results](#test)
  + [2.1 DTE enzyme expression system detection and DTE enzyme catalytic activity experimental results](#testa)
  + [2.2 Experimental results of the RNA-pepper-based allulose biosensor](#testb)
  + [2.3 Experimental results of the RNA-pepper-based DTE enzyme activity characterization system](#testc)

# Results

## 1. Construction Results1.1 Construction of DTE Enzyme Expression Systems  We constructed different DTE enzyme expression systems following this logic: ![Figure 3A](https://static.igem.wiki/teams/5890/wetlab-picture/figure-3a.webp) Schematic of DTE enzyme expression system construction (1: DTE-WT construction process; 2: DTE-1 construction process; 3: DTE-2 construction process)  We first obtained the target fragment via PCR, added BsaI restriction sites at both ends, then constructed the recombinant plasmid using Golden Gate technology. Following antibiotic screening, potential colonies were selected via colony PCR, and sequencing confirmed correct plasmid ligation and transformation outcomes. ![Figure 3B](https://static.igem.wiki/teams/5890/wetlab-picture/figure-3b.webp) Band PCR results (Target band sizes: p341-porin, 3500 bp; A1B, 1813 bp; WT, 903 bp; A2, 902 bp; B, 937 bp) ![Figure 3C](https://static.igem.wiki/teams/5890/wetlab-picture/figure-3c.webp) Colony PCR results (target band sizes: DTE2-bPCR, 2221 bp; DTE1-bPCR, 2200 bp; WT-bPCR, 1432 bp) ![Figure 3D](https://static.igem.wiki/teams/5890/wetlab-picture/figure-3d.webp) Colony growth after 12 hours of transformation  Colony clones expressing the three DTE enzyme systems were successfully obtained.  Colonies with appropriate fragment sizes were submitted for sequencing. The results are shown below, indicating normal sequences suitable for subsequent experiments. ![Figure 3E](https://static.igem.wiki/teams/5890/wetlab-picture/figure-3e.webp) Sequencing results schematic 1.2 Construction of an RNA-pepper-based DTE enzyme activity characterization system  Using electroporation, the DTE enzyme expression system and the RNA-pepper-based allulose biosensor were introduced into E. coli DH5α to construct an RNA-pepper-based DTE enzyme activity characterization system. The specific procedure is as follows:  Take overnight-cultured E. coli and transfer it into fresh medium for 4 hours of cultivation;  Collect the transferred E. coli, wash three times with 10% glycerol, and aliquot 100 μL into sterile centrifuge tubes to prepare E. coli competent cells.  In a laminar flow hood, add all constructed plasmids to the thawed competent E. coli. Gently mix, then transfer to a pre-chilled electroporation cup placed on ice. Wipe moisture from both sides of the cup before placing it in the electroporator for transformation.  Add 1 mL LB medium to the electroporation cup, vortex thoroughly, and transfer to centrifuge tubes. Incubate at 37°C, 200 rpm for 1 hour to allow recovery.  Centrifuge the recovered culture at 25°C and 4000 rpm for 2 minutes. Discard most of the supernatant, resuspend the remaining cells, and plate them onto LB solid agar plates containing the corresponding antibiotic. Invert the plates and incubate at 37°C for 16 hours.  Select moderately sized colonies growing on the corresponding antibiotic plates for colony PCR verification. Results are as follows: ![Figure 3F](https://static.igem.wiki/teams/5890/wetlab-picture/figure-3f.webp) Colony PCR results (target band sizes: DTE-WT: D: 2152 bp, P: 1886 bp; DTE2-bPCR: 2221 bp; DTE1-bPCR: 2200 bp; pepper: 1778 bp)  Some colonies yielded correct PCR verification results and were suitable for further experiments. With this, the construction of all three systems was completed, enabling progression to the subsequent efficacy characterization phase. 2. Test Results2.1 DTE enzyme expression system detection and DTE enzyme catalytic activity experimental results  We first measured the growth curves of strains expressing different DTE enzymes. We observed that the growth rates of different strains were largely similar, as the sizes of these three DTE enzymes were nearly identical. ![Figure 4A](https://static.igem.wiki/teams/5890/wetlab-picture/figure-4a.webp) Growth curves of colonies expressing DTE enzyme systems  To validate the system's ability to express DTE enzymes, we detected the expression levels of the three DTE enzymes via RT-qPCR. E. coli DH5α without the expression system served as the blank control. The detection results for all three expression systems were significantly higher than the control group. ![Figure 4B](https://static.igem.wiki/teams/5890/wetlab-picture/figure-4b.webp) Relative expression levels of three DTE genes  Next, we precisely quantified allulose content in the catalytic system using high-performance liquid chromatography (HPLC). The HPLC instrument configuration was as follows:  Column: Installed Waters Sugar-PAK I sugar-specific separation column.  Mobile phase: Wahaha purified water containing 50 mg/L ethylenediaminetetraacetic acid (EDTA), flow rate set at 0.4 mL/min.  Column temperature: Set to 80°C.  Detector: A refractive index detector was selected.  Injection volume: 5 μL per injection.  We prepared 50 mg/mL monosaccharide solutions of D-fructose and D-allulose to determine the retention times of their chromatographic peaks. As shown in the schematic diagram below, the retention time for D-fructose is 14.5 min, while that for D-allulose is approximately 15 min: ![Figure 4C](https://static.igem.wiki/teams/5890/wetlab-picture/figure-4c.webp) Chromatograms of fructose solution, allulose solution, and mixed fructose-allulose solution (1: Chromatogram of fructose monosaccharide solution; 2: Chromatogram of allulose monosaccharide solution; 3: Chromatogram of mixed sugar solution)  Next, to quantitatively calculate the catalytic efficiency of DTEK, we prepared mixed sugar solutions containing D-fructose and D-allulose at concentrations of 0.0313, 0.125, 0.25, 0.5, 0.75, and 1.5 g/L of D-fructose and D-allulose mixed sugar solutions. Standard curves for D-fructose and D-allulose were plotted with concentration on the x-axis and peak area on the y-axis. ![Figure 4D](https://static.igem.wiki/teams/5890/wetlab-picture/figure-4d.webp) Standard Curves for Fructose and Allulose Solutions  The standard curves exhibit good fit.  Finally, wild-type E. coli DH5α was selected as the control group, and recombinant E. coli DH5α (pSEVA341-DTE-WT) as the experimental group for separate cultivation.  After adjusting the bacterial density to be consistent, the cultures were incubated at 37°C for 2 hours. During this period, 300 mM fructose solution was added to the recombinant E. coli DH5α (pSEVA341-DTE-WT) culture medium for induction. After induction, supernatants were collected from both control and experimental groups. Each supernatant underwent 50-fold dilution to ensure the diluted fructose and allulose concentrations fell within the standard curve range (0.0313–1.5 g/L). Diluted samples were filtered through a 0.22 μm aqueous phase membrane before injection for analysis. Chromatograms were recorded. Calculate the concentrations of D-fructose and D-allulose in the samples based on the standard curve. &emspThe following formula was used to calculate D-allulose conversion rate, total D-fructose conversion rate, consumption rate, and other data reflecting the catalytic efficiency of DTE enzyme..  D-Allulose Conversion Rate (F) Calculation Formula \[ F = \frac{c\_a}{c\_{f\_0}} \times 100\% \]  Where: D = D-allulose conversion rate; ca = post-reaction D-allulose concentration (g/L); cf0 = initial D-fructose concentration (g/L).  D-Fructose Total Conversion Rate (D) Calculation Formula \[ D = \frac{c\_{f\_0} - c\_f}{c\_{f\_0}} \times 100\% \]  Where: D represents the total D-fructose conversion rate; cf₀ denotes the initial D-fructose concentration (g/L); cf indicates the D-fructose concentration in the supernatant after the whole-cell reaction (g/L).  Formula for D-fructose consumption rate (G) \[ G = D - F \]  The fructose consumption rate G represents the difference between the total D-fructose conversion rate and the D-allulose conversion rate, indicating the proportion of D-fructose diverted into alternative metabolic pathways.  However, liquid chromatography analysis revealed that both the control blank E. coli and E. coli expressing the DTE enzyme system exhibited indistinct allose peaks. Analysis indicated that insufficient bacterial biomass led to low allose production levels below the detection limit of liquid chromatography. ![Figure 4E](https://static.igem.wiki/teams/5890/wetlab-picture/figure-4e.webp) Chromatograms of different samples (1: DH5α; 2: DTE-WT; 3: DTE-1; 4: DTE-2) 2.2 Experimental results of the RNA-pepper-based allulose biosensor  Following construction and transformation of the pet28-porin-PsiRA-pepper plasmid, induction was performed at various allulose concentrations. The fluorescence intensity of the pepper-HBC dye complex was measured after 2 hours, yielding the following results: ![Figure 5A](https://static.igem.wiki/teams/5890/wetlab-picture/figure-5a-new.webp) Test results of the RNA-pepper-based allulose biosensor  It can be observed that as the concentration of the allulose inducer increases, the fluorescence intensity of the pepper-HBC dye complex also gradually increases. Furthermore, the fluorescence intensity exhibits an approximate S-shaped curve relationship with the inducer concentration, consistent with the expected results. 2.3 Experimental results of the RNA-pepper-based DTE enzyme activity characterization system  By transferring Pet28-porin-psiRA-pepper and pSEVA341-porin-DTE into DH5α and validating them, we successfully constructed the genetic circuit diagram shown below: As DTE enzyme catalyzes fructose conversion to allulose, exposure to PsiR inhibits pPsiA expression, leading to pepper expression. The fluorescence intensity of the pepper-HBC complex indirectly reflects DTE enzyme catalytic activity. ![Figure 5B](https://static.igem.wiki/teams/5890/dry-lab-modelling-pictures/figure-1.webp) RNA-pepper-based DTE enzyme activity characterization system  First, we explored the optimal operating conditions required for this activity characterization system. To determine the optimal fructose induction concentration, we incubated the cellular catalytic system at 100, 200, 300, 400, and 500 mM/L for 4 hours and measured fluorescence intensity. We observed that beyond 300 mM/L, further increases in fructose concentration did not significantly alter fluorescence intensity. Therefore, subsequent experiments employed 300 mM/L fructose for catalysis. ![Figure 5C](https://static.igem.wiki/teams/5890/wetlab-picture/figure-5c.webp) confirms the optimal fructose induction concentration.  Similarly, to determine the optimal reaction duration, we reacted the Pet28-porin-psiRA-pepper and pSEVA341-porin-DTE-WT systems at 300 mM/L for 2, 4, and 6 hours, obtaining fluorescence intensity data. Considering the degradation curve of pepper mRNA obtained in the dry experiment section, we concluded that fluorescence intensity beyond 6 hours of reaction no longer accurately reflects the actual allulose concentration. Therefore, in subsequent measurements, we will continue to monitor fluorescence intensity changes during the 6-hour catalytic process. ![Figure 5D](https://static.igem.wiki/teams/5890/wetlab-picture/figure-5d.webp) confirms the catalytic time.  To characterize the catalytic performance of different DTE enzymes, we measured the fluorescence intensity of strains expressing various DTE enzymes at 2h, 4h, and 6h under 300 mM/L fructose conditions, as shown below: ![Figure 5E](https://static.igem.wiki/teams/5890/wetlab-picture/figure-5e.webp) Comparison of catalytic results for different DTE enzymes   It can be observed that both the newly designed DTE1 and DTE2 exhibit catalytic activity. After 2 hours of catalysis, the activity differences among the various DTE enzymes are not significant. However, at 4 hours, the fluorescence intensity in both the DTE1 and DTE2 groups decreases to some extent. This may be because pepper itself is unstable, and the catalytic rates of DTE1 and DTE2 are not high. Additionally, converting fructose to allulose via DTE enzymes requires time before allulose interacts with the pepper expression inhibitor, leading to reduced pepper expression during this period. At 6 hours, fluorescence intensity increased across all groups. Since the DTE enzyme's conversion of fructose to allulose is reversible, it can be inferred that DTE1 likely exhibited higher conversion efficiency at this time point, while no significant activity difference was observed between DTE2 and the wild-type (WT).

![进度指示图片](https://static.igem.wiki/teams/5890/alo/12c905f248b198b39bf1495fd371412f.webp)

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