In our project, our team designed a total of 59 components, including 34 basic parts and 25 combination parts. Each component was meticulously crafted to align with our project objectives. Detailed information about each part can be found on the team part page or by clicking the corresponding component number below. Next, we will introduce our components through five key areas: flavin mononucleotide (FMN)-dependent fluorescent protein Bs2, tagatose-4-epimerase gene mining, studies on soluble expression, mutation analysis, and biosensors.
| part number | typology | name | description |
| BBa_K4119002 | Coding | Bs2 | A flavin mononucleotide (FMN)-dependent fluorescent protein |
| BBa_K5366071 | Regulatory | J23119 promoter | The "consensus" promoter sequence, which is the strongest member of the family |
| BBa_K5366072 | RBS | RBS (J23119 promoter) | ribosome binding site |
| BBa_K5366073 | Composite | J23119 promoter-RBS-Bs2-6×His-T7 termonator | The Bs2 expression plasmid with J23119 as the promoter |
In this study, we screened four protein sequences with potential tagatose-4-epimerase activity, specifically designated as AJC7, MBC, TET, and HDM. These sequences were then ligated into the pET-28a(+) vector for subsequent characterization experiments. The results revealed that, among the four sequences, wild-type AJC7 exhibited the highest enzyme activity.
| part number | typology | name | description |
| BBa_K5366016 | Coding | UxaE | Sequences of Thermotoga petrophila origin with tagatose-4-epimerase activity |
| BBa_K5366017 | Coding | AJC7 | Pseudothermotoga hypogea DSM-derived sequences with tagatose-4-epimerase activity |
| BBa_K5366018 | Coding | MBC | Sequences of Chloroflexota bacterium origin with tagatose-4-epimerase activity |
| BBa_K5366019 | Coding | TET | Sequences from the source of Candidatus Aerophobetes bacterium with tagatose-4-epimerase activity |
| BBa_K5366020 | Coding | HDM | Sequences of Thermotogales bacterium origin with tagatose-4- epimerase activity |
| BBa_K5366021 | Composite | T7 promoter-RBS-UxaE-6× His -T7 termonator | UxaE structural gene expression plasmid Candidatus Aerophobetes bacterium with tagatose-4-epimerase activity |
| BBa_K5366022 | Composite | T7 promoter-RBS-AJC7-6× His-T7 termonator | AJC7 structural gene expression plasmid |
| BBa_K5366023 | Composite | T7 promoter-RBS-MBC-6× His-T7 termonator | MBC structural gene expression plasmid |
| BBa_K5366024 | Composite | T7 promoter-RBS-TET-6× His-T7 termonator | TET structural gene expression plasmid |
| BBa_K5366025 | Composite | T7 promoter-RBS-HDM-6× His -T7 termonator | HDM structural gene expression plasmid |
| BBa_K5366053 | Tag | 6× His Tag | C-term His tag for purification and identification of protein of interest. |
| BBa_K5366068 | Regulatory | T7 promoter (lacI repressile) | A lac repressible T7 promoter. |
| BBa_K5366069 | Termonator | T7 terminator | Terminator for bacterial expression |
| BBa_K5366070 | RBS | RBS | Efficient ribosome binding site from bacteriophage T7 gene 10. |
To tackle the issue of protein aggregation during expression, we incorporated a pro-fusion tag sequence at the N-terminus of the target protein in order to promote soluble expression. This approach aimed to bind and separate the aggregation-prone folding intermediates of the target proteins. The experimental results demonstrated that for the soluble expression of the AJC7 protein, the pro-fusion tags NusA and MBP significantly enhanced solubility by 1.63-fold and 1.69-fold, respectively, compared to AJC7 without the tag.
| part number | typology | name | description |
| BBa_K5366026 | Protein_ Domain | MBP | Maltose-binding protein with wide solubilization range and high solubilization efficiency |
| BBa_K5366027 | Protein_ Domain | TrxA | Thioredoxin A, thermally stable, highly soluble |
| BBa_K5366028 | Protein_ Domain | NusA | Transcription anti-termination factor A, a highly soluble protein that binds and separates aggregation-prone folding intermediates of its target proteins |
| BBa_K5366029 | Composite | T7 promoter-RBS-MBP-linker-AJC7-6×His -T7 termonator | MBP pro-fusion labeling and AJC7 structural gene co-expression plasmid |
| BBa_K5366030 | Composite | T7 promoter-RBS-TrxA-linker-AJC7-6×His -T7 termonator | TrxA pro-fusion labeling and AJC7 structural gene co-expression plasmid |
| BBa_K5366031 | Composite | T7 promoter-RBS-NusA-linker-AJC7-6×His -T7 termonator | NusA pro-fusion labeling and AJC7 structural gene co-expression plasmid |
The DNA of AJC7 and MBP were both inserted into a plasmid, resulting in the construction of a recombinant plasmid. Subsequently, transcription and translation were carried out, leading to the fusion of NusA at the N-terminus of AJC7.
Among all the stacked mutations, we identified the optimal mutant, AJC7-S125D/T181A/I129T, which catalyzed the production of 37 g/L of tagatose from 100 g/L of fructose within 2 hours. This process achieved a maximum conversion rate of 37% and a yield of 18.5 g/(L·h) at a final enzyme concentration of 23 mg/mL. When compared to the previously reported best mutant, UxaE 5V, this performance was two-fold higher.
| part number | typology | name | description |
| BBa_K5366032 | Coding | AJC7/S125D | AJC7 single point mutant |
| BBa_K5366033 | Coding | AJC7/T181A | AJC7 single point mutant |
| BBa_K5366034 | Coding | AJC7/H342L | AJC7 single point mutant |
| BBa_K5366035 | Coding | AJC7/I129T | AJC7 single point mutant |
| BBa_K5366036 | Coding | AJC7/L140P | AJC7 single point mutant |
| BBa_K5366037 | Coding | AJC7/S125D/ T181A | AJC7 two-point mutant |
| BBa_K5366038 | Coding | AJC7/S125D/ H342L | AJC7 two-point mutant |
| BBa_K5366039 | Coding | AJC7/S125D/ I129T | AJC7 two-point mutant |
| BBa_K5366040 | Coding | AJC7/S125D/ L140P | AJC7 two-point mutant |
| BBa_K5366041 | Coding | AJC7/S125D/T181A/ I129T | AJC7 triple point mutant |
| BBa_K5366042 | Coding | AJC7/S125D/T181A/ L140P | AJC7 triple point mutant |
| BBa_K5366043 | Coding | AJC7/S125D/T181A/ H342L | AJC7 triple-point mutant |
| BBa_K5366044 | Coding | AJC7/S125D/T181A/ I129T/ L140P | AJC7 four-point mutant |
| BBa_K5366045 | Coding | AJC7/S125D/T181A/ I129T/L140P/ H342L | AJC7 five-point mutant |
| BBa_K5366054 | Composite | T7 promoter-RBS-AJC7/S125D-6×His-T7 termonator | AJC 7 single-point mutation expression plasmid |
| BBa_K5366055 | Composite | T7 promoter-RBS-AJC7/T181A-6×His-T7 termonator | AJC 7 single-point mutation expression plasmid |
| BBa_K5366056 | Composite | T7 promoter-RBS-AJC7/H342L-6×His-T7 termonator | AJC 7 single-point mutation expression plasmid |
| BBa_K5366057 | Composite | T7 promoter-RBS-AJC7/I129T-6×His-T7 termonator | AJC 7 single-point mutation expression plasmid |
| BBa_K5366058 | Composite | T7 promoter-RBS-AJC7/L140P-6×His-T7 termonator | AJC 7 single-point mutation expression plasmid |
| BBa_K5366059 | Composite | T7 promoter-RBS-AJC7/S125D/ T181A-6×His-T7 termonator | AJC 7 AJC 7 two-point mutation expression plasmid |
| BBa_K5366060 | Composite | T7 promoter-RBS-AJC7/S125D/ H342L-6×His-T7 termonator | AJC 7 AJC 7 two-point mutation expression plasmid |
| BBa_K5366061 | Composite | T7 promoter-RBS-AJC7/S125D/ I129T-6×His-T7 termonator | AJC 7 two-point mutation expression plasmid |
| BBa_K5366062 | Composite | T7 promoter-RBS-AJC7/S125D/ L140P-6×His-T7 termonator | AJC 7 two-point mutation expression plasmid |
| BBa_K5366063 | Composite | T7 promoter-RBS-AJC7/S125D/T181A/ I129T-6×His-T7 termonator | AJC 7 triple point mutation expression plasmid |
| BBa_K5366064 | Composite | T7 promoter-RBS-AJC7/S125D/T181A/ L140P-6×His-T7 termonator | AJC 7 triple point mutation expression plasmid |
| BBa_K5366065 | Composite | T7 promoter-RBS-AJC7/S125D/T181A/ H342L-6×His-T7 termonator | AJC 7 triple point mutation expression plasmid |
| BBa_K5366066 | Composite | T7 promoter-RBS-AJC7/S125D/T181A/ I129T/ L140P-6×His-T7 termonator | AJC 7 four-point mutation expression plasmid |
| BBa_K5366067 | Composite | T7 promoter-RBS-AJC7/S125D/T181A/ I129T/L140P/ H342L-6×His-T7 termonator | AJC 7 five-point mutation expression plasmid |
| part number | typology | name | description |
| BBa_K5366046 | Coding | GFP | Green fluorescent protein |
| BBa_K5366047 | Coding | ECOLIN_RS18700 tagR | Transcriptional factor |
| BBa_K5366048 | Regulatory | Feature 14 | Bidirectional promoter |
| BBa_K5366049 | Composite | pACYCDuet-1-tagR-P-RS-sfGFP1 | Sensor plasmids for deterrent protein and green fluorescent constructs |
| BBa_K5366050 | Coding | Gene Cluster | Includes tagatose transporter proteins, tagatose-responsive transcription factors and promoters |
| BBa_K5366051 | Composite | pCDFDuet-P1-sfGFP1 | Plasmids expressing green fluorescence |
| BBa_K5366052 | Coding | ECOLIN_RS18695 | Transcriptional factor |
Among our designed parts, BBa K5366017, BBa K5366021, and BBa K5366020 all demonstrated higher enzyme activity than the screening template, exhibiting superior tagatose-4-epimerase activity. Notably, BBa K5366017 showcased the highest enzyme activity; this part represents a newly identified Tagatose-4-epimerase sequence obtained through gene mining in this project, highlighting its significant potential.
Additionally, BBa K5366032 to BBa K5366036 are the five potential mutation sites identified through our mutagenesis approach. By rational design, we introduced mutations in the selected proteins that may enhance enzymatic activity. These sites were chosen based on their predicted ability to improve substrate binding and catalytic efficiency.
Figure 1 Molecular docking results of D-tagatose-4-epimerase (using AJC7 as an example)
These mutant elements are all elevated compared to the wild-type type
Figure 2 The concentrations of tagatose produced by Wild-type, S125D, T181A, H342L, I129T, and L140P with 100 g/L fructose as substrate for 5 h
BBa K5366037 to BBa K5366045 represent iterative mutations based on a single point mutation. Among these, BBa K5366041 demonstrated the most significant enhancement in enzyme activity, with its catalytic efficiency nearly tripling compared to BBa K5366041 (Fig. 3). At a final enzyme concentration of 23 mg/mL, it catalyzed the production of 37 g/L of Tagatose from 100 g/L of fructose within 120 minutes, achieving a 37% conversion rate and a yield of 18.5 g/(L·h). At the 80-minute mark, 33 g/L of Tagatose was produced, yielding a 33% conversion rate and an impressive yield of 24 g/(L·h), which represented the highest yield observed (Fig. 4). The values of Km (mM) and Vmax (mM/h) were determined using nonlinear regression with Origin software, yielding a Vmax of 27.309mM/h and a Km of 99.805mM (Fig. 5).
Figure 3 The concentration of tagatose produced by Wild-type, S125D, S125D/ T181A, S125D/ T181A/ I129T, and S125D/ T181A/ H342L, respectively, with 100 g/L fructose as the substrate for 5 h.
Figure 4 Conversion rate of AJC7-S125D/ T181A/ I129T under optimal conditions
Figure 5 Nonlinear regression equation for substrate concentration and reaction rate
To investigate the relationship between mutation sites during iterative mutation, we conducted molecular docking on the most effective mutants: S125D, S125D/T181A, and S125D/T181A/I129T. This analysis aimed to examine the role of the shared mutation site, S125D, across different mutants. The results of the docking studies are presented below:
Figure 6 Docking model of D-fructose in the S125D mutant
Figure 7 Docking model of D-fructose in the S125D/T181A mutant.
Figure 8 Docking model of D-fructose in the S125D/T181A/I129T mutant
Their results indicate that S125D plays a consistent role in all mutations due to the first step in the conversion of d-fructose to d-tagatose is the production of a glyceraldehyde intermediate.When serine is mutated to aspartic acid, the carboxy group of aspartic acid can interact with the terminal aldehyde group of the glyceraldehyde intermediate, promoting the protonation of the aldehyde and thus facilitating the catalysis and production of D-tagatose. Furthermore, the negatively charged aspartic acid may enhance interactions with the positively charged substrate and alter the charge distribution in the binding pocket, thereby improving the catalytic activity of the enzyme. The importance of the S125D mutation for increasing the activity of tagatose-4-epimerase is evident.
For the newly identified optimal mutant BBa K5366041 of the potential tagatose-4-epimerase sequence, we further investigated its enzymatic properties. This mutant catalyzed the conversion of fructose to tagatose with an optimal reaction temperature of 70 ℃(Figure 9) and an optimal pH of 9.0 (Figure 10). These findings enabled the iGEM team to conduct additional studies on its properties.
Figure 9 Effect of reaction temperature on the enzyme activity of AJC7-S125D/ T181A/ I129T.
Figure 10 Effect of pH on the enzyme activity of AJC7-S125D/ T181A/ I129T .
Upon reviewing the iGEM part library, we discovered that past teams, such as XJTU-China 2018, had also provided sequences for enzymes capable of catalyzing the generation of tagatose from fructose. However, these teams did not present any experimental data. In contrast, our team proposes an enzyme that catalytically generates tagatose from fructose, supported by wet experimental data for the first time in iGEM history. Furthermore, we modified this enzyme through protein engineering, and the three-point mutant of this enzyme resulted in a maximum conversion of 37%, achieving peak yield rates of 24 g/(L·h). While comparable data from previous iGEM entries is lacking, literature review indicates that our proposed enzyme remains competitive among others with similar functionalities. This advancement presents promising prospects for the industrial-scale production of Tagatose.
| part number | typology | name | description |
| BBa_K5366017 | Coding | AJC7 | Pseudothermotoga hypogea DSM-derived sequences with tagatose-4-epimerase activity |
| BBa_K5366022 | Composite | T7 promoter-RBS-AJC7-6× His-T7 termonator | AJC7 structural gene expression plasmid |
| BBa K5366032 | Coding | AJC7/S125D | AJC7 single point mutant |
| BBa_K5366054 | Composite | T7 promoter-RBS-AJC7/S125D-6×His-T7 termonator | AJC 7 single-point mutation expression plasmid |
| BBa K5366037 | Coding | AJC7/S125D/ T181A | AJC7 two-point mutant |
| BBa_K5366059 | Composite | T7 promoter-RBS-AJC7/S125D/ T181A-6×His-T7 termonator | AJC 7 two-point mutation expression plasmid |
| BBa K5366041 | Coding | AJC7/S125D/T181A/ I129T | AJC7 triple point mutant |
| BBa_K5366063 | Composite | T7 promoter-RBS-AJC7/S125D/T181A/ I129T-6×His-T7 termonator | AJC 7 triple point mutation expression plasmid |