This competition year we contributed to the iGEM’s Registry of Standard Biological Parts by adding a New Composite Part to the Registry:
pTRKH3 with Engineered N-terminal Secretion Signal Peptide pgxC Composite Part (Final Construct; BBa_252WDOMF–later modified to as BBa_25TXYOA2)
In creating this New Composite Part, we adhered to the following framework of workflow:
Assembling the New Composite Part
Building the New Composite Part
Quantifying the New Composite Part
Validating the New Composite Part
Composite Part #1: Assembling the Parts
Composite Part #1: Assembling the Parts
Working Logic Behind Assembly
To design our New Composite Part, we assembled the following parts:
ermB Promoter (BBa_K3183000)
N-terminal Secretion Signal Peptide of Usp45 (BBa_25KVZJA0)
pgxC Coding Sequence (BBa_252AGPHV)
Then, we designed a synthetic secretion signal fused directly to pgxC:
Engineered N-terminal Secretion Signal Peptide pgxC (BBA_2596ET3M)
Subsequently, assembling the promoter, secretion signal, coding sequence, and terminator into a single construct for expression, we obtained the following New Composite Part:
pTRKH3 with Engineered N-terminal Secretion Signal Peptide pgxC Composite Part (Final Construct; BBa_252WDOMF–later modified to as BBa_25TXYOA2)
(Figure 1. Schematic representation of the New Composite Part.)
The final construct, fused with pTRKH3 Plasmid Backbone (BBa_25GUIQHH), yields the final construct, which its sequence map is shown below:
(Figure 2. Schematic representation of the final plasmid construct.)
Besides the five new parts we added to the Registry, we added two additional parts to the Registry for engineering purposes. The additions are as follows:
*The pgxC signal peptide was modified by a silent PstI site removal to restore RFC 10 compatibility while maintaining the original amino acid sequence and secretion functionality.
Overview of the Parts
The characteristics of each part can be delineated by as below:
ermB Promoter:
Characteristics: The ermB promoter originates from the erythromycin resistance gene (ermB), which is commonly found in Gram-positive bacteria. It is known for its constitutive expression, robust expression, and antibiotic resistance association.
Why This Part: The ermB promoter not only avoids the need for costly or unstable inducers but also provides stable, continuous production of the desired enzyme, which is advantageous in food waste degradation settings where conditions are variable.
Application: In our iGEM project, the ermB promoter is used to drive constitutive expression of the signal peptide– modified pgxC gene in Lactobacillus reuteri. It ensures continuous production of polygalacturonase.
N-terminal Secretion Signal Peptide of Usp45:
Characteristics: The Usp45 secretion signal peptide originates from the major secreted protein Usp45 of Lactococcus lactis. It is one of the most widely used signal peptides for heterologous protein secretion in Gram-positive lactic acid bacteria systems.
Why This Part: The signal peptide is well-characterized and highly efficient for secretion in LAB such as L. lactis and L. reuteri and is compatible with a wide variety of heterologous proteins (enzymes, antigens, etc.).
Application: In our iGEM 2025 project, we fused the N-terminal Usp45 secretion signal peptide to the pgxC gene. It ensures that the pgxC enzyme is secreted outside of Lactobacillus reuteri rather than remaining intracellular.
pgxC Coding Sequence:
Characteristics: The pgxC gene encodes polygalacturonase, a pectinolytic enzyme belonging to the glycoside hydrolase family. The gene catalyzes the hydrolysis of α-(1,4)-glycosidic linkages in polygalacturonic acid (pectin backbone).
Why This Part: For our project, pgxC is specifically chosen because fruit peels contain high levels of pectin, and efficient degradation requires a robust polygalacturonase.
Application: In our iGEM construct, pgxC is placed under the control of the ermB constitutive promoter and fused with our engineered secretion signal peptide. It achieves extracellular secretion of polygalacturonase in Lactobacillus reuteri.
Engineered N-terminal Secretion Signal Peptide of pgxC:
Characteristics: Secretion signal peptides are short amino acid sequences that guide proteins into the Sec-dependent pathway, allowing them to be exported extracellularly.
Why This Part: By designing a synthetic secretion signal fused directly to pgxC, the project aims to maximize extracellular polygalacturonase yield in Lactobacillus reuteri, improving functional activity against fruit peel pectin within the context of our project.
Application: In our project, we designed an engineered N-terminal secretion signal peptide fused with pgxC for direct extracellular expression. We expect enhanced secretion yield, greater enzyme activity, and improved reproducibility.
pTRKH3 Plasmid Backbone:
Characteristics: The pTRKH3 plasmid is a shuttle vector designed for use in both Escherichia coli and lactic acid bacteria (LAB) such as Lactobacillus and Streptococcus.
Why This Part: Widely cited in literature for genetic engineering of Lactobacillus reuteri and Lactococcus lactis. This backbone is important because LABs are often used as food-grade hosts and require compatible shuttle vectors that can replicate efficiently.
Application: In our project, the pTRKH3 plasmid backbone effectively serves as the genetic platform to carry our engineered construct, which includes the promoter, gene of interest, secretion elements, and terminator.
Cumulation of the parts above yields the New Composite Part:
pTRKH3 with Engineered N-terminal secretion signal peptide pgxC Composite Part:
Characteristics: This part combines the ermB promoter, secretion signals, coding sequences, and terminators into a single construct for expression.
Why This Part: This composite design ensures stable and continuous product of polygalacturonase, direct secretion into the extracellular space for effective pectin degradation, and functional compatibility in the LAB chassis, a safe and food-grade host.
Application: In our iGEM project, the composite construct is the central innovation that enables engineered Lactobacillus reuteri to function as a fruit peel biodegradation agent. This composite part provides the functional blueprint for a scalable system in food waste management, composting, and potential bioreactor applications.
Assembly and Modeling of Parts
(Figure 3. Sequence design of composite part BBa_K5000001.)
The construct consists of the ermB constitutive promoter, followed by an EcoRI restriction site introduced at the 5′ end for cloning, the Usp45 secretion signal peptide, and the codon-optimized pgxC coding sequence. A downstream transcriptional terminator ensures stable expression. This design allows Lactobacillus reuteri to secrete active polygalacturonase for efficient fruit peel degradation.
(Figure 4. Sequence design of the 3′ region of codon-optimized pgxC.)
The terminal region of the pgxC coding sequence was engineered to include a BamHI restriction site at the 3′ end, allowing for flexible downstream cloning. Sequence alignment highlights the codon-optimized pgxC region, with annotated restriction sites (PpuMI, PciI, SexAI) for verification and modular assembly. This design ensures that pgxC can be efficiently integrated and expressed in the final plasmid construct for extracellular polygalacturonase secretion.
(Figure 5. Predicted 3D protein structure of engineered pgxC fused with USP45 peptide.)
The structure was modeled using the ESMFold program. The USP45 secretion signal peptide (left, red box) forms an extended flexible region, while the pgxC protein main body (right, blue box) adopts a stable β-sheet-rich fold characteristic of polygalacturonase enzymes. This fusion design facilitates extracellular secretion of the active enzyme by Lactobacillus reuteri.
Composite Part #2: Building the Composite Part
Composite Part #2: Building the Composite Part
Working Logic Behind Construction
To engineer L. reuteri for extracellular polygalacturonase (pgxC) secretion, we constructed a recombinant plasmid based on the shuttle vector pTRKH3. The construct was designed with restriction enzyme sites to enable efficient insertion of the codon-optimized pgxC gene fused to the Usp45 secretion signal peptide under control of the ermB constitutive promoter.
(Figure 6. Proposed engineered pgxC protein sequence fused with the USP45 secretion signal and cloned into the pTRKH3 backbone vector.)
Our work consisted of the following workflow framework:
Defining cloning strategy using restriction enzymes
Verification of cloning–gel electrophoresis
Verification of cloning–agar plates
Construction Results and Analysis
Part 1: Cloning Strategy Using Restriction Enzymes
The codon-optimized pgxC sequence was synthesized with EcoRI (5′) and BamHI (3′) restriction sites flanking the gene to facilitate cloning into the pTRKH3 plasmid vector:
EcoRI site: Positioned upstream of the promoter region, ensuring correct orientation and seamless ligation.
BamHI site: Placed downstream, allowing the pgxC insert to be stably joined with the vector backbone.
The pTRKH3 plasmid was linearized with these enzymes, and the pgxC fragment was ligated in-frame with the Usp45 signal sequence. This design ensures secretion of the expressed protein into the extracellular medium.
Schematic representation of the recombinant pTRKH3 plasmid (9585 bp) containing the ermB promoter, Usp45 secretion signal peptide, and the codon-optimized pgxC gene. Restriction enzyme sites EcoRI and BamHI (red boxes) were introduced to enable insertion of pgxC. The construct provides erythromycin resistance for selection in both E. coli and L. reuteri.
Part 2: Verification of Cloning–Gel Electrophoresis
(Figure 7. Verification of pgxC gene insertion into the pTRKH3 plasmid by restriction digestion. Agarose gel electrophoresis confirmed successful cloning of pgxC into the pTRKH3 backbone.)
The BamHI-only digest produced a single band at ~9585 bp, corresponding to the intact recombinant plasmid. The BamHI + EcoRI double digest generated two fragments: one corresponding to the vector backbone (8238 bp) and the other to the pgxC insert (1347 bp), consistent with the expected sizes of the engineered construct.
After ligation, the recombinant plasmid was first transformed into E. coli DH5α for propagation. Plasmid DNA was extracted and subjected to:
Restriction digestion analysis – BamHI-only digestion produced a single band (9585 bp), while BamHI + EcoRI double digestion produced two bands (8238 bp and 1347 bp), confirming correct pgxC insertion.
Agarose gel electrophoresis – Confirmed expected fragment sizes (Figure 7).
Sanger sequencing – Verified correct orientation and codon integrity of pgxC insert.
Once verified, the recombinant plasmid pTRKH3-pgxC was introduced into L. reuteri by electroporation, generating the strain for subsequent protein expression and functional assays.
Part 3: Verification of Cloning–Agar Plates
(Figure 8. Representation of agar plates after transformation of the pTRKH3-pgxC plasmid in L. reuteri (electroporation).)
The agar plate images illustrate the outcomes of transformation with the engineered pTRKH3-pgxC plasmid (Figure 8). The negative control plate (left, without plasmid) showed no colony growth, confirming the absence of background resistance. In contrast, the experimental plate (right, with plasmid) displayed abundant colony formation, indicating successful uptake and expression of the recombinant construct by L. reuteri competent cells. The observed colonies arose from bacteria harboring the pTRKH3-pgxC plasmid, which carries an antibiotic resistance marker, thereby allowing selective growth.
This result confirms the efficient introduction of the engineered plasmid into both E. coli and L. reuteri. Transformation of E. coli, used primarily as a cloning host, was achieved via the heat shock method, whereas L. reuteri, the intended expression host, was successfully transformed using electroporation. The ability to establish the construct in both bacterial systems demonstrates its broad host compatibility. The high density of colonies observed on the plasmid-containing plates highlights robust transformation efficiency, providing a solid foundation for downstream applications such as plasmid propagation, protein secretion analysis, and functional assays of the engineered pgxC enzyme.
Composite Part #3: Quantifying the Composite Part
Composite Part #3: Quantifying the Composite Part
Working Logic Behind Quantification
Following successful construction of the recombinant plasmid pTRKH3-pgxC, it was essential to evaluate whether the engineered Lactobacillus reuteri could express, secrete, and functionally utilize the pgxC enzyme. The rationale behind testing was to confirm that the designed genetic circuit not only worked at the DNA level but also translated into measurable protein production and functional pectin degradation. The specific objectives of the testing phase were:
To confirm expression of pgxC at the protein level by detecting the expected 48.5 kDa band using SDS-PAGE.
To quantify the amount of secreted protein in the engineered strain compared to controls using the Bradford assay.
Quantification of Results and Analysis
(Figure 9. Detection of secreted pgxC protein in Lactobacillus reuteri. (Left) Illustration showing separation of cell pellet and supernatant, with pgxC proteins detected in the extracellular fraction.)(Figure 10. Quantification of secreted proteins using the Bradford assay. (Top) BSA standard curve generated by measuring absorbance at 595 nm across a concentration range of 0–0.5 µg/µL. The linear regression equation (y = 0.401x + 0.091, R² = 0.94) was used to calculate protein concentrations in experimental samples. (Bottom) Protein concentrations in the culture supernatants of Lactobacillus reuteri with and without the pgxC plasmid. Samples with the plasmid showed a significantly higher protein concentration (0.10 ± 0.01 µg/µL) compared to the control without plasmid (0.04 ± 0.005 µg/µL), confirming enhanced extracellular protein expression.)
To verify that pgxC was expressed and secreted:
SDS-PAGE analysis revealed a distinct protein band at ~48.5 kDa in the culture supernatant of engineered L. reuteri, corresponding to the expected molecular weight of pgxC. This band was absent in the plasmid-free control strain.
Bradford assay quantified total secreted protein, showing significantly higher levels in engineered strains (0.10 µg/µL) compared to controls (0.04 µg/µL).
SDS-PAGE Analysis: The successful detection of pgxC in the extracellular fraction demonstrated that our engineered secretion system was functional, enabling L. reuteri to release the enzyme directly into the surrounding medium (Figure 9). This is a critical outcome for the project, as extracellular secretion eliminates the need for costly cell disruption or purification steps and allows the enzyme to act directly on fruit peel biomass. Since pectin is a major structural component of fruit peels, the secretion of active pgxC provides a practical foundation for efficient biodegradation of agricultural waste. Furthermore, the use of a probiotic chassis such as L. reuteri enhances the safety and scalability of this system.
Bradford Assay: The increase in protein concentration in plasmid-containing L. reuteri compared with the control confirmed that recombinant pgxC expression contributed to extracellular protein content (Figure 10). Although the total protein concentration measured included both secreted pgxC and native proteins, the relative increase in the plasmid-bearing condition is consistent with the SDS-PAGE results showing a clear 48.5 kDa pgxC band in the supernatant.
Composite Part #4: Validating the Composite Part
Composite Part #4: Validating the Composite Part
Working Logic Behind Validation
Following the successful quantification of the composite part, we validated the composite part with the working logic below, yielding a solid proof of concept of our project:
To validate the enzymatic activity of pgxC through a DNS assay that measures release of reducing sugars (D-galacturonic acid) from pectin substrates.
To demonstrate functional application of the engineered strain in degrading real fruit peel biomass (orange and apple), thereby linking molecular results to practical bioconversion outcomes.
Validation Results and Analysis
(Figure 11. Standard curve for D-galacturonic acid quantification using the DNS assay. The standard curve was generated by measuring absorbance at 540 nm for increasing concentrations of D-galacturonic acid (0–1.0 mg/mL).)(Figure 12. Quantification of D-galacturonic acid after 0, 12, 24, 48h incubation of supernatant produced from control (no pgxC) and engineered (pgxC) samples. Values are shown as mean ± SD from three replicates.)
The functional activity of pgxC was tested using the DNS (3,5-dinitrosalicylic acid) assay:
Engineered L. reuteri supernatants released significantly higher amounts of D-galacturonic acid from pectin substrate than control strains.
Absorbance at 540 nm was used to quantify reducing sugars, with engineered strains showing a ~2.5-fold increase in activity.
This demonstrated that the secreted pgxC retained enzymatic functionality.
To evaluate real-world utility, tests were performed using fruit peels (orange&apple):
(Figure 13. Comparison of D-galacturonic acid release from fruit peels after 24 h incubation with control and engineered L. reuteri supernatants. DNS assay was performed to quantify reducing sugars at 540 nm in extracts of banana, kiwi, pear, tomato, and watermelon peels.)
Homogenized fruit peel incubations – Engineered strains showed visible degradation, including liquefaction and reduction in peel particle size, compared to controls.
These results confirmed that engineered L. reuteri can degrade fruit peel components, validating the practical application of the construct.
DNS Assay: To evaluate the enzymatic activity of secreted pgxC, the DNS assay was performed to quantify the release of reducing sugars, specifically D-galacturonic acid, after incubating fruit peel pectin with culture supernatants. A standard curve generated using pure D-galacturonic acid (0–1.0 mg/mL) showed a strong linear correlation between concentration and absorbance at 540 nm (A = 1.207·C + 0.016, R² = 1.000, Figure 11).
To further assess the kinetics of pectin degradation, supernatants from control (no pgxC) and engineered (pgxC-expressing) Lactobacillus reuteri were incubated with pectin for 0, 12, 24, and 48 hours, and the release of reducing sugars was quantified by DNS assay (Figure 12). In the engineered strain, absorbance values at 540 nm increased steadily over time, reaching 0.35 at 12 h, 0.61 at 24 h, and 0.67 at 48 h, reflecting continuous production of D-galacturonic acid. By contrast, the control strain showed only minimal changes in absorbance throughout the incubation period, remaining below 0.1 even at 48 h. These results demonstrated that the pgxC-expressing strain actively degraded pectin in a time-dependent manner, while the control strain exhibited negligible enzymatic activity. Together, these findings confirm that secreted pgxC enabled effective extracellular breakdown of fruit peel pectin, validating the engineered system’s functional role in waste bioconversion.
Fruit Peel Assay: To evaluate applicability, we tested fruit peels from banana, kiwi, pear, tomato, and watermelon as substrates (Figure 13). After 24 h incubation, engineered L. reuteri supernatants produced significantly higher levels of D-galacturonic acid compared to controls, reflecting efficient peel degradation. Pear and tomato peels exhibited the greatest release (~0.42–0.51 mg/mL), followed by kiwi (~0.30 mg/mL) and banana (~0.22 mg/mL). Watermelon peels produced minimal sugars (<0.1 mg/mL), consistent with their lower pectin content.
Composite Part #5: Conclusions
Composite Part #5: Conclusions
Through our process, we reflected on the outcomes and identified strategies to optimize our construct and experimental design. The New Composite Part allowed us to evaluate the strengths and limitations, and to plan improvements for future iterations. There were two main insights garnered from our New Composite Part:
pTRKH3 with Engineered N-terminal Secretion Signal Peptide pgxC Composite Part (Final Construct; BBa_252WDOMF–later modified to as BBa_25TXYOA2)
We aimed to confirm whether Lactobacillus reuteri engineered with pTRKH3-pgxC could effectively secrete active (pgxC) and degrade fruit peel waste. Our tests showed:
Protein secretion was achieved, confirmed by SDS-PAGE with a visible 48.5 kDa band.
Protein yield was modest (0.10 µg/µL vs. 0.04 µg/µL in controls), indicating successful expression but suggesting room for higher secretion efficiency.
Enzymatic activity was validated through DNS assay, which showed ~2.5-fold more reducing sugar release in engineered strains compared to controls.
Functional fruit peel degradation occurred, but the process was slower than expected, suggesting that enzyme activity or secretion levels may not yet be optimal at large-scale.
In essence, our New Composite Part successfully underwent the Assembly → Construction → Quantification → Validating cycle. Furthermore, this confirmed that our synthetic biology approach successfully addressed the core challenge — enabling pectin degradation in a GRAS probiotic — with bottlenecks in yield and efficiency.
Learn #2: Next Steps and Improvements for Future Design Iterations
Based on these findings, we identified several improvements for the next engineering cycle:
Promoter Optimization
Replace ermB with stronger or inducible promoters to increase protein expression while balancing host growth.
Secretion Signal Variants
Test alternative signal peptides (SPuspA or SlpA) to enhance extracellular protein secretion compared to Usp45.
Codon Optimization Refinement
Re-analyze codon usage in L. reuteri and design optimized pgxC variants to improve translation efficiency.
Enzyme Engineering
Generate pgxC truncation variants or directed evolution mutants to improve activity and stability under acidic fruit waste conditions.
Adaptive Laboratory Evolution (ALE)
Expose L. reuteri to fruit peel-rich environments to select strains with improved fitness and degradation capacity.
System-level Integration
Explore co-expression of accessory enzymes (pectin methylesterase and or cellulases) to enhance complete biomass breakdown.
Summary of Learnings:
(Figure 14. The flowchart of Build, Design, Learn, and Test.)
Our engineered construct proved functional in secreting pgxC and initiating fruit peel degradation, validating our design. However, efficiency limitations highlighted opportunities for promoter engineering, secretion optimization, and enzyme enhancement. These insights will guide the next iteration of the New Composite Part cycle to achieve higher degradation efficiency and practical scalability.
References
References
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