Overview

Throughout our project, we read multiple research papers from previous iGEM teams. They not only equipped us with an abundance of information but also inspired us to explore new dimensions in our project. With the help of the experiences and insights from those who came before us, we made significant progress towards our goals.

We realized the importance of enriching the database for future iGEM participants. We hope that our discoveries on L-lactate production, RNAi technology, and gas diffusion rate investigation can provide knowledge and inspiration to support upcoming teams. We have educated high school students with synthetic biology skills and made a new hardware model for attracting climbing insects.

New part characterisation: Implementation of L-Lactate Dehydrogenase (L-LDH) for Pyruvate Conversion for Metabolic Flux Redirection

We have designed an overexpression system for the L-lactate dehydrogenase (L-LDH) encoding ldh gene with pBAD (BBa_K2442101), B0034 (BBa_B0034), the new part ldh (BBa_2550VFUD), B0015 (BBa_B0015) to build the new composite part BBa_25DJGY4C.

Characterising the Production of L-lactic Acid from E.coli

To confirm the enhanced production of L-lactic acid by the engineered E.coli strain, an L-lactic acid assay was performed. The absorbance of the L-lactate precipitate demonstrated significant yield compared to the negative control. This result validates the high efficiency of the inducible pBAD promoter in driving enhanced ldh expression.

We further explored the influence of the pBAD inducer, L-arabinose, and two different sugar substrates (glucose and fructose) on lactate production yield.

Arabinose Inducer Concentration and Sugar Substrate Efficiency

The concentration of the pBAD inducer, L-arabinose, was found to not significantly affect the overall lactate yield. This suggests that, within the tested range, the pBAD-driven expression level necessary for efficient lactate production has already reached, or that another factor is rate-limiting.

A comparative analysis of the two sugar substrates demonstrated that glucose is converted to lactate more efficiently than fructose. This difference in efficiency is likely attributable to the distinct metabolic pathways and energy requirements associated with the initial uptake and processing of these hexoses before they enter glycolysis. Glucose can directly enter the glycolytic pathway (as glucose-6-phosphate), whereas fructose typically requires extra enzymatic steps (via the fructose-1-phosphate pathway or phosphorylation to fructose-6-phosphate) to be utilized.

Glucose Concentration Analysis

Further examination of various glucose concentrations indicated that glucose has become excess at a concentration of 1%. This signifies that beyond this threshold, adding more glucose does not result in a proportionate increase in lactate production, suggesting that the system's capacity—such as enzyme concentration, transport rate, or co-factor availability—has been saturated or is acting as a bottleneck.

Investigation of Bioprocess Optimization

We investigated the critical trade-off between E.coli growth rate and L-lactic acid production yield under anaerobic fermentation conditions. This was achieved by systematically exploring differential oxygen-supplying methodologies within a precisely controlled bioprocess. Specifically, we characterized the impact of varying durations of aerobic versus anaerobic growth phases on overall L-lactic acid yield and cellular viability.

This approach strategically leverages the two metabolic states:

1. Aerobic Phase: To achieve optimal initial biomass accumulation (E.coli high growth rate). Aerobic condition is created by unlocking the lid of 15-ml tubes and incubating at 37°C with 250rpm shaking.
2. Anaerobic Phase (Fermentation): To shift the metabolic state for maximal flux towards the desired anaerobic fermentation product (L-lactic acid). Anaerobic condition is created by locking the lid of 15-ml tubes and incubating at 37°C without shaking or with 100 rpm shaking.

The results show that the oxygen supplying method of 3 hours aerobic growth and 24 hours anaerobic growth with 100 rpm shaking is more beneficial for L-lactic acid production.

The resulting data provides an optimized operational framework for future iGEM teams focused on the mass production of anaerobic organic chemicals using E.coli chassis strains, which is paramount for cost-effective and large-scale bioprocesses.

Potential Applications of L-lactic acid from Engineered E.coli

The successful production of L-lactic acid using the recombinant ldh system in E.coli provides a versatile platform with significant impact across various fields:

1. Biotechnological and Commercial Applications

• Pest Control and Insect Attractants: The availability of high-purity L-lactic acid is critical for the development of advanced insect control systems, such as specialized mosquito traps. As L-lactic acid is a key volatile component of human sweat, its production can facilitate the precise formulation of highly effective, bio-based attractants.
• Biopolymers and Biofuels: L-lactic acid is a valuable precursor for sustainable materials, including:
• Bioplastics (Poly-lactic acid or PLA): A high-demand, biodegradable polymer.
• Biofuels: As an intermediate in the production of sustainable energy sources.

2. Food and Cosmetic Industries

This robust production method promotes a broad range of investigations and commercial ventures in:

• Food Production: As an essential ingredient in the production of fermented foods, such as yogurt.
• Food Preservation: Utilizing its acidic properties as an effective, natural food preservative.
• Cosmetics and Skincare: Serving as a key component in skincare products due to its properties as an alpha-hydroxy acid (AHA).

Introducing RNAi Technology to Gene Silencing

Metabolic Engineering for Enhanced L-Lactic Acid Yield

To further enhance the final L-lactic acid yield, we implemented a metabolic engineering strategy leveraging RNA interference (RNAi) technology. The primary objective of this intervention was to minimize a competing metabolic pathway by reducing the expression of D-lactate dehydrogenase (D-LDH). This targeted knockdown is designed to redirect raw material (pyruvate) flux preferentially toward the overexpressed L-LDH pathway, thereby maximizing L-lactic acid productivity.

Antisense RNA (asRNA) Construct Design

To determine the most effective gene silencing agent, we designed and constructed multiple antisense RNA (asRNA) variants. These constructs were meticulously engineered to maximize their inhibitory specificity and efficacy by incorporating two key features:

1. Stem-Loop Structures: Inclusion of stem-loop motifs within the asRNA structure was employed to enhance the stability, processing, and overall potency of the interfering molecule.
2. Maximal Target Specificity: asRNA length was optimized based on established literature to include the longest possible sequence that maintains high specificity for the target mRNA of D-LDH-encoding ldhA, ensuring robust and targeted knockdown.

Validation and Characterization of the asRNA System with Stem Loop pt7

To quantitatively assess the gene repression efficiency of our asRNA constructs, we employed a standardized Green Fluorescent Protein (GFP) reporter system (BBa_252PPZTK). The gfp gene was placed under the control of the strong constitutive J23110 promoter to ensure a high baseline expression level.

Two specific asGFP constructs were generated to investigate the impact of structural elements: asGFP with (BBa_25AB9IW6) and without stem-loop pt7 structure. (BBa_25QLNT2S)

A crucial component of this validation was the inclusion of a negative control construct, asRFP (BBa_25ZT1479). This construct was specifically designed to target and repress Red Fluorescent Protein (RFP) mRNA. The use of asRFP ensures that any observed repression of gfp is sequence-specific but not because of mechanisms naturally present in E.coli.

The graph below illustrates the fluorescence/OD 600 in gfp-asRFP, gfp-asGFP and gfp-asGFP-pt7:

Contribution to the iGEM Community

The comprehensive characterization data generated from this system—comparing the repression efficiency of stem-loop vs. linear asRNA variants—establishes a robust and easily characterizable platform for the quantitative evaluation of asRNA-mediated gene repression efficiency in E. coli bioprocesses.

Applying the RNAi Technology to the ldh Circuit

With the data shown from the GFP testing, asRNA with a stem loop can significantly repress the expression of the target gene. After the testing, we have designed an asRNA construct for repression of D-LDH-encoding ldhA gene, optimised with the stem loop pt7.

Novel Diffusion Rate Determination

A novel, straightforward experiment has been designed to determine the diffusion kinetics of acidic gases. The method is intentionally accessible and economical, requiring only standard laboratory instruments like a pH meter and plastic containers.

Furthermore, we successfully determined the diffusion kinetics within different volumes. Our findings confirm a linear dependency, demonstrating that the time necessary to reach equilibrium is directly proportional to the volume of the diffusion environment.

This experimental design and resulting data provide a valuable framework for future iGEM teams, enabling them to precisely control the acidic gas diffusion rate. By exploring variables other than concentration, subsequent research can identify and manipulate additional factors that influence the kinetics.

Application of Hardware to All Climbing Infectious Insects

Our hardware design specifically targets the spread of bed bugs, emphasizing user safety and clarity in usage. Recognizing that lactic acid is an irritant and corrosive, we have utilized polycarbonate, an acid-resistant material, to securely contain the lactic acid within the kit. After using the kit, users can safely place it in the provided zipper bag to prevent both leakage of lactic acid and the escape of any captured bed bugs. Additionally, we include a comprehensive user manual to ensure clear and safe operation of the kit.

The safety measures and design principles of our kit can serve as a template for the future development of detection kits for other insects. For instance, silverfish, which damage books and clothing, and cockroaches, common household pests in Hong Kong that can carry various diseases, represent significant targets for detection. When adapting the kit for these insects, suitable pheromones or chemicals can replace L-lactic acid to effectively attract them.

Our detection kit provides a reference to safely and effectively attract flightless insects, thereby facilitating the detection of harmful species and preventing infestations. Future iGEM teams could enhance this design by modifying the lure used, adjusting the size of the components, optimizing the effective time frame, and determining the best placement for the kit to maximize detection efficiency.

Prepare High School Students to Become Future IGEMers

Our educational outreach was designed with the clear goal of inspiring and equipping high school students to become future contributors to the field of synthetic biology and the iGEM competition. We achieved this through a structured progression of initiatives that covered awareness, fundamental concepts, and practical skills.

We began with broad public awareness at our School Open Day, using our past project to demystify complex concepts and showcase the real-world problem-solving potential of synthetic biology. We then moved to targeted events: the collaborative HKSSC workshop provided secondary students with a compelling real-world context (the bed bug problem) for our research, demonstrating the societal relevance of iGEM. This was followed by the HKUST "Be an iGEMer" workshop, which was crucial for hands-on skill development. Here, participants were guided through fundamental lab techniques like cPCR and gel electrophoresis and concluded with a mini-research and presentation session, allowing them to experience the full scope of an iGEM project.

Finally, we ensured the long-term sustainability of our school's involvement through the Biology Research Team (BRT). By mentoring BRT members and conducting regular experiments with them, our iGEM team is actively passing on essential knowledge and technical skills, effectively cultivating and preparing the next cohort of iGEM participants. Through these tiered efforts, we are consistently building enthusiasm, providing practical training, and securing the future of synthetic biology engagement in our community.

Educating and Engaging the Public in the Bed Bug Problem

Our education efforts were a dynamic, multi-faceted strategy focused on engaging the public in the bed bug problem and simultaneously inspiring future scientists. We leveraged creative media to ensure maximum reach and engagement across diverse age groups. Our primary educational tool was the multilingual leaflet, which provided scientifically validated information on bed bug habits and effects, compiled from peer-reviewed literature and a Cambridge medical expert, and distributed strategically to community centers to ensure accessibility across linguistic groups. For the younger audience, we created an original Comic Book and a hands-on Card Game, transforming a serious public health issue into approachable, interactive learning experiences for children and secondary students, respectively. This creative content was brought to life during our Primary School Outreach, where we used a dramatization of the comic and provided an accessible introduction to synthetic biology, demonstrating its real-world problem-solving potential. This comprehensive approach ensured that we not only raised widespread public awareness about bed bugs but also actively cultivated interest in science and research among the next generation.