L-Lactic acid production by the overexpression of ldh gene

Cycle 1: L-LDH Overexpression Under Strong Promoter J23100

Design

This circuit is utilized to drive the overexpression of the ldh gene, which encodes for L-lactate dehydrogenase (L-LDH), facilitating the conversion of pyruvate into L-lactate. In our initial design, we chose to use a strong constitutive promoter (BBa_J23100), strong ribosome binding site (BBa_B0034), L-LDH encoding gene ldh (BBa_2550VFUD), and a double terminator (BBa_B0015). The decision to utilize the strongest constitutive promoter was based on the premise that it would lead to maximum expression levels, thereby optimizing the production yield of L-lactate.

Build

Despite successful plasmid synthesis and verification, the absence of transformant colonies suggests that the ldh overexpression under the strong constitutive promoter (BBa_J23100) has a negative effect on the growth of E. coli. We suspect that the reason behind this is the continuous conversion of pyruvate to L-lactate, thereby depleting pyruvate, a key metabolic intermediate. Pyruvate is essential for E. coli survival, particularly under anaerobic conditions where it is critical for mixed-acid fermentation and the regeneration of NAD⁺. This metabolic disruption and subsequent toxic buildup of lactate likely proved lethal to the host cells, preventing the formation of selectable colonies.

Learn

To reduce the risk of E. coli mortality prior to the induction of ldh overexpression, an inducible promoter should be employed for ldh expression.

Cycle 2: Using Inducible Promoter pBAD for ldh Overexpression

Design

We implement an inducible promoter pBAD (BBa_K2442101) in the genetic circuit as a replacement for promoter J23100. The pBAD promoter, a common promoter in E. coli, is specifically induced by L-arabinose. It is recognized as a strong promoter with low basal expression, thereby reducing the likelihood of leaky expression.

Build

We transformed the pBAD-ldh into DH5α E. coli and the formation of colonies indicate a success in transformation. We picked colonies to do cPCR and conducted gel electrophoresis. From the cPCR result, we can conclude that we have the correct plasmid constructed and transformed into DH5α E. coli for ldh expression.

Test

After the successful transformation, we focused on measuring the concentration of L-lactate produced by our modified strain.

Effect of anaerobic respiration on E.coli growth

L-Lactate, the target product, is generated via anaerobic respiration. Consequently, we investigated the effect of an anaerobic environment on the growth rate of E.coli (wild type).

The experimental data indicates that aerobic growth (unlocked lid with 200 rpm shaking) yields the highest E. coli growth rate, while both anaerobic methods (sealed lid with or without shaking) significantly reduce the growth rate.

Based on these findings, we concluded that a 24-hour period of continuous anaerobic incubation would result in a lower overall L-lactate yield due to a significantly reduced biomass concentration.

1. Biomass Accumulation Phase: 3 hours of aerobic respiration (high growth rate).
2. L-Lactate Production Phase: 24 hours of anaerobic respiration (product generation).

To optimize the L-lactate yield during the 24-hour anaerobic production phase, we designed a follow-up experiment to evaluate the effects of shaking on the L-lactate yield under anaerobic conditions. This is necessary because while agitation generally promotes E. coli growth, it may negatively affect the L-lactate production rate due to the reduced anaerobic respiration. The new experiment aims to determine whether anaerobic respiration with or without shaking provides the optimal balance for L-lactate yield.

L-lactate measurement with ldh E.coli

A static anaerobic condition (no shaking + sealed lid) was tested against an agitated condition (100 rpm + sealed lid). We concluded first aerobic fermentation for 3 hours, then anaerobic fermentation for 24 hours with 100rpm shaking as our standard protocol to grow E. coli to produce L-lactate.

To produce L-lactate, the raw material for respiration was also added, and L-arabinose was supplied to induce the pBAD promoter. Normally, glucose can be used to make L-lactate, but according to research in 2014 (Simcikova et al., 2014,), glucose may repress the downstream expression of pBAD promoter(p. 1). Therefore, we found another sugar—fructose as another carbon source, as suggested in the paper (Mulok et al., 2009) for L-lactate production.The culture of ldh+ and Empty pET was grown with the standardised oxygen supplying protocol to compare the yield.

This is our set-up used for testing the production of L-lactate with glucose, fructose, and L-arabinose:

Transformed E. coli were initially cultured in LB broth for 3 hours. Subsequently, various specified concentrations of glucose, fructose, and arabinose were introduced, and the cells were then incubated for an additional 24 hours under anaerobic conditions with continuous agitation at 100 rpm.

After the culturing, we measured the yield of L-lactate produced from the recombinant bacteria with a L-lactate assay (details on “experiment” page) on the culture medium.

A: L-arabinose G: Glucose F: Fructose

This graph illustrates the concentration of produced L-lactate divided by OD 600 of the nutrient broth. Overall, the broth with 1% glucose shows a significantly higher conc./OD600 compared to the negative control empty pET plasmid. In contrast, the addition of fructose had a negligible effect on the lactate yield. This suggests that fructose might not be a suitable carbon source in this metabolic pathway.

The result indicates an unexpected discovery — the existence of L-arabinose does not seem to have a significant inducing effect on producing L-lactate even though the inserted promoter, pBAD, is said to be induced by L-arabinose. We suspected that the leaky expression of ldh synthesizes enough L-LDH so that it was no longer a limiting factor on producing L-lactate. We suspected that the concentration of glucose is the limiting factor. To verify our assumption, we tried these sets to compare the yield of L-lactate from broth with different glucose concentration:

A: L-arabinose G: Glucose

The results show that increasing the concentration of glucose were not able to significantly enhance the production yield, which indicates that glucose is also not a limiting factor on the yield of L-lactate.

Learn

Our investigation into the expression of ldh under the control of the pBAD promoter has revealed constitutive-like activity, contrary to its expected L-arabinose-inducible nature. This behavior indicates a potential failure in the AraC-mediated repression mechanism, which is essential for silencing the promoter. Such a regulatory failure could stem from a complete absence of a functional AraC protein, which might occur if the gene was omitted from the plasmid construct used in a non-complementing AraC-host strain like DH5α. Alternatively, the issue may be an insufficient AraC concentration to repress all promoter copies—a phenomenon known as repressor titration, which is common when using high-copy-number plasmids. A final possibility is the presence of a mutation within the AraC gene itself, rendering the protein non-functional and unable to form the DNA loop required for repression. Any of these scenarios would result in significant basal transcription, explaining the observed constitutive expression that is not suppressed by high glucose concentrations.

Interestingly, while the pBAD promoter's leakage was significant, its basal expression level was still measurably lower than that of J23100. This unforeseen characteristic proved advantageous for our project. In a previous design iteration utilizing a stronger constitutive promoter, we observed significant cell death, likely due to the metabolic burden imposed by high-level, unregulated ldh expression. The moderately high, but not excessive, leakage from the pBAD promoter provided a level of enzyme expression sufficient for our pathway while avoiding the toxicity observed in our initial attempts. This highlights how a component not functioning as specified can inadvertently provide a solution to a separate engineering challenge.

In the future, our engineering cycle will focus on repairing the pBAD promoter system to achieve true inducibility. The immediate next step is to definitively diagnose the cause of the leakiness by systematically testing our plasmid in a known AraC host strain and transferring the genetic circuit to a low-copy backbone to address potential repressor titration. Once tight repression is restored, we will re-characterize the promoter's induction curve to identify a L-arabinose concentration that yields the desired, non-toxic level of ldh expression. This approach will allow us to retain the tunable control that the pBAD system offers, leading to a more robust and sophisticated final design that meets our original project specifications.

Future testing with pBAD-gfp

To test for our assumption and characterise the pBAD expression, we have designed a construct to test for how L-arabinose affect the expression of pBAD with the reporter gene GFP.

RNAi-Driven Optimization of L-Lactic Acid Biosynthesis: A Fluorescent Reporter-Based Study

Cycle 1: Gene Repression of ldhA with RNA interference (RNAi) by Co-transformation

Further Enhancement of the Production Yield with RNAi Gene Silencing

To further increase the yield of L-lactate, we aim to reduce competition for pyruvate between D-lactate and L-lactate by silencing the expression of the ldhA gene, which encodes the enzyme D-lactate dehydrogenase (D-LDH) responsible for converting pyruvate into D-lactate.

Exploring the Way to Silence Gene

In our project, selecting a method for gene regulation required careful consideration of cellular viability and metabolic function. While powerful, gene editing tools like CRISPR-Cas9 cause irreversible gene knockouts. The permanent deletion of a protein like D-lactate dehydrogenase could negatively impact the growth rate of E. coli, as D-lactate production is critical during certain metabolic phases.

For this reason, we chose a reversible approach: RNA interference (RNAi). This strategy allows for temporary and controllable gene silencing, offering significant advantages over permanent gene ablation.

A key benefit of our RNAi system is the ability to control when gene silencing occurs. The silencing mechanism relies on an anti-sense RNA that binds to and inhibits the target messenger RNA (mRNA). By placing the gene that produces this asRNA under the control of an inducible promoter (e.g. pLAC system), we can turn silencing on at the most optimal time.

This temporal precision is crucial. D-lactate serves as a vital electron sink for regenerating Nicotinamide Adenine Dinucleotide (NAD⁺), an essential cofactor, during the initial stages of fermentation. Our system allows the cells to grow and establish a robust metabolic state first. Only then do we induce the promoter to suppress D-lactate production, avoiding the lethal consequences that a permanent knockout would cause.

Beyond controlling the timing, our RNAi system allows us to modulate the strength of the gene knockdown. We can fine-tune the repression of the ldhA gene by modifying key aspects of the asRNA construct, such as the length of the complementary RNA sequence or the strength of the promoter used to drive its expression.

In summary, the use of an inducible RNAi system provides a significant strategic advantage for our metabolic engineering goals. The combination of precise temporal control and tunable silencing efficacy makes it a superior choice over irreversible methods like CRISPR-Cas9. This advanced level of regulation allows us to strategically redirect metabolic flux toward our desired product, L-lactate, without compromising the essential metabolic functions that ensure cell survival and robust growth.

Design

Basic asRNA Design

We first aimed to test the specificity and effectiveness of using RNAi to knockdown the gene. To investigate the underlying mechanism, we constructed a plasmid that expresses GFP and an anti-sense RNA of gfp (asGFP) to repress gfp expression. Efficiency of RNAi knockdown can be easily determined by the GFP fluorescence intensity.

The design of asRNA, typically involves selecting a target region that efficiently inhibits gene expression, such as a 20-400 bp fragment encompassing the ribosome binding site (RBS) and extending to the 5' end of the gene, to ensure hybridization with the mRNA near the initiation site and promote degradation or translation inhibition. The use of approximately 400 bp is based on its proven effectiveness in providing sufficient complementarity to induce targeted silencing while minimizing off-target effects (Magistro et al., 2018); this length strikes a balance between specificity and stability of the antisense molecule. Proper design also involves computational prediction of secondary structures to avoid unintended folding that could reduce binding efficiency. In our design, we use RNAfold web server to predict the structure. Once cloned into an inducible or constitutive promoter vector, the asRNA is expressed in the host, leading to specific repression of the target gene. For all RNAi constructs, a uniform length of 400 base pairs was chosen to enhance target specificity. A strong constitutive promoter (J23100) was utilized for the expression of asRNA.

GFP expression

A medium-strength constitutive promoter (BBa_J23110) was selected for the expression of green fluorescent protein (GFP)(BBa_E0040), while a strong constitutive promoter (BBa_J23100) was utilized for the expression of asRNA. This design ensures that the asRNA is in excess, a condition hypothesized to maximize gene silencing efficiency. The GFP expression plasmid was engineered with upstream prefix and downstream suffix sites, enabling the facile cloning of various inserts.

pt7 Stemloop Testing: asGFP (400bp) and asGFP (400bp)-pt7 Against gfp

Two RNAi regions are designed to be cloned into an expression vector, with or without stabilizing features like paired termini or stem-loop structures (e.g., the pt7 stemloop) to enhance asRNA stability and accumulation within the host cell. The stem-loop not only increases the half-life of the antisense molecule but may also improve its binding affinity and effectiveness in silencing the target gene. To evaluate the effect of secondary structure on gene repression, we compared linear asRNA designs with those incorporating a stem-loop structure.

Controllable System for asRNA Expression Under Inducible Promoter pLAC

To achieve tunable RNAi repression, we engineered another construct featuring asGFP gene under the transcriptional control of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible Lac promoter. Our objective is to precisely modulate asGFP expression using varying IPTG concentrations. This approach is necessitated by observations from the previous cycle, which indicated that driving the ldh gene with a strong constitutive promoter may inhibit E. coli colony formation. We hypothesize that this inhibition is due to the complete cessation of D-lactic acid production, one of the essential components for survival. Therefore, to ensure host viability, we will implement this IPTG-inducible system to regulate the copy number of the antisense RNA that represses the ldhA gene, rather than achieving complete transcriptional knockdown.

Negative Control: asRFP (400bp) Against gfp

A non-targeting asRNA against red fluorescent protein (asRFP) served as a negative control to confirm the specificity of the RNAi mechanism.

The RNA Folding Prediction Models

Because RNA is single stranded, it can form stable intramolecular structures through complementary base-pairing, potentially inhibiting the formation of the required asRNA-mRNA duplex but also decreasing the likelihood of being degraded by RNase. Therefore, understanding the predicted conformation of various asRNA candidates can facilitate the rational selection of an optimal asRNA length to balance between binding efficiency and structural stability.

The structure of our designed is predicted with RNAfold web server

Build

We transformed the plasmids into DH5α E. coli successfully.

Then we extract asRNA plasmids with miniprep. PCR was conducted to verify the presence of correct plasmids.

Test

To evaluate RNAi-mediated gene silencing in E. coli, we used a co-transformation method with two plasmids: one expressing GFP and another expressing anti-sense GFP. While this approach is much less complicated than cloning, it can be less precise because the copy numbers of the two plasmids can be imbalanced within the cells, skewing the fluorescence data. To account for this variability and get a more accurate picture of the silencing effect, we analyzed three independent colonies from each co-transformation, measuring both their fluorescence and optical density to normalize the reporter gene's expression to cell density

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

Learn

The experimental data reveal that the efficacy of RNAi is critically dependent on the structural stability of the asRNA transcript. Constructs lacking the pt7 stemloop sequence failed to produce a statistically significant reduction in GFP fluorescence. Conversely, the incorporation of the pt7 stem-loop, which is presumed to enhance transcript stability and prevent degradation, mediated a significant repression of reporter gene expression. These findings collectively demonstrate that a 400 bp asRNA stabilized by a terminal stem-loop structure is an effective molecular tool for achieving targeted protein knockdown in E. coli. Due to time constraints, we were unable to complete the functional characterization of the plac-asGFP-pt7 construct. Future work is planned to rigorously assess the effect of antisense RNA (asRNA) expression rate on target gene expression across a range of IPTG concentrations.

Applying the Design on L-lactate Production

After the test of the RNAi technology, we aimed to enhance our design for L-lactate production by incorporating asRNA targeting ldhA. This modification aims to repress the genomic expression of D-lactate dehydrogenase, thereby optimizing the production of L-lactate. The addition of asldhA should be able to repress the production of D-lactate, but D-lactate is a factor that affects the growth of E. coli, so an inducible promoter is necessary to prevent E. coli death before producing L-lactate.

Cycle 2: Cloning the Gene Repression Constructs

Design

In parallel with the co-transformation procedure, we executed the cloning steps required to merge the antisense RNA (asRNA) sequence with the gfp expression under control of medium promoter(J23110). This process yielded a single construct designed to co-express both asGFP and gfp, which is crucial for ensuring an identical expression rate for both the reporter (gfp) and asGFP. By standardizing the transcription rate, this construct validates the assay design, thereby making the results across different asGFP variants comparable and reliable. We used the plasmids verified in cycle 2 for cloning, except we used another J23110-gfp with kan resistant backbone for cloning, where prefix and suffix are designed at the end of the construct.

Build

We have successfully transformed the plasmid into DH5α E. coli.

A double restriction digestion is conducted for J23110-gfp(kan), asGFP, asGFP-pt7, asRFP. For the first trial, the protocol for digestion is below:

1. Mix the following components together:

2. incubate for 2h

After running the agarose gel, the results show unsuccessful digestion. All of the constructs have only one band.

To validate both the experimental protocol and the functionality of our equipment, we extended the incubation duration to two hours and confirmed enzyme activity using the standard J23110-gfp(chl) plasmid, which has been previously verified by others. Specifically, we performed single restriction digests to verify the efficacy of the enzymes cutting EcoRI, SpeI, and PstI. The resulting data confirmed that enzyme denaturation had not occurred.

Using the optimized incubation duration, we proceeded to perform a double restriction digest. Subsequent gel electrophoresis analysis confirmed the successful digestion, as evidenced by the observation of two distinct bands for each antisense RNA (asRNA) construct and a single band for the medium promoter J23110 backbone construct(kan).

Consequently, the subsequent step involved gel purification to isolate the target DNA fragments, specifically the antisense RNA (asRNA) insert and the J23110-gfp backbone, utilizing a QIAGEN kit. We then performed a ligation reaction to construct the complete plasmid, which incorporated the two genes encoding the asRNA and GFP. Finally, the ligated product was transformed into DH5α E. coli and incubated overnight.

Learn

After the modification of RE digestion protocol and several trials of cloning, we are still trying to transform E.coli containing asRFP, asGFP, asGFP-pt7, and pLAC-gfp-pt7. Due to time constraints, we have not yet obtained the final construct for testing, but we will continue the process in the future.

L-lactic acid Diffusion Rate Measurement

Cycle 1: Diffusion Rate Measurement Tool Determination

After L-lactic acid production, we have used commercial L-lactic acid to test its diffusion rate, characterising it for our proposed application–detecting bed bugs in a small area.

Design

The pH change will serve as a metric to quantify the diffusion rate of L-lactic acid gas within sealed containers. For practical implementation, two readily available and straightforward methods for pH measurement are the pH meter and pH paper.

A pH meter is an electronic device that provides a quantitative, numerical reading of pH, often to two decimal places (e.g., 6.85). It can provide high precision and objective pH value. It measures the electrical potential difference between two electrodes, which is directly related to the hydrogen ion concentration. The digital display eliminates human error. Though with such advantages, the main disadvantages are the time and effort required for calibration with buffer solutions and the fact that it is more expensive than pH paper.

pH paper can solve the problem of the lengthy calibration duration and does not require a battery for running. It also eliminates the duration of L-lactate dissolution in water, producing a more realistic value for diffusion rate. However, it has low precision, limited to a resolution of 0.5-1.0 pH units and the pH obtained is highly dependent on the subjective colour of paper.

Build

Both comparative experimental designs—the pH meter protocol and the pH indicator paper protocol—utilize a common apparatus and set of controlled variables to ensure valid comparison.

The fundamental setup involves a plastic box to serve as the enclosed environment, which is sealed using masking tape to prevent any potential leakage of gaseous compounds.

Using pH paper as Measurement Tool

The procedure for this experiment is described as the following:

1. Diffusion Interval: The L-lactic acid will be placed inside the sealed plastic box and allowed to diffuse for several predetermined time intervals.
• a. At predetermined time intervals (e.g., 1 day, 2 days, 3 days), remove one setup from the series.
• b. For example, to obtain measurements after three consecutive days, three separate, identical setups were needed.
2. Moisture Application: After the diffusion interval, the paper will be moisturized with 2.0 mL of distilled water.
3. Color Development: The paper will be allowed to stand for 30 minutes to allow for the dissolution of adsorbed ions and the subsequent color change. Note: As observed, the color change will only occur post-hydration.
4. pH Determination: the pH paper will be removed from the plastic box and the resulting color will be immediately compared against a standard color chart provided by the manufacturer. The corresponding pH value will be recorded to the nearest 0.25 pH unit.

Using pH meter as Measurement Tool

1. Calibration: calibrate with 3 buffer solutions(pH 4.01, 6.86, 9.18) to ensure the value obtained from pH meter is accurate
2. Preparation: Add 20 mL of L-lactic acid in one 50 mL beaker and 20 mL of distilled water in another 50 mL beaker.
3. Initial Measurement: Measure and record the initial pH of the distilled water. This serves as the baseline pH for all experimental trials.

4. Experimental Setup:

   • Place the beakers of lactic acid and distilled water in opposite corners of an enclosed box.

5. Diffusion and Measurement:

   • Repeat the setup with multiple identical sealed boxes to create a series of independent trials. This is a crucial step to avoid opening the container during the experiment.
   • At predetermined time intervals (e.g., 1 day, 2 days, 3 days), remove one setup from the series.
   • For example, to obtain measurements after three consecutive days, three separate, identical setups were needed.
   • Open the container and measure the pH of the deionized water sample inside.
   • Record the time elapsed and the corresponding final pH value.

6. Data analysis:

   • Calculate the pH change of each water sample and plot of graph of pH change over time.

Test

Accurate pH values could not be reliably determined using pH paper because of observer bias in color assessment and the minor magnitude of the pH change. For this reason, we did not plot a pH trend graph. Nevertheless, the results definitively indicate that the diffusion of lactic acid resulted in a detectable pH alteration

Conversely, the determination of the equilibrium time is readily achieved through the use of pH meter. This methodology effectively illustrates the relationship between the pH changes and time allowed for diffusion. As demonstrated in figure 30, the system reaches equilibrium between day 3 and day 4. The precise point of equilibrium can be ascertained through rigorous data analysis, specifically by utilizing differentiation to locate the minimum point.

Learn

Because the measured pH change is too subtle, the lack of precision from the use of pH paper would render the experiment's results unreliable and make equilibrium time difficult to be measured. Moreover, subjective colour observation and different lighting conditions will make the pH inaccurate. While pH paper is quick and inexpensive, the pH meter is the only instrument that will provide the reliable, quantitative data needed for this experiment to be scientifically valid. Therefore, we chose to use a pH meter as a measurement tool for further testing.

One difficult situation we faced when using pH meter is that it requires a lot of boxes, since one box can only be used to make one pH change data (e.g. change after 4 days), but not a set of data (e.g. the entire pH trend from day 1-6), making this experiment time consuming.

Cycle 2: Exploring Different Measurement Method of Diffusion Rate

Design

The previous model required the use of multiple identical boxes to collect a single set of results over time. At each predetermined time interval, a separate box had to be opened to measure the pH, making this a time-consuming and materially inefficient process. Another issue was the variability introduced by using different containers for each trial. Even after thorough cleaning, the use of separate beakers for the water samples led to minor variations in the initial pH.

To overcome these challenges, a new model has been developed. This redesigned apparatus will allow for all measurements to be conducted within a single, controlled environment, which can enhance the material efficiency and accuracy of our data by using the same sample for the entire set of data.

Build

We have built a model that has plastic bags fixed on the box to control the pH meter inside, so that we can measure the pH change with only one box without interrupting the sealed environment.

Instruments involved in the new set up is the following:

• pH meter: to measure the change in pH value of water.
• Electrolyte solution: to ensure a pH probe is kept hydrated when the probe is not in use
• Distilled water: simulates bed bug, used to quantify the evaporated l-lactic acid with its pH change
• L-lactic acid: the smell was used as a chemical lure to attract bed bugs
• distilled water in a bottle: used to clean the chemical on the pH meter probe after measurement of slightly acidic water
• Lens paper: used to remove liquid remained on the pH probe to ensure that the results are not affected by contamination
• Tape: sealed up all the gaps between the table and the container

Test and learn

While a more accurate model was available, its associated experimental procedures proved overly complex and inconvenient, particularly concerning the manipulation of the pH meter. This challenge led to inconsistent and unreliable results. Consequently, we made the strategic decision to adopt the simpler timepoint model for the remainder of the investigation.

Despite being able to determine the diffusion rate in standard conditions, we acknowledge the inherent limitations of the timepoint model for the real world application proposed by our team–using our designed hardware to detect the presence of bed bug in a luggage, but due to strict time constraints, further methodological refinement was not feasible.

1. Difference in evaporation mechanism of the experiment and hardware

   In the original setup, lactic acid evaporates directly from the beaker's surface. However, the proposed model introduces a different mechanism: the absorption of lactic acid onto a paper material, followed by evaporation from the paper's surface. The rate of evaporation from a saturated paper surface will differ from the rate of evaporation directly from a liquid in a beaker. As a result, any equilibrium time obtained from this experiment will be an estimation rather than a precise measurement of diffusion rate from our hardware.

2. Wider range of concentration can be included

   To fully characterize the relationship between lactic acid concentration and its diffusion rate, it is recommended to expand the experimental scope. By including a test with 25% lactic acid concentration, the data will provide a clearer understanding of how concentration influences the equilibrium time, allowing for better manipulation of the diffusion rate.

3. The assumptions behind the experiment: dissolution duration

   This experimental model operates under the assumption that the time required for lactic acid gas to dissolve into the water is negligible. Consequently, the actual time to reach equilibrium would be shorter than the value observed in the experiment.

   A more precise measurement of the diffusion rate could be achieved by using an acid gas detector, which would directly measure the concentration of gas in the air. This approach eliminates the variable of gas-liquid dissolution, providing more accurate data.

4. Equilibrium time of lactic acid may be reached at a later stage due to the blockage of items when applied in reality

   The original experimental design, which used an empty box, does not account for the absorption and obstruction effects of items found in real-world scenarios, such as clothing in luggage. The presence of these materials would slow down the diffusion rate of lactic acid gas. As a result, the time required for the system to reach equilibrium would be considerably longer than observed in a controlled, empty environment.

   • Absorption: Porous materials like fabrics can absorb the gas molecules into their fibers and onto their surfaces. This removes the gas from the air, lowering its concentration.
   • Physical Obstruction: The presence of objects creates physical barriers, forcing the gas molecules to navigate a longer and more complex path.

Designing an Effective Hardware Model

Cycle 1: “Attract and Kill” Bed Bug Trap

Design

For our team’s 1st implementation, we want to create a kit that could be used in households. Our kit would have a lure to attract bed bugs, and the bugs would be trapped in the kit which later be easily killed. We chose L-lactic acid as a lure to attract bed bugs to the kit, as L-lactic acid is proven to show attraction abilities towards bed bugs[5]. After a user returns from a place with high risk of bed bugs infestation, multiple kits could be placed around the house. When any bed bug brought home goes toward the kit to approach the lure, they fall into the indented well. The well is designed to have a smooth surface which bed bugs are unable to escape by climbing up. When the user spots any bed bug present in the well, they could remove the well and put it in 70℃ hot water for 10 minutes to ensure all the bed bugs are killed. The well could be connected back to the kit for repeated usage. If the lure is used up, it could be replaced and a new one could be put back on the disc.

Test and Learn

Our 1st prototype is presented to Mr. Francisco Pazos, a bed bug control specialist, as our team would like to collect professional feedback from people working in the pest control industry. Mr. Pazos stated that if the trap is not immediately applied after the bed bugs got into the home, the trap could not exterminate 100% of the bed bug population. This is due to the high reproduction rate of bed bugs, if not all bed bugs are attracted to the kit, the remaining ones would reproduce rapidly, resulting in recurring bed bugs infestation. Therefore, the effectiveness of our trap may not work well if users do not put out the traps right after they get home. In addition, he stated that currently, there is no chemical that could attract the whole bed bug population. In large areas such as houses, the attracting effect would be minimal. If our kit needs to be guaranteed to have the ability to attract all bed bugs in an apartment, it would simply be impossible.

The insights from Mr. Pazos has led us to conclude that the “attract and kill” method used in households could not be used, and we have to find a different approach.

Cycle 2: Attraction and Detection Bed Bug Kit

Design and Build

We have discovered that not many services are available for early detection of bed bugs. From further research, we found out that bed bugs usually hide in confined and small spots, such as furniture, luggages and bags. Due to the fact that bed bugs are primarily spread by ‘hitchhiking’ on carriers[6], our team wants to provide an early detection kit that could check for presence of bugs in luggages before any of them could spread out. The kit lures out bed bugs hiding in clothes or gaps and users would know they have to sterilize their luggages if they see bed bugs present in the kit.

We would have to modify the design of our kit, as our original prototype could only be used when put on grounds.

We made various adjustments to accommodate the usage of kit in luggages. The updated design of our kit is split into two main parts: the lure container and the diffuser.

The acid-resistant lure container only allows the entry of the diffuser, which covers the connection parts.

The diffuser consists of 3 main parts: the support, the cardboards and the chromatography papers . Firstly, the support of the diffuser is made from wood. Secondly, there are layers of cardboards to provide shelter for the bed bugs. When the diffuser is connected to the lure container, the chromatography papers absorb the lactic acid and allow lactic acid to diffuse and attract bed bugs.

Users could use a flashlight to facilitate their checking of bed bugs in the kit. If any is found, the luggage may contain more of them and require immediate sterilization. The used diffuser could be put into a zip lock bag provided to prevent bed bugs escaping, while the container with remaining lure could be reused upon inserting with a new diffuser.

As L-lactic acid may have potential harm, our team wants to guarantee our users could use our kit safely and clearly. With the above reason, we have designed a user manual to provide guidance on using the kit.

The second design is effective at all orientations compared to our original design. Our improved prototype could be applied better in luggages, with the reusable lure container and user manual, our team offers a safer, more convenient, and cost-effective solution to prevent the spread of bed bugs.

Test and Learn

From different testings on our new prototype, we have discovered some room for improvement. However, due to constrained time and materials, we could not adjust our design accordingly at this moment, and we hope to enhance our model in the near future.

• 1. materials: The diffuser could be made from more cost effective materials, such as recycled cardboards for harborage. For repeated usage of the liquid container, a lid is required to cover it. Other than using plastic film, we aim to utilize materials which are more environmentally-friendly.
• 2. Leakage: Slight leakage occurs as testing liquid (water) seldom leaks through the holes of chromatography paper, and liquid seeps through the four edges of the container when put sideways. For future design, rubber rings could be added in the interior to prevent leakage.
• 3. Outlook: The current model utilizes hot-melt adhesive to connect different parts, leaving unpleasant looking stains. In the future, we would make use of silicon rubber to make a cleaner look.

Reference

[1]Mulok, T. E. T. Z., Chong, M. L., Shirai, Y., Rahim, R. A., & Hassan, M. A. (2009). Engineering of E. coli for increased production of L-lactic acid. African Journal of Biotechnology Vol. 8, 8(18), 4597–4603. https://doi.org/10.5897/ajb09.614

[2]Simcikova, M., Prather, K. L., Prazeres, D. M., & Monteiro, G. A. (2014b). On the dual effect of glucose during production of pBAD/AraC-based minicircles. Vaccine, 32(24), 2843–2846. https://doi.org/10.1016/j.vaccine.2014.02.035

[3]Nakashima, N., Tamura, T., & Good, L. (2006). Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli. Nucleic Acids Research, 34(20), e138. https://doi.org/10.1093/nar/gkl697

[4]Magistro, G., Magistro, C., Stief, C. G., & Schubert, S. (2018). A simple and highly efficient method for gene silencing in Escherichia coli. Journal of Microbiological Methods, 154, 25–32. https://doi.org/10.1016/j.mimet.2018.10.003

[5]Singh, Narinderpal & Wang, Changlu & Cooper, Richard & Liu, Chaofeng. (2012). Interactions among Carbon Dioxide, Heat, and Chemical Lures in Attracting the Bed Bug, Cimex lectularius L. (Hemiptera: Cimicidae). Psyche. 2012. 10.1155/2012/273613.

[6]Doggett SLDwyer DE, Peñas PF, Russell RC.2012.Bed Bugs: Clinical Relevance and Control Options. Clin Microbiol Rev 25:.https://doi.org/10.1128/cmr.05015-11