Part 1: L-lactate production
Successful Transformation of pBAD-ldh
We transformed the pBAD-ldh into DH5a E. coli, and the formation of colonies indicates 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 DH5a E. coli for ldh expression.
(fig 1, pBAD-ldh design)
(fig 2, succesful transformation of pBAD-ldh)
(fig 3, cPCR of pBAD-ldh, expected size of the insert is 1486bp)
Measuring the L-lactate production yield in our modified E. coli
Calibration curve
A standard curve was generated by measuring the absorbance of standard L-lactate solutions at concentrations ranging from 1 to 7 μmol/mL, with measurements taken at 1 μmol/mL intervals. The resulting linear regression plot yielded a coefficient of determination (R2) of 0.981, indicating a very strong and highly reliable linear relationship between L-lactate concentration and absorbance.
(fig 4, calibration curve of absorbance against L-lactate concentration)
Oxygen supply method to take a balance between growth rate and L-lactate yield
To optimize our two-stage fermentation, we tested the anaerobic production phase under both static and agitated (100 rpm) conditions. We found that agitation improved the L-lactate yield per cell. We concluded first aerobic fermentation for 3 hours, then anaerobic fermentation for 24 hours as our standard protocol to grow E. coli to produce L-lactate
| Oxygen supplying method | Lactate conc. | OD600 | conc./OD600 |
| Aerobic 3h + anaerobic 24h without shaking | 15.4 | 0.43 | 35.8 |
| Aerobic 3h + anaerobic 24h with 100rpm shaking | 16.3 | 0.437 | 37.3 |
(fig 1, comparison of concentration of L-lactate per cell with two culturing methods)
Measuring L-lactate yield in E. coli in different nutrient
To produce L-lactate, the raw material for respiration was also added, and L-arabinose 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. Therefore, we found another sugar—fructose as another carbon source, as suggested in the paper (Mulok et al., 2009) for L-lactate production.
This is our set-up used for testing the production of L-lactate with glucose, fructose, and L-arabinose:
| Plasmid transformed in E. coli | Added sugar |
|---|---|
| pBAD-ldh | 1% L-arabinose + 1% Fructose |
| 1% L-arabinose + 1% Glucose | |
| 1% Fructose | |
| 1% Glucose | |
| Empty pET plasmid (Neg. control) | 1% L-arabinose + 1% Fructose |
| 1% L-arabinose + 1% Glucose |
Transformed E. coli were initially cultured in LB broth for three hours. Subsequently, various specified concentrations of glucose, fructose, and L-arabinose were introduced, and the cells were then incubated for an additional 24 hours under anaerobic conditions with continuous agitation at 100 rpm.
After culturing, we measured the yield of L-lactate produced from the recombinant bacteria with a L-lactate assay (details on “measurement” page) on the culture medium and cell lysate.
A: L-arabinose G: Glucose F: Fructose
(fig 5, concentration. of L-lactate/OD 600 with different concentrations of glucose, fructose and L-arabinose. A=L-arabinose, F=fructose, G=glucose)
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:
| Glucose | L-arabinose |
|---|---|
| 0% | 1% |
| 1% | |
| 2% | |
| 3% | |
| 4% |
A: L-arabinose G: Glucose
(fig 6, concentration of L-lactate/OD 600 with different glucose concentrations)
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.
Part 2: RNAi gene silencing of GFP
Introduction
To further enhance the final L-lactic acid yield, we plan to implement RNA interference (RNAi) to suppress competing pathways.
To compare how different designs of RNAi affect the repression rate, we design three different asRNA expression plasmids, respectively, to test which asRNA design gives the strongest repression on GFP intensity.
One of our main goals is to test the difference between RNAi with or without pt7 stemloop, which are used to stabilize the RNAi. Another thing for us to test is the length of RNAi. In this experiment, all the RNAi designs have a length of 400 bp.
(For detailed design, refer to “Engineering Cycle” page)
Successful co-transformation of GFP-asGFP, GFP-asGFP-pt7 and GFP-asRFP
We transformed the plasmids into DH5a E.coli successfully.
(1)asGFP (kan) (BBa_25QLNT2S)
(2)asGFP pt7 (kan) (BBa_25AB9IW6)
(3)asRFP (kan) (BBa_25ZT1479)
(4)J23110-GFP (chl) (BBa_25YO6YTU)
(5)pLac-asGFP-pt7 (kan) (BBa_25SMWT82)
(table 1, successful transformation of plasmids asGFP, asGFP pt7, asRFP, J23110-GFP(chl) and pLAC-asGFP-pt7 into DH5α E.coli)
Then we made a starter culture for all of the transformed E.coli and extract the plasmids with miniprep. PCR was conducted to verify the presence of correct plasmids.
(fig 7, The verification of plasmid size using PCR with expected results)
Next, we proceed to co-transformation of GFP-expressing plasmid (Chl) J23110-GFP with asGFP, asGFP-pt7, and asRFP (kan).
(fig 8, Successful co-transformation of GFP expressing plasmid(chl) with GFP-asGFP or GFP-asGFP-pt7 orGFP-asRFP observed under blue light)
An initial starter culture was incubated for 24 hours. The culture was then diluted into a fresh growth medium and subcultured for an additional 24 hours, after which fluorescence/OD 600 were measured.
The graph below illustrates the fluorescence/OD 600 in GFP-asRFP, GFP-asGFP and GFP-asGFP-pt7:
(fig 9, RNAi test flu/OD 600 graph **<0.005, n.s. = non-significant)
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.
Part 3: Diffusion properties of L-lactate in a closed system
By characterizing the relationship between the volume of a closed system and the time taken for L-lactate gas to equilibrium, we can determine the kit's functional lifespan in various volumes. This data will allow us to create a rough usage guide for customers, advising them on the optimal duration for the detection treatment based on the size of their luggage.
Volume of boxes used for testing
| Size of boxes | Volume (cm3) | Equilibrium time (day) |
| S | 7680 | 2.87 |
| M | 15000 | 3.54 |
| L | 22678 | 4.35 |
L-lactic acid will undergo evaporation and gaseous diffusion through an enclosed air volume, and this diffusion will be quantifiable by measuring the progressive decrease in the pH of an open-surface distilled water sample placed within the same sealed plastic box. The pH of the water will decrease as the acidic gas dissolves into the water and dissociates into hydrogen ions and lactate ions. When the pH value stabilises at its lowest value, it is considered equilibrium.
The following graphs illustrate the effect of varying volume on the equilibrium time for a 50% L-lactate solution:
Blue line: pH change of water with L-lactate in the box
Red line (Neg. control): pH change of water without L-lactate in the box
(fig 10, pH change against day in S size box)
(fig 11, pH change against day in M size box)
(fig 12, pH change against day in L size box)
For each dataset, a regression curve was fitted to the plotted points using computational software. Differential calculus was then employed to determine the minimum point of each curve. The equilibrium time, defined as the x-coordinate of this minimum, was subsequently indicated on the respective graph.
Subsequently, a graph of equilibrium time versus box volume was plotted:
| Size of boxes | Volume (cm3) | Equilibrium time (day) |
| S | 7680 | 2.87 |
| M | 15000 | 3.54 |
| L | 22687 | 4.35 |
(fig 13, Equilibrium time against volume with 50% L-lactate)
The analysis reveals a strong positive linear relationship between equilibrium time (t) and box volume (v). This relationship is described by the linear regression equation: t = 9×10-5v + 2.09
The model's high reliability is substantiated by a coefficient of determination (R²) of 0.998. This value indicates that 99.8% of the variance in equilibrium time is predictable from the volume, confirming the model's excellent predictive power. With this equation, we can calculate and instruct users the usage duration of our final bed bug traps of different sizes.
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