Introduction

Here are the experiments we have done:

1. L-lactate production in modified E.coli
2. Silencing gfp gene with RNAi in E.coli
3. Characterization of L-lactate diffusion properties

The results of these experiments are indicated in the “result” page.

Protocols

Production of L-Lactic acid by Genetically Modifying E.coli

Measurement of L-lactate Concentration

In our project, we aim to produce L-lactate in our modified E.coli. To monitor the yield of L-lactate, we used an optical method to measure the concentration of L-lactate in the bacterial cell lysate. We used a L-lactate assay kit (solarbio cat. BC2230 50T/48S) to extract and measure the concentration of L-lactate.

Principle of the Functional Assay for L-lactate with Enzymatic Reactions

A significant challenge in the direct turbidimetric measurement of L-lactate is its inherent absorption in the near-infrared spectrum. To circumvent this issue, an enzymatic assay was employed. Following extraction, L-lactate is oxidized to pyruvate by the enzyme L-lactate dehydrogenase (L-LDH), generating NADH as a byproduct.

The enzymatic oxidation of L-lactate to pyruvate by L-LDH is an equilibrium-limited reaction that strongly favors L-lactate, which could lead to incomplete conversion. To drive the reaction to completion, the generated NADH is continuously consumed in a subsequent reaction. In this process, NADH reduces the chromogenic substrate MTT to formazan in the presence of the electron mediator 1-mPMS. The resulting formazan is then quantified by its distinct absorption peak at 570 nm, ensuring a reliable and complete measurement of the initial L-lactate concentration.

Material and Method

Using Inducible Promoter pBAD for ldh Overexpression

We implement an inducible promoter pBAD in the genetic circuit. 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.

Balancing Growth Rate and Lactate Production

E.coli growth requires oxygen for growth while lactate production requires anaerobic conditions. To make a balance between these two, we tested with 2 different oxygen supplying methods: growing aerobically for 3 hours and then anaerobically with either shaking or no shaking. Then the L-lactate production yield (abs/OD600) is determined.

Protocol is below:

Making starter culture

1. Pick an isolated colony of transformed E.coli with ldh gene from the agar plate(kan)
2. Grow aerobically overnight

Making subcultures

1. Dilute the culture into 1:10 ratio.
2. Grow for 3 additional hours aerobically by unlocking the tube lid and shaking with 200 rpm.
3. Add excessive L-arabinose (1%) and glucose (4%) to the culture.
4. Grow anaerobically for 24 hours by locking the tube lid, either without shaking or with 100rpm shaking.

Measurement: refer to the protocol for L-lactic acid measurement with commercial assay kit.

Varying Nutrients Provided for Lactate Production

To produce L-lactate, the raw material for anaerobic respiration is needed, and L-arabinose is needed 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 an alternative 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:

Making starter culture

1. Pick an isolated colony of transformed E.coli with ldh gene from the agar plate(kan)
2. Grow aerobically overnight

Making subcultures

1. Dilute the culture into 1:10 ratio.
2. Grow for 3 additional hours aerobically by unlocking the tube lid and shaking with 200 rpm.
3. Add corresponding concentrations of L-arabinose and glucose to the culture.
4. Grow anaerobically for 24 hours by locking the tube lid and with 100 rpm shaking.

Measurement: refer to the protocol for L-lactic acid measurement with commercial assay kit.

Discovering the Relationship between Glucose Concentration and L-lactic Acid Yield

Because the result indicates the existence of L-arabinose does not seem to have a significant inducing effect on producing L-lactate, we suspected that the concentration of glucose is the limiting factor. Therefore, we tried to increase the glucose concentrations to compare the yield of L-lactate:

All the procedures were the same as the last experiment set.

Gene Repression of gfp with RNA interference (RNAi)

Further Enhancement of the Production Yield with RNAi

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.

The use of an inducible RNAi system provides a significant strategic advantage for our metabolic engineering goals. 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.

Testing the RNAi System with gfp Reporter Gene

To better understand the optimal expression of antisense RNA, we have designed several plasmids that expresses GFP and an anti-sense RNA of GFP (asGFP) to test for how the asRNA and stem loop repress its target gene expression. Efficiency of RNAi knockdown can be easily determined by the GFP fluorescence intensity.

Co-transformation for the Expression of gfp(chl) and asGFP(kan)

The protocol for co-transformation is the following:

1. Thaw 50 - 100 μL competent cell on ice
2. Mix 1 - 10ng of both plasmids with the cells
3. Incubate on ice for 10 - 30 minutes
4. Heat shock at 42°C for 45 seconds
5. Incubate on ice for 2 minutes
6. Add 800 μL LB(no antibiotic) to the cells
7. Incubate for 1 hour at 37°C for recovery
8. Centrifuge the tubes for 30 seconds and remove most supernatant by flipping it upside down to leave ~100 μL inside
9. Resuspend the pellet by pipetting up and down
10. Spread all the 100 μL cell to agar plates with the 2 antibiotics (chl and kan) that the 2 plasmids can develop resistance
11. Incubate for 2 days at 37°C

Then, the successfully co-transformed E.coli is used to compare the fluorescence intensity. The protocol is the following:

Making starter culture

1. Pick 3 isolated colonies of transformed E.coli into 3 separate broths (chl + kan)
2. Grow aerobically overnight

Measurement of fluorescence intensity/OD600

1. Vortex the culture
2. Extract 100ul and transfer to 96-well plate
3. Measure the optical density with micro-platereader to obtain the absorbance and fluorescence intensity.
4. Analyse the data by dividing fluorescence over absorbance and do statistical tests.

Experiment to Characterize L-lactic Acid Diffusion Properties

By determining the equilibrium time for different concentrations and volumes, we can ensure that the kit's functional lifespan is maximized. This will allow for the L-lactic acid to reach a sufficiently attractive concentration for bed bugs, without the system reaching equilibrium too quickly and becoming ineffective.

The Influence of Enclosed Volume on Lactic Acid Diffusion Kinetics

Introduction

The time it takes for a gas to reach an equilibrium state is directly influenced by the volume of that space. By characterizing this relationship, 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.

Method and materials

Volume of boxes used for testing:

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.

Procedure

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 50% 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.
   • Secure the box with masking tape to create an airtight environment.
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.
   • Repeat the whole process with another size of boxes.
6. Control Setup:
   • Prepare a separate container with two beakers, both containing 20 mL of distilled water.
   • Seal this container and place it alongside the experimental setups.
   • At each measurement interval, open a control container and measure the pH of one of the water samples to account for any changes due to the container or atmospheric carbon dioxide.
7. Data analysis:
   • Calculate the pH change of each water sample and plot a graph of pH change over time

Characterizing the Relationship Between Concentration of L-lactic acid and Diffusion

The diffusion rate of lactic acid gas is a critical factor for the effectiveness of our detection kit. Two key factors can influence this rate: environmental temperature and the concentration of the lactic acid source. While temperature significantly affects diffusion, manipulating the air temperature within luggage or bags is not a practical or user-friendly method for customers. Therefore, we have identified that varying the concentration of the lactic acid source is the most convenient way to control the rate of diffusion. This approach provides a reliable and simple means to manage the kit's performance and longevity.

Material and Methods

Concentration of lactic acid used for testing: 50% and 75% v/v L-lactic acid.

All testing methods are the same as that of the volume set up, except the concentration of L-lactic acid varies in the same volume of box.