Overview

As we cannot directly conduct functional validation of contact lenses in the human body, we plan to verify the effectiveness of our project in preventing and treating myopia through step-by-step experiments. The first step is the production module, where we need to ensure that our engineered bacteria can produce Rhodiola glycoside under experimental conditions. Building upon this, we will continue to verify the effectiveness of the dormancy switch, focusing on the RNA thermometer to ensure its proper functioning at the set temperature of 37℃, as the light-controlled and AND gate have already been successfully validated. Lastly, we need to verify whether the engineered bacteria can be encapsulated within liposomal vesicles to demonstrate the feasibility of the hardware.

Verification I Production of salidroside

As an anti-hypoxic drug, salidroside can downregulate the expression of hypoxia-inducible factors and slow down the progression of myopia.

In order to prove that our engineering bacteria can produce salidroside under experimental conditions, we conducted basic fermentation and improved fermentation experiments. We used three sets of plasmids respectively: ①pSA-ARO10-adh6 ②pSA-ARO10-adh6, pET-UGT85A1 ③pSA-ARO10-adh6, pET-UGT85A1, pCS-pgm-galu to construct three fermentation engineering bacteria. Among them, group ① only introduced the gene for tyrosol production. Group ② introduced a complete metabolic pathway for the production of salidroside. Group ③ also introduced auxiliary genes that can regulate metabolic flux. We used these three different engineering bacteria to conduct basic fermentation experiments.

After sampling, liquid chromatography analysis showed that salidroside was detected in both groups ② and ③, and the yield of group ③ was significantly greater than that of group ②. Comprehensive analysis of each set of data shows that our engineered bacteria can successfully produce salidroside with good yields.

Verification II RNA Thermometer

In our design, the RNA thermometer and light control switch together with the AND gate form a suspend switch. We need to allow the engineering bacteria to recover after being worn by the human body, which means we need to make the RNA thermometer have better temperature sensing around 37°C.

In order to prove that the RNA thermometer can meet the design requirements of our engineering bacteria, we constructed three types of E. coli (U6, U7, U8) with EGFP and RNA temperature elements. We drew a standard curve over time to determine 16h as the optimal sampling time.

Through the analysis of fluorescence intensity, we found that at different temperatures, the fluorescence intensity of U0 group will decrease as the temperature decreases. However, at 30°C and 37°C, the fluorescence intensity of groups U6, U7 and U8 was significantly higher than that at 17°C and 22°C. This proves that our RNA temperature element has good sensing capabilities near 37°C and can meet our temperature control conditions.

Fig.1 RFU/OD-Temperature Curve

Fig.2 A quick comparison of RNA thermometer constructs by temperature-controlled EGFP expression in E. coli

Verification III Liposome vesicles

In order to effectively combine our engineered bacteria with contact lenses, we plan to use liposome vesicles to encapsulate E. coli. Liposome vesicles play an important role in encapsulating bacterial storage media and blocking metabolic waste.

In order to verify the feasibility of encapsulating E. coli with liposome vesicles, we used LB liquid culture medium containing E. coli instead of water for hydration to prepare liposome vesicles encapsulating E. coli. According to the DLS results, when we hydrated with pure water, we obtained liposomes with a size around 1200nm. When hydrated with LB medium containing E. coli, in addition to the peak at 1200-1500 nm, we also found a peak around 5500 nm, indicating that we may have obtained liposomes containing E. coli. And we also observed E. coli wrapped in liposome vesicles under the microscope.This proves that our idea of using liposome vesicles to encapsulate engineering bacteria is feasible.

Fig.3 DLS result of lipsome

Fig.4 E. coli encapsulated in liposome vesicles