In order to verify the functions of our biomarker sensing system, we designed three steps to prove our concept of the project. The three approaches include in vivo functional test, device monitoring, and kill switch construction.
We combined SaeRS with a fluorescent protein gene, fluorescent signal of which can indicate the activation of SaeRS. The function of the SaeRS system then can be verified by OD600 and fluorescent intensity measurements.
To ensure the biosafety both in vivo and in the environment, we constructed a kill switch in the engineered plasmid. By changing the temperature, the CcdB protein will be expressed to eliminate the bacteria.
With these approaches, we are confident that “VigilaGuard” is capable of biosensing and disease prevention, which is more than just a concept but is feasible in the future.
In order to assess the sensitivity of our modified E. coli, we devised a practical experiment. We cultivated the modified E. coli in solutions with varying concentrations of Zn2+ and monitored the fluorescence density emitted by the bacteria. We quantified both the fluorescence intensity and the OD600 readings of the E. coli to examine the correlation between Zn2+ concentration and the expression levels of the fluorescent genes.
The Zn2+ test was performed twice, with data points having OD600 measurements below 0.2 being excluded to mitigate potential errors stemming from the limited bacterial population. We suspect that the growth issues observed in high Zn2+ concentration groups are likely due to elevated osmotic pressure.
We noted that for Zn2+ concentrations below 1mM, there is no distinct trend in fluorescent density. However, within the range of 1 to 1.4mM Zn2+ concentration, there is a noticeable correlation between fluorescent density and Zn2+ concentration. Nonetheless, it's important to mention that our experiment only measured fluorescent density up to 1.4 mM Zn2+.
To check whether the result of our device is correct and is prepared for practical use, we compare the data collected by wet lab with our data measured by our device. The correlation of fluorescent protein emission light intensity inhibit by different concentration of zinc ion can be found in both graph below.
After observing the relationship between light intensity at different wavelengths and the concentration of fluorescent proteins, we determined the optimal measurement range to be 425nm to 465nm. Therefore, the final device was designed to measure these specific wavelengths. We conducted a comparison verification using the same samples, consisting of SaeS bacteria with varying zinc ion concentrations, with both the device and a plate reader. Higher zinc ion concentrations suppress the expression of the fluorescent protein gene, resulting in reduced fluorescence intensity. The first graph below represents the results measured using our hardware device, while the second graph below shows the theoretical values obtained using a wet lab plate reader. The matching trends between the two graphs validate the functionality of our device.
After a series of experiments and validations, our final device has indeed demonstrated its capability to accurately measure the variations in zinc ion concentration and fluorescence light intensity. As the zinc ion concentration increases, the light intensity decreases, showing a consistent downward trend. Moreover, the trend aligns with the results obtained by the wet lab group using the sophisticated instrument, the Infinite 200 Pro Plate Reader. This serves as evidence of the effectiveness of our device.
To demonstrate the capability of our EET system device to accurately measure different concentrations of Zn2+ ions in a solution of lysogeny broth + Chloramphenicol + Ampicillin, and to prepare it for practical use, our team conducted tests using known concentrations of Zn2+ solution. We employed multiple CV plots overlaid on top of each other (Figure 4A. and Figure 4B.) and established calibration curves (Figure 5.) to observe the coefficient of determination (R2) as a means of verification and validation.
To enhance the accuracy and credibility of CV (Cyclic Voltammetry) plots and results, the scan rate was set to a slower 100mV/s to avoid distortion of the results and all the CV plots we used were from the fourth cycle of each measurement in the IviumSoft software. By referencing the reduction potential of the reference electrode in the standard electrode potential table and the reduction potential of zinc ions (Zn(OH)4 2⁻＋2e⁻ ⇌ Zn(s)＋4OH⁻, E°(V)=-1.199), we ultimately set the scanning range from -2V to +2V. The study and discussion of the CV plot for Zn2+ and the creation of the calibration curve were ultimately conducted with the reduction peak at -1V.
Ignoring the small noise peaks, which may be due to redox reactions of trace substances or contaminants in the solution, it is evident from Figure 4A. and Figure 4B. that at -1V, there is a consistent trend among different concentrations. Higher concentrations of Zn2+ exhibit more pronounced reduction peaks, with their CV plot lines appearing lower. For example, the highest concentration of 1.8mM Zn2+ is represented by the bottom purple line in Figure 4B. In summary, the CV overlay plots in this test have successfully and comprehensively validated the performance of Zn2+ at different concentrations, demonstrating a consistent and favorable trend.
From Figure 5., we can see that our trendline has a positive correlation, with stronger currents as the Zn2+ concentration increased. The formula of the trendline is y= 0.185*x + 0.256, with a coefficient of determination R2 of 0.971. The coefficient of determination for our trendline is above the threshold of 0.95, which makes it reliable in predicting Zn2+ concentration when we have the current of the solution at -1V.
In conclusion, the EET device system developed by our team has successfully demonstrated its capability to measure concentrations ranging from 0mM to 1.8mM of Zn2+ in an LB+CM+Amp environment. This is supported by the high coefficient of determination (R2) of 0.971 achieved with our established calibration curve. By designing this EET device system, we have created something for everyone to use and can prepare it for practical use.
Environmental hazard is a crucial concern when engineering bacteria. In addition, Our project is designed to detect biomarkers in the human body. Therefore, it is essential to focus on biosafety both in vivo and in the environment. We constructed a kill switch into the engineered plasmid, aiming to achieve the goal of biosafety.
We utilized part BBa_K115002, an RNA thermometer which initiates translation at 37°C, as the promoter. We also constructed the ccdB coding region BBa_K145151. Also, we added gene sequences of the promoter and the operator of lac operon to the plasmid. The complete sequence of the kill switch is illustrated in Figure 6.
of the kill switch is illustrated in Figure 1. At 37°C, the thermometer will be activated, promoting the translation of lacI repressor (from BBa_C0012). Subsequently, the promoter BBa_R0011 will be inhibited, preventing the expression of the downstream gene ccdB.
At room temperature, the thermometer won’t be activated, which means that ccdB will be expressed and lead to the production of CcdB protein. The bacteria then die because of the damage caused by CcdB protein.
We tested the function of the kill switch in two mediums: LB+CM broth and LB+CM agar plates. In each medium, we divided the samples into three groups: Plasmid with the kill switch and lacI (Group 1), Plasmid with the kill switch but without lacI (Group 2), and Plasmid without the kill switch and lacI (Group 3).
Initially, Group 1 and Group 3 were incubated at 37°C, while Group 2 was left to grow at room temperature overnight. Subsequently, we divided each group into two parts, denoted as Group 1-1, 1-2, 2-1, 2-2, 3-1, and 3-2. Group 1-1, 2-1, and 3-1 were incubated at 37°C, while Group 1-2, 2-2, and 3-2 were kept at room temperature.
To assess the bacterial growth status, we measured the fluorescence density to create growth curve graphs for bacteria in LB+CM broth and recorded the colony sizes of the groups on agar plates. By analyzing the growth curve and changes in colony size, we could determine whether the designed kill switch was effective.
The expected results differ between Group 1 and Group 2. Group 1 might survive at 37°C and die at room temperature, whereas Group 2 might survive at room temperature and die at 37°C. As for Group 3, due to the absence of the kill switch, we expected that the bacteria might survive at both 37°C and room temperature.
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