Part:BBa_M50499:Experience

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Applications of BBa_M50499

For this project, we sought to create a synthetic DNA-based glucose biosensor in E. coli to eventually be paired with insulin-actuator system to treat elevated BGLs for type 1 diabetes. To do this, we re-engineered E. coli’s natural glucose-repressed lac operon to create a glucose-inducible promoter. We designed Plasmid E and hypothesized that placing the CAP binding site downstream from a constitutive promoter would create a glucose-inducible system that could be assayed by expressing GFP. Our experiments aimed to establish a dynamic range for Plasmid E that demonstrates clear glucose-induced fluorescence.

We resuspended bacteria with each plasmid in 10 mL of LB broth overnight. For each plasmid, we measured fluorescence over glucose concentrations ranging from 17.78 mM to 0.03 mM using two-fold dilutions and 0 mM to characterize a dynamic range.

The construct potentially demonstrates a dynamic range from 0 mM to 0.28 mM, albeit with overlapping error bars. This dynamic range does not extend to higher glucose concentrations, as the positive association between fluorescence and glucose concentration does not hold above a concentration of 0.28 mM.

We observed glucose-dependent expression at sub-millimolar glucose concentrations, but this relationship was not present at higher, physiologically relevant levels of glucose. This dynamic range corroborates previous research with the CAP binding site. From this experiment, we concluded that the current construct is not glucose-inducible at physiological levels.

"Time-averaged dose-response curves from initial time point to three hours with sampling every 15 minutes. Glucose concentrations ranged from 0.035 mM to 17.78 mM with each concentration doubling the previous. Normed fluorescence levels at a glucose concentration of 0 mM (not shown on semi-log plot) fell below the error bars of the fluorescence level for 0.035 mM for Plasmid E. Mean ± standard error shown for each point."

Next, due to the clear lack of a trend in fluorescence beyond 0.56 mM for all plasmids and the high variability in results, we chose to repeat this experiment with a narrower range of seven glucose concentrations (1.11 mM to 0.035 mM with two-fold dilutions and 0 mM). Within this smaller range of glucose values, the glucose dose-response curve demonstrates clear glucose-induced fluorescence. Thereby, we corroborated the existence of a dynamic range from 0.007 to 0.28 mM glucose.

"Time-averaged dose-response curves from initial time point to three hours with sampling every 15 minutes. Glucose concentrations ranged from 0.035 mM to 1.11 mM with each concentration doubling the previous. Normed fluorescence levels at a glucose concentration of 0 mM (not shown on semi-log plot) fell below the error bars of the fluorescence levels for 0.035 mM. Mean ± standard error shown for each point."

The first two experiments do not reveal the time course of the sensor’s response to changing glucose concentrations, as the LB culture media does not have a defined glucose concentration. Therefore, we performed another dynamic range experiment by culturing the bacteria overnight in EZ-Rich Medium without glucose and transferring them to higher glucose concentrations. This allows us to determine the time course of the sensor’s equilibration to an increase in glucose concentration. We tested a greater number of glucose concentrations by performing two-fold dilutions from 1.11 mM to 0.002 mM, along with 0 mM. By increasing the number of dilutions, we aimed to establish the lower limit of the sensor’s dynamic range.

Although the overlapping error bars complicate analysis, the dynamic range began between 0.002 and 0.0035 mM and shifted to 0.007 to 0.28 by 150 minutes. This final dynamic range appears to remain relatively constant after 150 minutes.

"Panel showing glucose dose-response plots over time from 0 to 210 minutes, with sampling every 30 minutes. Glucose concentrations ranged from 0.002 mM to 1.11 mM with each concentration doubling the previous. Bacteria were cultured in EZ-Rich Medium without glucose. Mean ± standard error shown for each point."

We performed a complementary experiment to our last by incubating bacteria transformed with Plasmid E in a glucose-rich environment (0.28 mM) and transferring them to glucose concentrations ranging from 0.002 mM to 0.56 mM. This allows us to analyze the sensor’s equilibration to a decrease in glucose concentration.

After transfer to glucose-rich media, the bacteria began to show glucose-dependent expression at 60 minutes and maintained this glucose-induced fluorescence relationship consistently for all subsequent measurements. This response occurred faster than for the bacteria that incubated in glucose-starved media and also appeared to be more precise, as indicated by the narrower error bars. The sensor appeared to exhibit a clear dynamic range over 0.014 to 0.28 mM, which roughly corresponds to the range determined previously.

"Panel showing glucose dose-response plots over time from 15 to 120 minutes, with sampling every 15 minutes. Glucose concentrations ranged from 0.002 mM to 1.11 mM with each concentration doubling the previous. Bacteria were cultured in EZ-Rich Medium with 0.28 mM concentration of glucose. Mean ± standard error shown for each point."

Our findings suggest that our construct works successfully as a glucose-inducible promoter at low glucose concentrations and responds actively to changing glucose concentrations.

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