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Tri Color Glucose Sensing System

Methodology

Overall Experimental Design

To construct our plasmid, we had to create the three different promoters and chromoproteins separately. In addition, we designed 4 MLC parts based on literature and had them synthesized as g-Blocks. The 4 MLC's differed in strength of promoter, and placement of promoter vs. binding site. This was to determine if we could alter the sensitivity to glucose of the MLC parts based on these differences. We originally attempted 3A assembly of the inducible, repressible, and MLC promoters with the chromoproteins, but since the promoters were very small in size (<50 bp), we would often lose the promoter during the digestion. Therefore, we decided to have the promoter and chromoprotein synthesized together as a gBlock. The next step was to insert the gBlock into the backbone, but we were only able to successfully insert three different MLC's plus yellow chromoprotein, and the inducible plus blue chromoprotein. The parts that we had successfully created were then characterized using UV-vis spectroscopy. The parts were grown in liquid cultures of varying glucose concentration ranging from 3 mg/dL - 500 mg/dL. Using the Nanodrop and 1 cm pathlength micro-cuvettes, the absorbance was taken at the maximum absorbance wavelength of 504 nm for the yellow chromoprotein (according to 2013 Braunschweig) after 24 hours of cell growth. The machine was blanked with a liquid culture of colorless cells that were grown at the same time. The inducible part did not express any blue color after 24 hours of growth. We suspect this was due to the glucose concentrations being too low.

Results

Results

Shown here are the different intensities of yellow chromoprotein expression with respect to varying glucose concentrations. The parts were grown in LB media of varying glucose concentration ranging from 3mg/dL to 500 mg/dL. These values of glucose concentration were chosen because they were within the range of blood glucose levels. MLC's were expected to behave as glucose inducible promoters; however, from our results, the data appears to show the expression of the chromoprotein decrease as the concentration of glucose increases. All three MLC's seemed to behave in the same fashion and are all sensitive to glucose levels.

This picture shows a clearer image of the decreasing yellow color intensity in MLC 4 as glucose concentration is increased. Since the LB media itself is yellow in color, it is diffucult to distinguish between the chromoprotein and the background color of the culture. The decrease in expression can be more clearly seen in the absorbance vs. concentration plots below.

Data

Absorbance plots for the three MLC parts attached to the yellow chromoprotein. These plots show that instead of behaving as a glucose inducible promoter, the MLC parts instead act like glucose repressible promoters by decreasing in protein expression. In addition, the accurate range of measurement appeared to stop at 200 mg/dL of glucose. After that concentration, the absorbances were inconsistant and negative suggesting the rate of cell growth was impacted, or some other mechanism was present.

Future Work

Although we created a glucose repressible promoter instead of an inducible, our theoretical construct would still work, as long the sensitivity to glucose differs between the three promoters. We hope to further test the sensitivity of the MLC part and compare it to other repressible promoters in the registry. In addition, we will continue working on piecing the entire construct together and design parts with restriction sites around the promoters to allow for easy exchange of promoters for other teams to customize this tricolor system to their needs. We are looking forward to meeting all these goals at next years iGEM!