Difference between revisions of "Team:UNC-Chapel Hill/Project"
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<p id="methods" style="color:#56A0D3; font-size:30px"> Methodology</p> | <p id="methods" style="color:#56A0D3; font-size:30px"> Methodology</p> | ||
<h2 style="color:#56A0D3"> Overall Experimental Design</h2> | <h2 style="color:#56A0D3"> Overall Experimental Design</h2> | ||
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<p style = "text-align: center"><img src = "https://static.igem.org/mediawiki/2015/3/34/Unc-methodology.png" width = "600px"></p> | <p style = "text-align: center"><img src = "https://static.igem.org/mediawiki/2015/3/34/Unc-methodology.png" width = "600px"></p> | ||
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| + | <p>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. </p> | ||
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<p> 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.</p> | <p> 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.</p> | ||
<p style = "text-align: center"><img src = "https://static.igem.org/mediawiki/2015/e/e1/Unc-cuvettes.png" width = "900px"></p> | <p style = "text-align: center"><img src = "https://static.igem.org/mediawiki/2015/e/e1/Unc-cuvettes.png" width = "900px"></p> | ||
| − | <p> 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. </p> | + | <p id = "data"> 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. </p> |
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| − | <p> | + | <h2 style="color:#56A0D3">Data</h2> |
| + | <p style = "text-align: center"><img src = "https://static.igem.org/mediawiki/2015/e/e8/Unc-mlc2graph.png"><img src = "https://static.igem.org/mediawiki/2015/9/90/Unc-mlc3graph.png"><img src = "https://static.igem.org/mediawiki/2015/4/4e/Unc-mlc4graph.png"></p> | ||
| + | <p id = "future"> 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.</p> | ||
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| − | <p id="references" style="color:#56A0D3; font-size:30px"> | + | <p id="references" style="color:#56A0D3; font-size:30px"> Future Work</p> |
| − | + | <p>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! </p> | |
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Latest revision as of 03:46, 19 September 2015
| Tri Color Glucose Sensing System | ||||
| Abstract | Methodology | Results | Data | Future Work |
|
Abstract This project introduces a novel glucose sensing system in which glucose-responsive promoters drive the expression of three reporter chromoproteins. We designed four novel glucose-sensitive promoters and tested their ability to drive expression of reporter chromoproteins at various glucose concentrations. In conjunction with existing glucose sensitive promoters from the Parts Registry, we used our novel promoters to design a biological device that expresses different combinations of the three different chromoproteins in response to glucose in Escherichia coli. As such, this device can detect a larger dynamic range of concentrations of selected molecules (e.g., glucose). Our project aims to provide a cheaper alternative for diabetics than current, more expensive, glucose-monitoring systems. While driven by this initial problem, continuing work has shown that our approach may have its greatest potential as a more general molecular sensing platform, capable of being easily customized for the sensing of a broad range of relevant compounds. |
|
|
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! |