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Team:Carnegie Mellon/Met-Results
From 2012.igem.org
Dosage Curves of Spinach and FAP with Their Respective Dyes
In order to understand binding kinetics of both Spinach and FAP with their respective fluorogens, we collected dosage curves of the biosensors by adding different concentrations of fluorogens to bacteria that express both Spinach and FAP. Based on the dosage curves, we calculated dissociation constants (KD) of each biosensor-fluorogen complex and maximum saturation dose of fluorogen.
Figure 1: Fluorescence intensities of Spinach-DFHBI at a fixed concentration of Spinach and different concentrations of added DFHBI . The measured KD of the Spinach-DFHBI complex is 537nM [2]. Our measured KD is 100 times smaller than the published KD. We hypothesized that this could be due to magnesium levels inside bacteria because it has been shown that the binding of DFHBI by Spinach is sensitive to magnesium concentration. We also measured the dosage curves at both 10th and 60th minute timepoint. We did not observe significant difference in the fluorescence levels, suggesting that DFHBI diffusion into bacteria and its binding to Spinach occurs rapidly. Lines are drawn as guide of eyes. Please refer to the Protocols page for details of experiments.
Figure 2: Fluorescence intensities of FAP-MG at a fixed concentration of FAP and different concentrations of added MG . The measured KD of the FAP-MG complex is close to the published value of 320nM [1]. A line is drawn as guide of eyes. Please refer to the Protocols page for details of experiments.
RNA and Protein Expression Levels of T7Lac Promoters
We aim to compare expression levels of three new T7Lac promoters with the wild-type T7Lac promoter, when either 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) or Malachite Green (MG) was added. DFHBI is a specific fluorogen that binds to Spinach and MG is a specific fluorogen that binds to FAP. Therefore, we assume that there is a positive correlation between fluorescence values and the amount of either RNA and proteins in bacteria.
Figure 3: Spinach fluorescence over time. Fluorescence values increase over time with all promoters. Mutant I closely parallels the wild-type promoter in terms of magnitudes and expression rates of Spinach. Mutant II exhibits significantly lower fluorescence levels than the wild-type promoter, indicating a slower mRNA transcription rates with time. Fluorescence levels of mutant III seems to be increasing at an accelerating rate as compared to the wild-type promoter and reach a significantly higher fluorescence level at the end of the experiment. All fluorescence values are normalized by the corresponding OD600 readings. Error bars indicate one standard error of two replicates. Please refer to the Time-Lapse protocol in the Protocols page for the full experimental details.
Figure 4: FAP fluorescence over time . Fluorescence values increase over time with all promoters. The fluorescence level of Mutant I increases more rapidly than the other constructs, indicating that this promoter indirectly increases the translation rate of mRNA from the promoter. Fluorescence levels of mutant II closely parallels the wild type fluorescence levels, being only slightly lower in magnitude. Mutant III's fluorescence levels increase very rapidly at first, but seems to be leveling off after one hour. This may indicate that bacteria have adapted host machinery to compensate for the metabolic burden. All fluorescence values are normalized by the corresponding OD600 readings. Error bars indicate one standard error of two replicates. Please refer to the Time-Lapse protocol in the Protocols page for the full experimental details.
[1] Szent-Gyorgyi, Christopher, Brigitte A. Schmidt, Yehuda Creeger, Gregory W. Fisher, Kelly L. Zakel, Sally Adler, James A J. Fitzpatrick, Carol A. Woolford, Qi Yan, Kalin V. Vasilev, Peter B. Berget, Marcel P. Bruchez, Jonathan W. Jarvik, and Alan Waggoner. "Fluorogen-activating Single-chain Antibodies for Imaging Cell Surface Proteins." Nature Biotechnology 26.2 (2007): 235-40. Print.
[2] Paige, J. S., K. Y. Wu, and S. R. Jaffrey. "RNA Mimics of Green Fluorescent Protein." Science 333.6042 (2011): 642-46. Print.
