Team:Carnegie Mellon/Met-Results


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Time course experiment with Spinach and FAP

The two figures below are plots of Spinach and FAP fluorescence over time. The figures compare the fluorescence 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. All fluorescence values are normalized by the corresponding OD600 readings. Please refer to the Time-Lapse protocol in the Protocols page for the full experimental details. For both the Spinach-DFHBI and FAP-MG plots, fluorescence values increase over time with all promoters. This makes intuitive sense, as we expect the amount of transcribed RNA (reported by Spinach-DFHBI) and translated protein (reported by FAP-MG) to increase with time after inducing cells with IPTG.

The fluorescence level of Mutant I increases more rapidly than the other constructs, indicating that this promoter significantly increases the translation rate of mRNA from the promoter. Mutant II closely parallels the wild type fluorescence level, being only slightly lower in magnitude. Mutant III's fluorescence level increases very rapidly at first, but seems to be leveling off after one hour. This may indicate that bacteria have adapted host machinery to the metabolic burden.

Mutant I closely parallels the wild-type promoter in terms of magnitudes and expression rates of FAP fluorescence levels. Mutant II exhibits significantly lower fluorescence levels than the wild-type promoter, indicating a slower mRNA translation 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.

Dosage curves of Spinach and FAP with their respective dyes

Dosage curves for both MG and DFHBI were obtained to determine whether the dissociation constant (Kd) values matched those found in literature, and also to determine the maximum saturation dose. The experiment was carried out according to the Dosage curve protocol in the Protocols page. <\p>

The measured Kd is close to the published value of 320nM [1] for one of the similar FAPs, which verifies that we were truly measuring the fluorescence of the FAP-MG construct and that FAP was successfully expressed by our construct.

The measured Kd for the Spinach-DFHBI complex is 537nM [2]. Our measured Kd is around a 100 times smaller in magnitude.
[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. What the figure is ploting significant results Brief summary of methodology