Part:BBa_K1391108:Experience
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<a name="ex4"></a>
Experiment 4:
Quantifying cleavage levels with non-activated receptorA  "> <img width="95%" src=""> </a> |
B  "> <img width="95%" src=""></a> |
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Non-activated cells were examined to determine basal TEVp cleavage levels. eBFP was used as a transfection marker in all samples, and TEVp cleavage was measured via Gal4UAS:mKate activation (Gal4UAS is a Gal4VP16-inducible promoter and mKate production results in red fluorescence). Black lines indicate a control containing Gal4UAS:mKate, TRE:Syk-TEVp (inducible Syk-TEVp), and all components of the BCR, where neither CD79A nor CD79B is fused to Gal4VP16. Cleavage in non-activated cells was examined for three different variations of transcription factor (Gal4VP16) placement: (A) CD79A-Gal4VP16 and CD79B, (B) CD79A and CD79B-Gal4VP16, and (C) CD79A-Gal4VP16 and CD79B-Gal4VP16. Red lines indicate controls for each of these variations where no Syk-TEVp was transfected. Blue lines indicate samples where Syk-TEVp was transfected (and its expression induced in the rtTA/TRE system using 2000nM doxycycline). |
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To determine the frequency of non-specific activation of our system, we tested our system's output in cells that were not activated (they were incubated with neither anti-IgM antibodies nor beta-amyloid oligomers). We used constitutive eBFP (blue fluorescence) as a transfection marker and Gal4UAS:mKate (red fluorescence) as a reporter for system activation. Gal4UAS is a mammalian promoter whose activation is dependent on binding by Gal4VP16, the transcription factor that we fused to CD79A and CD79B components of the BCR, meaning that the production of a red fluorescent signal should be related to cleavage of the transcription factor from the receptor by Syk-TEVp. Three different transcription factor arrangements were examined: CD79A-Gal4VP16 and CD79B, CD79A and CD79B-Gal4VP16, and CD79A-Gal4VP16 and CD79B-Gal4VP16.
As expected, across most variations of transcription factor placement (such as B, C), the highest levels of red fluorescence that we observed occurred in samples where Syk-TEVp was expressed at high levels (with 2000nM doxycycline induction under the regulation of rtTA and TRE). Also unsurprisingly, we observed almost no red fluorescence in cells where the BCR components did not contain any fusion proteins with our transcriptional activator (i.e., where CD79A and CD79B were used without any fusions to Gal4VP16). To our surprise, however, we saw some red fluorescent output in cells that were not transfected with Syk-TEVp (in A and C, at levels comparable to those observed under high Syk-TEVp induction). This suggests that, in some cases, TEV protease cleavage is not required for our system to produce an output. To explain this phenomenon, it may be possible that, rather than localizing to the cell membrane, fusion proteins of CD79A and CD79B with Gal4VP16 are recruited to activate Gal4UAS:mKate.
From this non-activated receptor experiment, we learned that high levels of Syk-TEVp result in system activation even in the absence of stimulus and that our system can produce high levels of output even in the absence of TEV protease. While investigating these phenomena in more detail could produce useful information for the optimization of our system, we were not able to pursue this line of inquiry given our limited time frame.
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<a name="ex5"></a>
Experiment 5:
Cleavage levels in active versus non-activated receptor
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Cells activated with anti-IgM antibodies show lower levels of fluorescent output relative to non-activated cells. Red lines indicate cells activated with anti-IgM antibodies and blue lines indicate non-activated cells. Red fluorescent output was used to quantify system activation based on signaling between Gal4VP16 and Gal4UAS:mKate. tagBFP was used as a transfection marker. Various conditions were assayed which involved transfecting different masses of the B-cell receptor components and varying levels of Syk-TEVp (achieved by adding differing levels of doxycycline). Some of the conditions that were tested included: (A) 25ng of each receptor component, 10nM doxycycline; (B) 12.5ng of each receptor component, 1nM doxycycline; (C) 6.25ng of each receptor component, 0nM doxycycline; (D) 6.25ng of each receptor component, 0nM doxycycline. |
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Our last experiment compared output from activated (using anti-IgM antibodies) and non-activated versions of our system. This experiment used a similar design to that of Experiment 4 (including the same transfection marker (eBFP) and fluorescent output (mKate); however, the only variation of transcription factor placement that was tested was CD79A-Gal4VP16 and CD79B. To find the best signal to noise ratio for our system, we varied two different parameters: the mass of receptor components that was transfected and the amount of Syk-TEVp present (which was altered using different doxycycline concentrations). A sampling of these cases are presented here (A,B,C,D). In every case that we tested, the amount of system output was higher for the non-activated cells than for the activated cells, which was the opposite of what we were expecting. Though the exact mechanism behind this discrepancy remains unclear, it is possible that activation of our receptor causes secondary, unintended effects that affect the cell's ability to produce our output. Further investigation will be required to determine the mechanism behind this effect.
Citations
[1] Gilad Barnea, Walter Strapps, Gilles Herrada, Yemiliya Berman, Jane Ong, Brian Kloss, Richard Axel, Kevin J. Lee.The genetic design of signaling cascades to record receptor activation. PNAS (2007) Print
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