Team:MIT/BCR

From 2014.igem.org

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<p style="font-size:15px"> <b> Experiment 1: </b> &nbsp; <i>Localization of receptor to the cell membrane</i> </p>
<p style="font-size:15px"> <b> Experiment 1: </b> &nbsp; <i>Localization of receptor to the cell membrane</i> </p>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In the first preliminary experiment, we aimed to determine if the engineered B-cell receptor components (CD79A, CD79B, IgM Heavy Chain, and Kappa Light Chain) were able to assemble to form the receptor complex and localize to the cell membrane. This is important to ascertain since the receptors would be used to detect beta-amyloid oligomers in the extracellular matrix of the brain. The system must therefore be able to detect the oligomers outside the cell and relate this information inside the cell.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;To determine localization of the receptors, we used IgM specific antibodies to immunostain for the receptors. We analyzed the immunostained samples in two ways. The first was through flow cytometry analysis which would allow us to determine if the antibodies, and in turn the receptors, were on the cell surface since the cells were not permeabilized. We also confocal microscopy to look at the immunostained samples in order to visualize membrane localization and determine subcellular localization, if any, in permeabilized cells.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;For samples that were to be analyzed by flow-cytometry, we transiently transfected HEK293 cells with plasmids encoding constitutive expression of the engineered B-cell receptor components under the hEF1a promoter along with hEF1a:mKate2 as a transfection marker. We then treated cells with a anti-IgM antibodies conjugated to a yellow AlexaFluor which allowed us to detect them using the flow cytometer.
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<br><br>INSERT FIRST FLOW CYTOMETRY DATA
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In our initial trial of this experiment, we saw a significant increase in yellow fluorescence between untransfected cell populations and transfected ones. The interesting result was that we saw similar amounts of yellow fluorescence between cells that were transfected with just hEF1a:mKate2 and those transfected with both hEF1a:mKAte2 and the receptor DNA and that the data showed a very strong one-to-one correlation between yellow and red fluorescence. This led us to believe that our results were actually stemming from bleedthrough of the mKate2 fluorescent protein into the FITC channel used to detect yellow fluorescence.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;To address this problem, we decided to not use a transfection marker since all of the fluorescent proteins that we had available to us would produce the same, if not a greater, bleedthrough effect.
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<td width="70%" align=center><a href="https://static.igem.org/mediawiki/2014/0/01/MIT_BCR_blind_plots.png"><img width="90%" src="https://static.igem.org/mediawiki/2014/0/01/MIT_BCR_blind_plots.png"></a></td>
<td width="70%" align=center><a href="https://static.igem.org/mediawiki/2014/0/01/MIT_BCR_blind_plots.png"><img width="90%" src="https://static.igem.org/mediawiki/2014/0/01/MIT_BCR_blind_plots.png"></a></td>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;In our second method, we looked for membrane localization through confocal microscopy in order to visualize membrane localization and other, subcellular localization, if any. To do this we, again, transfected HEK293 cells with DNA encoding constitutive expression of the receptors and hEF1a:eYFP as a transfection marker. Our choice of transfection marker here was not important since any fluorescence would be quenched when the cells were fixed. We used the transfection marker to determine if the transfection efficiency was high enough before we proceeded with the immunostaining. After transfecting, we then fixed the samples and stained them with the same selection of antibodies we used for the flow cytometry analysis as well as DAPI to stain the nucleus for better visualization of the cells.
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Revision as of 14:38, 17 October 2014

 


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Experiments


Experiment 1:   Localization of receptor to the cell membrane

      In the first preliminary experiment, we aimed to determine if the engineered B-cell receptor components (CD79A, CD79B, IgM Heavy Chain, and Kappa Light Chain) were able to assemble to form the receptor complex and localize to the cell membrane. This is important to ascertain since the receptors would be used to detect beta-amyloid oligomers in the extracellular matrix of the brain. The system must therefore be able to detect the oligomers outside the cell and relate this information inside the cell.
      To determine localization of the receptors, we used IgM specific antibodies to immunostain for the receptors. We analyzed the immunostained samples in two ways. The first was through flow cytometry analysis which would allow us to determine if the antibodies, and in turn the receptors, were on the cell surface since the cells were not permeabilized. We also confocal microscopy to look at the immunostained samples in order to visualize membrane localization and determine subcellular localization, if any, in permeabilized cells.
      For samples that were to be analyzed by flow-cytometry, we transiently transfected HEK293 cells with plasmids encoding constitutive expression of the engineered B-cell receptor components under the hEF1a promoter along with hEF1a:mKate2 as a transfection marker. We then treated cells with a anti-IgM antibodies conjugated to a yellow AlexaFluor which allowed us to detect them using the flow cytometer.

INSERT FIRST FLOW CYTOMETRY DATA

      In our initial trial of this experiment, we saw a significant increase in yellow fluorescence between untransfected cell populations and transfected ones. The interesting result was that we saw similar amounts of yellow fluorescence between cells that were transfected with just hEF1a:mKate2 and those transfected with both hEF1a:mKAte2 and the receptor DNA and that the data showed a very strong one-to-one correlation between yellow and red fluorescence. This led us to believe that our results were actually stemming from bleedthrough of the mKate2 fluorescent protein into the FITC channel used to detect yellow fluorescence.
      To address this problem, we decided to not use a transfection marker since all of the fluorescent proteins that we had available to us would produce the same, if not a greater, bleedthrough effect.
Flow cytometry demonstrates synthetic B-cell receptor membrane localization in HEK293 cells. Cells were stained with Alexa Fluor 488 conjugated anti-IgM antibodies. Typical HEK293 cells do not express B-cell receptors whereas Ramos cells are derived from B cells and do express B-cell receptors. (A) HEK293 transfected with dummy DNA, stained; (B) HEK293 transfected with synthetic B-cell receptor, stained; (C) Ramos, unstained; (D) Ramos, stained

      In our second method, we looked for membrane localization through confocal microscopy in order to visualize membrane localization and other, subcellular localization, if any. To do this we, again, transfected HEK293 cells with DNA encoding constitutive expression of the receptors and hEF1a:eYFP as a transfection marker. Our choice of transfection marker here was not important since any fluorescence would be quenched when the cells were fixed. We used the transfection marker to determine if the transfection efficiency was high enough before we proceeded with the immunostaining. After transfecting, we then fixed the samples and stained them with the same selection of antibodies we used for the flow cytometry analysis as well as DAPI to stain the nucleus for better visualization of the cells.
Fluorescent microscopy suggests membrane localization of synthetic B-cell receptor in HEK293 cells. Cells were stained with Alexa Fluor 488 conjugated anti-IgM antibodies. (Left) Untransfected HEK293 control, stained; (Right) HEK293 transfected with synthetic B-cell receptor, stained

Experiment 2:   Beta-amyloid binding to the receptor


Experiment 3:   Evaluating relative levels of Syk-TEVp and endogenous Syk


Experiment 4:   Quantifying cleavage levels with non-activated receptor

A

B

C

Non-activated cells were examined to determine basal cleavage levels. Transfection marker: eBFP. Black lines indicate control without CD79A-Gal4VP16 or CD79B-Gal4VP16 fusion proteins. Red lines indicate controls with no Syk-TEV protease fusion proteins. Blue lines indicate Syk-TEV protease expression (Tre promoter induced with 2000nM doxycyclin). (A) CD79A-Gal4VP16 (B) CD79B-Gal4VP16 (C) both CD79A-Gal4VP16 and CD79B-Gal4VP16


Experiment 5:   Cleavage levels in active versus non-activated receptor

A

B

C

D

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. 15-16, 17-18, 21-22, 23-24 (A) Conditions (B) Conditions (C) Conditions (D) Conditions


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