Team:MIT/BCR

From 2014.igem.org

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Outcome information <br />
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<h2>Experiments</h2><a name="experiments" ></a>
<h2>Experiments</h2><a name="experiments" ></a>
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<p style="font-size:15px"> <b> Experiment 1: </b> &nbsp; <i>Localization of receptor to the cell membrane</i> </p>
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<p style="font-size:15px"> <a name="ex1"></a><h3> Experiment 1: </h3> &nbsp; <i>Localization of receptor to the cell membrane</i> </p>
<br>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.
<br>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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<p style="font-size:15px"> <b> Experiment 2: </b> &nbsp; <i>Beta-amyloid binding to the receptor</i> </p>
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<p style="font-size:15px"> <a name="ex2"></a><h3> Experiment 2: </h3> &nbsp; <i>Beta-amyloid binding to the receptor</i> </p>
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<br>In this experiment we aimed to determine whether or not the B-cell derived receptor in our system was, in fact, binding to beta-amyloid oligomers. To do this, we transfected HEK293 cells with plasmids encoding our receptor components and hEF1a:eBFP2 as a transfection marker. We then treated the cells with biotinylated beta-amyloid oligomers and red AlexaFluor-conjugated streptavidin. If the receptor bound to the beta-amyloid, the streptavidin would, in turn, bind to the biotin on the beta-amyloid oligomers leading to a higher level of red fluorescence. Similarly to the first experiment, we analyzed the cells using both flow cytometry and confocal microscopy, looking for increased red fluorescence in cell populations that were transfected with the receptors.
<br>In this experiment we aimed to determine whether or not the B-cell derived receptor in our system was, in fact, binding to beta-amyloid oligomers. To do this, we transfected HEK293 cells with plasmids encoding our receptor components and hEF1a:eBFP2 as a transfection marker. We then treated the cells with biotinylated beta-amyloid oligomers and red AlexaFluor-conjugated streptavidin. If the receptor bound to the beta-amyloid, the streptavidin would, in turn, bind to the biotin on the beta-amyloid oligomers leading to a higher level of red fluorescence. Similarly to the first experiment, we analyzed the cells using both flow cytometry and confocal microscopy, looking for increased red fluorescence in cell populations that were transfected with the receptors.
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<p style="font-size:15px"> <b> Experiment 3: </b> &nbsp; <i>Evaluating relative levels of Syk-TEVp and endogenous Syk</i> </p>
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<p style="font-size:15px"> <a name="ex3"></a><h3> Experiment 3: </h3> &nbsp; <i>Evaluating relative levels of Syk-TEVp and endogenous Syk</i> </p>
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<br>In this experiment, we wanted to compare the levels of endogenous cofilin and Syk-TEV protease (TEVp) expressed under an inducible promoter with different levels of dox induction. This will give us an indication as to what level of expression of cofilin would lead to the best signal:noise ratio of exogenous cofilin to endogenous cofilin. To do this we transfected HEK293 cells with DNA encoding inducible expression of our Syk-TEVp fusion construct and hEF1a:eYFP as a transfection marker as well as hEF1a:rtTA. We added different concentrations of doxycycline. We analyzed the cell lysates by probing for cofilin in a Western blot analysis.  
<br>In this experiment, we wanted to compare the levels of endogenous cofilin and Syk-TEV protease (TEVp) expressed under an inducible promoter with different levels of dox induction. This will give us an indication as to what level of expression of cofilin would lead to the best signal:noise ratio of exogenous cofilin to endogenous cofilin. To do this we transfected HEK293 cells with DNA encoding inducible expression of our Syk-TEVp fusion construct and hEF1a:eYFP as a transfection marker as well as hEF1a:rtTA. We added different concentrations of doxycycline. We analyzed the cell lysates by probing for cofilin in a Western blot analysis.  
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<br>We used an antibody specific to Syk to probe for both Syk and Syk-TEVp. The difference in size between the endogenous Syk and the exogenous Syk-TEVp allow us to distinguish between the two on the Western blot, and hence compare their relative quantities. We also probed for GAPDH and eYFP in the Western blot. We used GAPDH as a loading control to allow us to normalize for the amount of protein that was loaded in each lane. In a similar way, probing for eYFP allowed us to normalize for the transfection efficiency.
<br>We used an antibody specific to Syk to probe for both Syk and Syk-TEVp. The difference in size between the endogenous Syk and the exogenous Syk-TEVp allow us to distinguish between the two on the Western blot, and hence compare their relative quantities. We also probed for GAPDH and eYFP in the Western blot. We used GAPDH as a loading control to allow us to normalize for the amount of protein that was loaded in each lane. In a similar way, probing for eYFP allowed us to normalize for the transfection efficiency.
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<p style="font-size:15px"> <b> Experiment 4: </b> &nbsp; <i>Quantifying cleavage levels with non-activated receptor</i> </p>
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<p style="font-size:15px"> <a name="ex4"></a><h3> Experiment 4: </h3> &nbsp; <i>Quantifying cleavage levels with non-activated receptor</i> </p>
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<p style="font-size:15px"> <b> Experiment 5: </b> &nbsp; <i>Cleavage levels in active versus non-activated receptor</i> </p><table>
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<p style="font-size:15px"> <a name="ex5"></a><h3> Experiment 5: </h3> &nbsp; <i>Cleavage levels in active versus non-activated receptor</i> </p><table>
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Revision as of 20:09, 17 October 2014

 


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B-cell receptors (BCRs) are multiprotein immune receptors found exclusively on the surface of B cells. The BCR multiprotein complex is centered around a membrane-bound IgM antibody. When the antibody binds to an extracellular antigen, receptors dimerize resulting in the phosphorylation of the intracellular tails of CD79A and CD79B by the tyrosine-protein kinase Lyn. In response, another cofactor, spleen tyrosine kinase (Syk), is recruited to the receptor and phosphorylated, initiating a signalling cascade that results in the proliferation of the activated B cells. This receptor is important in clonal selection of B cells during human immune response.

For this project, we engineered a BCR to respond to beta-amyloid plaques, the hallmark of Alzheimer's disease. This task was accomplished by using a beta-amyloid specific variable region [derived from Gantenerumab] in the membrane-bound IgM antibody. Our design was based on that of the Tango system [1], which capitalizes on the interaction between TEV protease (TEVp) and its cleavage site (TCS), an amino acid sequence for which the protease has a high affinity. A TEV cleavage site was used to link a transcriptional activator (Gal4VP16) to the intracellular tails of BCR accessory proteins CD79A and CD79B, and the receptor’s cofactor, Syk, was fused to TEV protease. Thus, when the modified receptor activates upon binding its antigen, beta-amyloid, Syk-TEVp fusion protein is recruited, bringing TEVp in close proximity to its cleavage site. This proximity of TEVp to TCS results in the cleavage of the transcriptional activator from the receptor releasing it to activate downstream gene circuits.

The engineered BCR we developed binds beta amyloid with high specificity and releases a transcriptional activator upon binding, making it an extremely valuable tool in the detection of Alzheimer’s Disease. More importantly, the IgM antibody that determines what the receptor binds can be easily swapped out as can the transcription factor the receptor releases. This means that the receptor we developed can bind to any molecule that an antibody can be produced against and it can release any transcription factor in response to the binding of the target molecule. This modularity allows this receptor to be generalized to almost any extracellular sensing making it an invaluable part of any synthetic biologists toolkit.

Outcome

Outcome information

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.

A

B

C

D

Determining membrane localization was complicated by bleedthrough from mKate into the yellow fluorescence channel. Samples were stained with Alexa fluor 488 (yellow) conjugated anti-IgM antibodies and evaluated using flow cytometry. Red fluorescence refers to the amount of mKate (transfection marker) in the cells and yellow fluorescence measures the amount of anti-IgM binding (a measure of the presence of B-cell receptors on the cell surface). (A) Untransfected, unstained HEK293; (B) HEK293 transfected with dummy DNA, stained; (C) HEK293 transfected with mKate, stained; (D) HEK293 transfected with mKate and BCR components, stained.


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


In the resulting images, we see a clear increase in yellow fluorescence between cells that were transfected with the receptors and those that were not. However, we do not see any clear localization to the membrane. Results of further experiments suggest that the receptors might be getting overexpressed, given the mass of receptor DNA we were transfecting and the fact that we were using a strong constitutive promoter to express the receptors.

Experiment 2:

  Beta-amyloid binding to the receptor



In this experiment we aimed to determine whether or not the B-cell derived receptor in our system was, in fact, binding to beta-amyloid oligomers. To do this, we transfected HEK293 cells with plasmids encoding our receptor components and hEF1a:eBFP2 as a transfection marker. We then treated the cells with biotinylated beta-amyloid oligomers and red AlexaFluor-conjugated streptavidin. If the receptor bound to the beta-amyloid, the streptavidin would, in turn, bind to the biotin on the beta-amyloid oligomers leading to a higher level of red fluorescence. Similarly to the first experiment, we analyzed the cells using both flow cytometry and confocal microscopy, looking for increased red fluorescence in cell populations that were transfected with the receptors.

Flow cytometry inconclusive about beta-amyloid binding. Cells were incubated with biotinylated beta-amyloid and Alexa fluor (red) conjugated streptavidin. Red fluorescence indicates beta-amyloid binding and blue fluorescence is the transfection marker (eBFP). (A) HEK293 transfected with eBFP; (B) HEK293 transfected with eBFP, stained with beta-amyloid and streptavidin; (C) HEK293 transfected with eBFP and synthetic B-cell receptor, stained with streptavidin; (D) HEK293 transfected with eBFP and synthetic B-cell receptor, stained with beta-amyloid and streptavidin

Experiment 3:

  Evaluating relative levels of Syk-TEVp and endogenous Syk



In this experiment, we wanted to compare the levels of endogenous cofilin and Syk-TEV protease (TEVp) expressed under an inducible promoter with different levels of dox induction. This will give us an indication as to what level of expression of cofilin would lead to the best signal:noise ratio of exogenous cofilin to endogenous cofilin. To do this we transfected HEK293 cells with DNA encoding inducible expression of our Syk-TEVp fusion construct and hEF1a:eYFP as a transfection marker as well as hEF1a:rtTA. We added different concentrations of doxycycline. We analyzed the cell lysates by probing for cofilin in a Western blot analysis.

A

B

Exogenous Syk-TEVp is expressed at comparable levels to endogenous Syk in HEK293 cells under high doxycycline induction. Transfection marker: eYFP. (A) Blocked in 5% BSA (B) Blocked using Odyssey Blocking Buffer


We used an antibody specific to Syk to probe for both Syk and Syk-TEVp. The difference in size between the endogenous Syk and the exogenous Syk-TEVp allow us to distinguish between the two on the Western blot, and hence compare their relative quantities. We also probed for GAPDH and eYFP in the Western blot. We used GAPDH as a loading control to allow us to normalize for the amount of protein that was loaded in each lane. In a similar way, probing for eYFP allowed us to normalize for the transfection efficiency.

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


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

Parts

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