Difference between revisions of "Team:EPFL/Measurement"

 
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            <h1 class="text-center">Measurement</h1>
 
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                <p class="lead">At the heart of CAPOEIRA is our Encapsulin vaccine system. Since the beginning of our project, we sought to have a characterization of our Encapsulin vaccine as extensive as time and resources would allow for. The first characterizations
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                  we did were sequence verifying our plasmid constructs, and this is detailed on our results and registry pages. Then, we characterized the protein productivity of our TX-TL cell free expression system. Once our Encapsulin-based protein
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                  constructs were expressed in our cell free TX-TL system, we characterized this expression using standard denaturing SDS-PAGE, fluorescent SDS-PAGE, and native gel. The standard denaturing SDS-PAGE gels were the first indication for the
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                  correct expression of our protein constructs. However, we went further through the expression of our protein with lysine BODIPY to ensure correct expression. Moreover, we ran a native gel to characterize our protein in its native state.
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                </p>
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                <p class="lead">After gel-based characterizations that indicate the approximate expressed protein weight, we figured that we should also be characterizing the structure of our protein. Thus, we performed DLS size measurement to characterize the diameter
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                  of our expressed Encapsulin.
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                </p>
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                <p class="lead">We thus have strong evidence that we are truly expressing our intended constructs. Still, we wanted a definite proof of the correct expression of Encapsulin, and Encapsulin with the standard immunogenic peptide OT-1. We thus prepared and
 +
                  submitted our expressed proteins to mass spectrometry facility at EPFL that confirmed the expression of both Hexahistidine Encapsulin and Hexahistidine Encapsulin-OT-1. This is as decisive an evidence as we can get! One other characterization
 +
                  of our vaccine that we wished to do, was TEM measurements; however, time and resource limitations prevented us to achieving it.
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                </p>
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                <br>
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                <p class="lead">Following this characterization of the correct expression of our vaccine constructs, we proceeded to validate the efficiency of our vaccine construct. We first set to demonstrate the uptake of Encapsulin by dendritic cells. We thus approached
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                  a unique technology available near EPFL campus, NanoLive(fundamental patent US. 8,937,722 & EU WO 2011/121523). This technology allows the 3D imaging of biologically relevant objects, with the option to overlay fluorescent 2D images
 +
                  in. We were indeed able to show that dendritic cells cultured in 3D Matrigel internalize our fluorescently labeled Encapsulin nanoparticles. Multiple 3-D images with 2-D fluorescent overlays were acquired. Moreover, a video of a 3D reconstruction
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                  of a mature dendritic cell after vaccine production was produced.
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                </p>
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                <br>
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                <p class="lead">Finally we aimed to validate neoantigen presentation on dendritic cells following delivery on Encapsulin surface. For this, we expressed Encapsulin/Encapsulin-OT-1, presented to cultured murine dendritic cells, and then did immunostaining/FACS
 +
                  to check for OT-1 presentation on MHC-I complexes on dendritic cells. Unfortunately, our results were not positive. However, we aim to repeat this measurement, as previous literature has indeed shown that Encapsulin can deliver neoantigens
 +
                  for dendritic cell display on MHC-1 complexes.
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                </p>
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              </figure>
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Latest revision as of 20:34, 7 December 2018

iGEM EPFL 2018

Measurement


At the heart of CAPOEIRA is our Encapsulin vaccine system. Since the beginning of our project, we sought to have a characterization of our Encapsulin vaccine as extensive as time and resources would allow for. The first characterizations we did were sequence verifying our plasmid constructs, and this is detailed on our results and registry pages. Then, we characterized the protein productivity of our TX-TL cell free expression system. Once our Encapsulin-based protein constructs were expressed in our cell free TX-TL system, we characterized this expression using standard denaturing SDS-PAGE, fluorescent SDS-PAGE, and native gel. The standard denaturing SDS-PAGE gels were the first indication for the correct expression of our protein constructs. However, we went further through the expression of our protein with lysine BODIPY to ensure correct expression. Moreover, we ran a native gel to characterize our protein in its native state.

After gel-based characterizations that indicate the approximate expressed protein weight, we figured that we should also be characterizing the structure of our protein. Thus, we performed DLS size measurement to characterize the diameter of our expressed Encapsulin.

We thus have strong evidence that we are truly expressing our intended constructs. Still, we wanted a definite proof of the correct expression of Encapsulin, and Encapsulin with the standard immunogenic peptide OT-1. We thus prepared and submitted our expressed proteins to mass spectrometry facility at EPFL that confirmed the expression of both Hexahistidine Encapsulin and Hexahistidine Encapsulin-OT-1. This is as decisive an evidence as we can get! One other characterization of our vaccine that we wished to do, was TEM measurements; however, time and resource limitations prevented us to achieving it.


Following this characterization of the correct expression of our vaccine constructs, we proceeded to validate the efficiency of our vaccine construct. We first set to demonstrate the uptake of Encapsulin by dendritic cells. We thus approached a unique technology available near EPFL campus, NanoLive(fundamental patent US. 8,937,722 & EU WO 2011/121523). This technology allows the 3D imaging of biologically relevant objects, with the option to overlay fluorescent 2D images in. We were indeed able to show that dendritic cells cultured in 3D Matrigel internalize our fluorescently labeled Encapsulin nanoparticles. Multiple 3-D images with 2-D fluorescent overlays were acquired. Moreover, a video of a 3D reconstruction of a mature dendritic cell after vaccine production was produced.


Finally we aimed to validate neoantigen presentation on dendritic cells following delivery on Encapsulin surface. For this, we expressed Encapsulin/Encapsulin-OT-1, presented to cultured murine dendritic cells, and then did immunostaining/FACS to check for OT-1 presentation on MHC-I complexes on dendritic cells. Unfortunately, our results were not positive. However, we aim to repeat this measurement, as previous literature has indeed shown that Encapsulin can deliver neoantigens for dendritic cell display on MHC-1 complexes.