Team:MIT/Project

From 2013.igem.org

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The MIT iGEM team sought to create a new mode of engineered intercellular communication for use in synthetic biology by modifying the contents of existing exosomes through the use of naturally occurring miRNA and the protein domain Acyl-TyA. We built on existing research targeting proteins to exosomes to enable intercellular communication by targeting signal proteins into exosomes and into HEK 293 receiver cells. <br><br>
The MIT iGEM team sought to create a new mode of engineered intercellular communication for use in synthetic biology by modifying the contents of existing exosomes through the use of naturally occurring miRNA and the protein domain Acyl-TyA. We built on existing research targeting proteins to exosomes to enable intercellular communication by targeting signal proteins into exosomes and into HEK 293 receiver cells. <br><br>
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<h3>Exosomal Cell-Cell Communication with miRNA</h3>
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<h3>(1) Exosomal Cell-Cell Communication with miRNA</h3>
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Jurkat T cells are known to produce a large number of exosomes which naturally contain high levels of miRNA 451. Using this natural system, our initial goal is to create a miRNA 451/Exosome sensor to begin our work with Exosomal communication.
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Jurkat T cells are known to produce a large number of exosomes which naturally contain high levels of miRNA 451. Using this natural system, our initial goal is to create a miRNA 451/Exosome sensor to begin our work with Exosomal communication.<br><br>
 +
 
 +
<h3>(2) Creating/Testing miRNA Sensor</h3>
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We constructed an EYFP fluorescent gene with four miRNA 451 target sites which will allow the EYFP reporter to be repressed by the miRNA 451. By expressing our reporter along with synthetic siRNA 451, we saw repression of our reporter.<br><br>
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 +
<h3>(3) Exosomes + Sensor</h3>
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After seeing our sensor work with siRNA 451, we then isolated exosomes from Jurkat T cells and used them to treat HEK 293 expressing our reporter. We observed similar repression of our reporter.<br><br>
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<h3>(4) Jurkat T Cells + Sensor</h3>
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With our reporter sensing isolated exosomes, we proceeded to coculture both Jurkat T cells producing exosomes with HEK 293 cells transfected with our reporter. We observed repression of our reporter indicating that we have achieved CELL-CELL COMMUNICATION!<br><br>
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<li>Demonstrated Acyl-TyA targeting proteins to the cell membrane and into exosomes
<li>Demonstrated Acyl-TyA targeting proteins to the cell membrane and into exosomes

Revision as of 00:18, 26 October 2013

iGEM 2012

Overview

  • Project Overview

miRNA Signal

  • Overview
  • siRNA Characterization
  • Exosome Isolation and Co-Culturing
  • Cell-Cell Co-Culturing

Protein Signals

  • Overview
  • GFP
  • rtTA3
  • Cre
  • L7Ae
  • Cas9-VP16

Novel DNA Sensor: Cas9 Split Venus Fusion

  • Overview
  • Leucine Zipper Fusion
  • DNA Sensing

Our BioBricks

  • Favorites
  • All BioBricks

Attributions

  • Attributions

The MIT iGEM team sought to create a new mode of engineered intercellular communication for use in synthetic biology by modifying the contents of existing exosomes through the use of naturally occurring miRNA and the protein domain Acyl-TyA. We built on existing research targeting proteins to exosomes to enable intercellular communication by targeting signal proteins into exosomes and into HEK 293 receiver cells.

(1) Exosomal Cell-Cell Communication with miRNA

Jurkat T cells are known to produce a large number of exosomes which naturally contain high levels of miRNA 451. Using this natural system, our initial goal is to create a miRNA 451/Exosome sensor to begin our work with Exosomal communication.

(2) Creating/Testing miRNA Sensor

We constructed an EYFP fluorescent gene with four miRNA 451 target sites which will allow the EYFP reporter to be repressed by the miRNA 451. By expressing our reporter along with synthetic siRNA 451, we saw repression of our reporter.

(3) Exosomes + Sensor

After seeing our sensor work with siRNA 451, we then isolated exosomes from Jurkat T cells and used them to treat HEK 293 expressing our reporter. We observed similar repression of our reporter.

(4) Jurkat T Cells + Sensor

With our reporter sensing isolated exosomes, we proceeded to coculture both Jurkat T cells producing exosomes with HEK 293 cells transfected with our reporter. We observed repression of our reporter indicating that we have achieved CELL-CELL COMMUNICATION!

  • Demonstrated Acyl-TyA targeting proteins to the cell membrane and into exosomes
  • Designed a number of reporter constructs to assay for our signals:
    • rtTA3
    • L7Ae
    • Cas9-VP16
    • Cas9-Split Venus
    • Cre Recombinase
  • Designed Acyl-TyA fusion proteins with our signals:
    • rtTA3
    • L7Ae
    • Cas9-VP16
    • Cas9-Split Venus
    • Cre Recombinase
  • Demonstrated native exosomal microRNA repression with isolated exosomes and Jukat/HEK293 coculture experiments.
  • Demonstrated activation of a reporter using the trans activator Cas9-VP16.
  • Demonstrated DNA sensing using Cas9-Split Venus reconstitution.

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