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Exosome Mediated Mammalian Cell-Cell Communication

Overview

miRNA Signal

Protein Signals

Novel DNA Sensor: Cas9 Split Venus Fusion

Our BioBricks

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!

(5) Exosomal Cell-Cell Communication with Proteins

Proteins have been shown to be targeted into exosomes with the addition of a high-order oligomerizing protein Acyl-TyA. By fusing a protein signal to an Acyl-TyA domain, we could send the protein from cell to cell through exosomes.

(6) Protein Targeting

We began by fusing Acyl-TyA to GFP and observing colocalization of the Acyl-TyA-GFP with the membrane stain Rh-PE, which has been shown to be targeted to the site of exosomal biogenesis. Then, we demonstrated through a western blot that our protein signal exists within the exosome rich media.

(7) Testing our Protein Signal

We demonstrated retained functionality of our protein signal after fusion with Acyl-TyA (Acyl-TyA-rtTA3). In addition, we tested our reporter construct (TRE-tight_mkate), which allowed us to assay for the function of our protein signal.

(8) Testing with Isolated Exosomes

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(9) Protein Signal Coculture!

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(10) Application - Endogenous Gene Activation

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(11) Testing Cas9-VP16

We tested our Cas9-VP16 fusion protein by transfecting it into HEK 293 cells along with a guide RNA which will target the Cas9-VP16 to our reporter construct and activate the expression of EYFP. We showed that our Cas9-VP16 fusion is indeed functional.

(12) Crispersomes

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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.