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!

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.