Team:Berkeley/Project/Overview

From 2010.igem.org

Revision as of 05:58, 26 October 2010

This year our goal was to engineer bacteria to serve as a vector to deliver proteins to the cytoplasm of choanoflagellates. We start with a bacteria that expresses a Payload, either proteins, nucleic acids, or a combination both.
Choanoflagellates naturally eat bacteria, so during normal digestion the choanoflagellate envelopes bacteria in a phagocytotic vesicle.
This vesicle is then transported to choanoflagellate's food vacuole, where it merges with the vacuole. At this point the contents of the vesicle are exposed to the inside of the food vacuole and everything is destroyed, including the bacteria and all its contents.
In order to avoid being digested by the choanoflagellate our bacteria must be able to deliver the payload in the short time between ingestion and digestion. When the bacteria is in the phagocytotic vesicle, there are two barriers between our payload and the cytoplasm of the choanoflagellate, the bacteria's own membranes, and the vesicle membrane itself. Once our bacteria is engulfed by the choanoflagellate, and an inducer is added, it lyses itself using the Self-Lysis device derived from the 2008 UC Berkeley iGEM team.
At this point, the proteins being expressed by the bacteria will be released into the vesicle. Included in these proteins is the Vesicle-Buster device, which punctures the food vesicle membrane and releases the payload into the cytoplasm of the choanoflagellate.

    Once both barriers have been broken, we will have successful delivery of the payload to the cytoplasm of the choanoflagellate. Future work involves targeting the payload to the nucleus in order to genetically modify the choanoflagellate. A transposon/transposase device will splice DNA in or out of the genome. Although we tested our constructs on choanoflagellates, the devices are general enough to be applied to any phagocytic organism.