"

From 2010.igem.org

---

 We demonstrated the functionality of the the self-lysis and vesicle buster device by delivering GFP to the cytoplasm of the choanoflagellate. 

We are engineering bacteria to serve as a vector to deliver payload into choanoflagellates. We start with a bacteria that expresses a payload. The payload can consist of either protein, nucleic acids, or a combination of the both.

Since choanoflagellates naturally eat bacteria, our bacteria easily enters the choanoflagellate through phagocytosis. During normal digestion the choanoflagellate envelopes a bacterium 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 exists 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, it is programmed to open itself using a Self-Lysis device derived from the 2008 UC Berkeley iGEM team.

At this point, proteins being expressed by the bacteria will be released and ready to act. A Vesicle-Buster device will open the small food vesicle and release our payload into the cytoplasm.

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.