Team:Berkeley/Project Overview

From 2010.igem.org

Abstract
The ability to manipulate the DNA of an organism is vital to many modern fields of biology. Although we have perfected this in common research species such as E. coli, yeast and mammalian cells, it is still impossible to transform many other species. Lower metazoans in particular have several advantages over pre-existing chassis but remain out of reach because of an inability to deliver protein and DNA. We aim to develop the tools that synthetic biologists will need to work with these organisms.

For our project, we focused on delivering protein to a family of single-celled organisms called choanoflagellates. Choanoflagellates were chosen to demonstrate proof-of-concept because they are extremely easy to culture, proliferate quickly and are applicable to evolutionary research. In a lab culture, choanoflagellates eat dead bacteria, live in sea water, survive at room temperature and do not need to be aerated. In addition, these species are interesting to researchers because they are the closest living relative to our microbial ancestor that became the first multicellular animal. Developmental biologist who study these single-celled eukaryotes are hindered by the inability to genetically manipulate them.

We are engineering bacteria to serve as a vector to deliver payload into the choanoflagellate. Since Choanoflagellates naturally eat bacteria, our bacteria easily entered the choanoflagellate. 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. The payload can consist of either protein, nucleic acids, or a combination of the both. We demonstrated the functionality of the the self-lysis and vesicle buster device by delivery GFP to the cytoplasm of the choanoflagellate. Future work involves targeting protein and DNA to the nucleus in order to genetically modify the choanoflagellate. A transposon/transposase device will move to the nucleus and 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.