Team:Berkeley/Project Overview


Choa's Choa's Delivery Service!

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 to these cells. From the perspective of synthetic biology, lower metazoans hold the promise of harnessing new biological pathways and developing complex biological machinery. Compared to bacteria and yeast, they are both evolutionarily closer to animals and much easier to culture. Today, however, they are generally avoided due to a lack of genetic engineering techniques. E. coli and S. cerevisia continue to dominate the field not because of their genetic or biological value, but because they were readily accessible to our predecessors. Our project was to expand synthetic biology to new lower metazoan chassis by designing a general device that can deliver protein and DNA to any phagocytic eukaryotes.

We are engineering bacteria to serve as a vector to deliver payload into choanoflagellates. Since choanoflagellates naturally eat bacteria, our engineered E. Coli 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.

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 our lab, they were grown in petri dishes with artificial sea water and stored in a desk drawer. They are also incredibly resilient to heat, UV damage and other natural stresses.

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. It is believed that choanoflagellates can help reveal some of the key events that were necessary for multi-cellularity to arise. The ability to knock in and knock out genes allows researchers to study the function of each gene individually and access its contribution to the development of multicellularity. Developmental biologists who study these single-celled eukaryotes, however, are hindered by the inability to genetically manipulate them. Attempts at transfection, electroporation, and retroviral infection have all failed in the past. A mechanism to genetically manipulate choanoflagellates would open an entirely new area of research for developmental biologists and allow choanoflagellates to become a new model organism. From a synthetic biology perspective though, choanoflagellates' close relationship to animals makes them superior tools compared to bacteria or yeast.

Choanoflagellates, as seen in blue above, consist of a head and tail region. They can either be found swimming freely, grouped as colonies, or, as seen in the picture above, attached to a substrate. The 'choano' in their name refers to the actin filaments that forms around their long flagellum. Choanoflagellates beat their flagellum to push water and the bacteria they feed on, into their collar. Their voracious appetite for bacteria makes them an easy target for phagocytosis-based infection. The image below shows a choanoflagellate in the process of engulfing its prey.