1. Synthetic Chloroplasts Shannon Duffy

2. Origin of the Chloroplast The chloroplast was originally a photosynthetic prokaryote A eukaryotic cell containing a mitochondria engulfed the prokaryote, and an endosymbiotic relationship was formed The prokaryote evolved to become the chloroplast organelle

3. Palma A. Silver Leader of the team researching an endosymbiosis between Synechococcus elongatus and zebrafish embryos Also experimented on the possibility of endosymbiosis with E. coli Endosymbiosis: o Symbiosis where one organism lives within the other

4. Synechococcus elongatus and Zebrafish Synechococcus elongatus pcc 7942: o Cyanobacteria that produces energy through photosynthesis and naturally fluorescent Zebrafish embryos: o Eukaryotes in the earliest stage of development o Produce no pigment, allowing light to pass through o Easy to microinject and well studied

5. Insertion into Zebrafish Embryos Three ways to insert the bacteria into an animal cell in a lab o Inject live into the animal cell o Engineer the bacteria to be able to invade the animal cell o Engineer the bacteria to be digested by the cell

6. Injection While the zebrafish were in the one-cell stage, they were microinjected with live S. elongatus or E. coli The development of the embryos could be monitored with a fluorescence dissecting microscope

7. Results Red fluorescent bacteria were found in cell throughout the development with no appearance of affecting the growth The S. elongatus survived for twelve days until the experiment was terminated However, the E. coli killed the zebrafish within two hours even when the E coli were killed prior to the experiment with UV rays

8. Results

9. Invasion The Synechococcus elongatus were engineered with invasin from Yersinia pestis, and Listeriolysin O from Listeria monocytogenes Invasin: a protein that causes an uptake in bacterial cells Listeriolysin O: hemolysin that allows bacteria to enter the cytoplasm after the uptake

10. Results 4.8% of the mammalian cells were positive for red autofluorescence The cells were sorted based on their fluorescence, and it was determined that there was approximately one bacteria per cell

11. Phagocytosis Bacteria can also enter cells through phagocytosis, but for symbiotic growth, the bacteria also needs to escape digestion by the lysosome Plates were incubated with macrophages and either E. coli or S. elongatus

12. Results Similar to the zebrafish embryos, the E. coli that were engulfed quickly killed the macrophage, and those expressing listeriolysin could kill the macrophage even faster But, the S. elongatus could remain inside the macrophage for up to two days with little effect After two days the macrophage died containing both Synechococcus with an empty vector and those expressing invasin and listeriolysin

13. Results

14. Additional Research Kwang W. Jeon researched creating an endosymbiotic relationship between an amoeba and its infectant, a naturally occurring parasitic bacteria Over time, some of the surviving amoebae became dependent on the bacteria within their cells This is because the amoebae no longer produced a protein that was required for survival, because the bacteria were providing that protein So if the bacteria were removed, the amoebae’s nucleoli were damaged, because the amoebae could no longer produce the protein

15. Conclusion It was established by Jeon that an endosymbiotic relationship can be created in a lab in only a few years Through her experiments, Silver took the first step in making an endosymbiotic relationship, and with more time, the zebrafish could become reliant on the photosynthetic bacteria If the endosymbiotic theory is correct, then after the zebrafish becomes dependent on the Synechococcus elongatus, the bacteria could evolve into an organelle with a similar function to chloroplast, and therefor a synthetic chloroplast