Synthetic Chloroplasts

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 Synechococcuselongatusand zebrafish embryos • Also experimented on the possibility of endosymbiosis with E. coli • Endosymbiosis: • Symbiosis where one organism lives within the other

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

5. Insertion into Zebrafish Embryos • Three ways to insert the bacteria into an animal cell in a lab • Inject live into the animal cell • Engineer the bacteria to be able to invade the animal cell • 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 Synechococcuselongatuswere 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. elongatuscould 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 Synechococcuselongatus, the bacteria could evolve into an organelle with a similar function to chloroplast, and therefor a synthetic chloroplast