Synthetic ChloroplastsShannon Duffy

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

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

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 developmento Produce no pigment, allowing light to pass througho Easy to microinject and well studied

Insertion into Zebrafish Embryos• Three ways to insert the bacteria into an animal cell in a

labo Inject live into the animal cello Engineer the bacteria to be able to invade the animal cello Engineer the bacteria to be digested by the cell

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

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

Results

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

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

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

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

Results

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

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