Difference between revisions of "Team:NYMU-Taipei/Nitrogen starvation"

　　With the development of global economy in the latest demands of energy in world, Taiwanese person produce 2.58 billion tons of carbon emission a year. The carbon emission of each person in Taiwan is far ahead China, Japan and South Korea. So in our project, we want to use biofuel as an alternative to fossil fuel in our project. We choose Microalgae as biofuel because it will produce more oil during the period of nitrogen starvation.

　　One research indicates nitrogen starvation promotion fuel accumulation in microalgae. Under nitrogen starvation, de novo synthesis of triacylglycerol from acyl-CoA increases. Then acyl moieties from the degradation of membrane lipids recycle into triacylglycerol and finally increase carbon flux towards glycerol-3 -phosphate and acyl-CoA for fatty acid synthesis. Therefore, the oil accumulation under nitrogen starvation will increase.

　　How to reach nitrogen starvation? In the past, people cultivate microalgae in closed pond. Give microalgae no nitrogen mediate and extract nitrogen. But this method is consumes lots of energy like closed pond cultivation need electricity to maintain the temperature, nutrition, light etc. So, we want to develop a new method to make microalgae reach nitrogen starvation and evaluate oil production in open pond.

　　NrtA protein sticks to the periplasmic membrane by a flexible linker and it can capture nitrite or nitrate in the periplasm. Then delivery to the transmembrane complexed that made by NrtB. In our project, we try to transform NrtA gene from cyanobacteria Synechosistis PCC 6803 to E.coli. Engineering E.coli will be capable of clutching nitrite or nitrate in the environment. They will not intake nitrate or nitrite since the gas accumulation may be lethal to cells. But the amount of cells that contain nitrite will decrease. Therefore, the microalgae will undergo nitrogen Starvation and produce oil more efficient.

　　After building up the nitrogen starvation and extract the oil from microalgae, we need to kill E.coli to prevent contamination. So we plan to use endolysin and holin for cell lysis, which is similar to the mechanism used by team Pecking (2014iGEM Beijing). Holin can trigger the formation of holes on cell membrane. When the holin successfully triggers holes on cell membrane, endolysin can pass the membrane through holes and decompose peptidoglycan. E.coli is lysed after the cell membrane and cell wall are destroyed. To control the suicide timing, we design an inducible promoter for holin, so that we can induce E.coli suicide at the exactly time we want.

PCC 6803 gDNA extraction

NrtA expression

endolysin construct

holin construct

endolysin-holin construct

endolysin-holin-NrtA construct

*See our parts: click

*See our experiments protocols: click

PCC 6803 gDNA extraction