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

　　With the development of global economy in the latest demands of energy in world, an average Taiwanese produces around 2.58 billion tons of carbon emission a year. This number far surpasses those of China, Japan and South Korea. Our project, as a result, works to use biofuel as an alternative fossil fuel to reduce the current energy crisis. In our lab, we use Microalgae as the source of biofuel since they have the greatest capability of producing large amount of oil.

　　Our project aims to increases oil accumulation through nitrogen starvation, which was brought up in a research we came upon._{[ref 1,2]} Under nitrogen starvation, de novo synthesis of triacylglycerol from acyl-CoA increases. Acyl moieties derived from the degradation of membrane lipids then recycle into triacylglycerol, increasing carbon flux towards glycerol-3 -phosphate and acyl-CoA for fatty acid synthesis. As such, the oil accumulation under nitrogen starvation will increase.

　　There are currently two types of cultivating systems: open-pond and closed bioreactors. While open-pond costs way lower than closed bioreactors, open-pond cultivate microalgae with significantly lower oil contents. Many microalgae farms today cultivate microalgae in closed ponds, where regulations are made to keep the nitrogen level low. This method, though, consumes lots of energy due to the need to maintain proper temperature, nutrition, light, and other growing factors. We want to develop a new method that allows microalgae to reach nitrogen starvation in open-pond, and thus reaching the same effectiveness of closed bioreactors with the affordable price of open-pond.

　　NrtA protein sticks to the periplasmic membrane through a flexible linker to capture nitrite or nitrate in the periplasm. Then delivery to the transmembrane complex that made by NrtB. In our project, we try to transform NrtA gene from cyanobacteria Synechocystis PCC 6803 to E.coli. Engineered E.coli will be capable of clutching nitrite or nitrate present 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 efficiently.

　　After building up the nitrogen starvation and extracting 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 (2014 iGEM Beijing). Holin can trigger the formation of holes on cell membrane. When holin successfully triggers holes on cell membrane, endolysin can pass through the membrane to decompose peptidoglycan. E.coli is lysed after the cell membrane and cell wall are destroyed. To control the suicide timing, we designed an inducible promoter for holin, so that we can induce E.coli suicide at the exact 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