Difference between revisions of "Team:Nanjing-China/Design"

Liying Wang et. demonstrated a minimal nitrogen fixation gene cluster from Paenibacillus sp.WLY 78 which was compried of Pnif promoter and nine structural genes. Inspired by this study, we transferred this gene cluster to E.coli to create engineered E.coli cells which are capable of producing active nitrogenase. We achieved this by extracting the gene cluster from Paenibacillus sp.WLY78, connecting it to plasmid Psb1C3 and transformed it to E coli cells.In order to ensure the expression of this gene cluster in E coli,first we verified the transcriptional activity of Pnif promoter in E coli cells by conducting control experiments.In the test group,we replaced the native T5 promoter on pQE80L vector with Pnif,connected it to Dronpa fluorescent protein gene and transformed the new vectors to E.coli cells.In the control group, pQE80L vectors with T5 promoter and Dronpa gene were transformed to E coli cells. The comparable level of fluorescence intensity of the two groups indicated that Pnif promoter is transcriptional active in E coli cells.

Simultaneously, in order to achieve in site synthesis of CdS nanocrystals on cell surfaces,we transformed the vector with opmA-PbrR gene to E.coli cells.This gene encodes opmA-PbrR protein complex,which can be fixed on cell surface by outer membrane protein (OMP) opmA.The function of PbrR protein is to adsorb Cd2+ in the environment and further form CdS nanocrystal on cell surface,a key component of this light-harvesting system. When this system is exposed by light, electrons from electron donor conduct transition while CdS nanocrystals on cell surfaces transfer high-energy electrons to Mo-Fe protein subunit of nitrogenase.Mo-Fe protein then utilizes the energy from these electrons rather than ATP to reduce dinitrogen into ammonia. With the method mentioned above, we successfully constructed a whole-cell light-driven nitrogen fixation system.

In later period of our study, in order to elevate the electron transfer efficiency, we introduced Ag2S to realize Ag2S CdS cocatalysis. We respectively introduced ompA-spy tag Ag oligopeptide complex and ompA-spychcatcher-PbrR complex to E.coli cells. In that way, two types of E coli cells--with Ag2S or CdS nanocrystals adsorbed on surfaces specifically bind with each other through covalent bonds between spytag and spycatcher. Eventually, nitrogen fixation efficiency of our system showed a remarkable increase