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

Based on the existing part complete line of nif cluster, BBa_K1796015, which contains essential components for nitrogen fixation: nif Promoter, nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV from the Paenibacillus sp. WLY78. We choose a new nitrogen fixation gene cluster from more common strain Paenibacillus polymyxa CR1, to comprise the nitrogen fixation system in our project.

In our this year’s project, we intends to establish a sound and ideal whole-cell photocatalytic nitrogen fixation system. We use the engineered E. coli cells to express nitrogenase(Fig 1) and in-situ synthesize of CdS semiconductors in the biohybrid system. Instead of ATP-hydrolysis, such system is able to photocatalytic N2(nitrogen) to NH3(ammonia). The biohybrid system based on engineered E. coli cells with biosynthesis inorganic materials will likely become an alternative approach for the convenient utilization of solar energy.

[[File:T--Nanjing-China--011part-design.png|800px|thumb|center|Fig 1. Design of our project: Engineered E. coli cells with nitrogenase]]

So, certainly we need not only a powerful solar energy transition system but also a strong nitrogen fixation system to improve the efficiency of our whole-cell photocatalytic nitrogen fixation system. According to the above requirements, we choose a different nif gene cluster from Paenibacillus polymyxa CR1 to test its expression level compared with the BBa_K1796015 from Paenibacillus sp. WLY78.

 

In order to test the expression efficiency of the nif cluster,firstly we measured the transcriptional activity of nif promoter by combining it with the gene of fluorescent protein Dronpa,with T5 (IPTG Inducible) Promoter BBa_M50075 as a positive control(Fig 2).

[[File:T--Nanjing-China--11part.png|800px|thumb|center|Fig 2：Expression efficiency of Pnif]]

Comparison of the expression efficiency of Pnif and T5 (IPTG Inducible) Promoter.
T5 (IPTG Inducible) Promoter BBa_M50075; Pnif: nif promoter BBa_K1796001.

As demonstrated above, nif promoter is quite strong,however, how capable it is in our nitrogen fixation system remains an unclear question. So we also detected the expression level of the essential components in our system by conducting Real-time Quantitative PCR(QPCR),using 16S DNA as an internal reference.The results are shown in Fig3.
After we compare the result with the ideal expression ratio in Paenibacillus CR1 and model the transcription, we plan to optimize the nif gene cluster by adding promoters or altering the position of genes.

[[File:T--Nanjing-China--QPCR1.jpg|800px|thumb|center]]
[[File:T--Nanjing-China--QPCR2.jpg|800px|thumb|center|Fig 3. The qPCR results for components of nitrogen fixation system]]

Nitrogenase can not only reduce dinitrogen to ammonia but also reduce ethylene to acetylene. Therefore, we use gas chromatography to detect the amount of acetylene reduced, and indirectly detect its nitrogen fixation activity.