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From 2014.igem.org

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<img src=""> Fig 1: EcoRI,PstI Double digest check of biobricks,K1314000,K1314013,K1314014 and K1314015

The expression level of the gene under this promoter was found to be highest when nitrogen is depleted in A. vinelandii.(Trinity L. et al. , 2011) With a sufficiently low nitrogen level, the expression level from nifH promoter protein expression system is expected to be higher than that in tranditional E. coli T7 protein expression system. (2)

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Bba_K1314001 is comprised of nifH promoter and Bba_E1010. It is designed for testing nifH promoter. The right tube shows the expression of RFP using ptep promoter in Dh5a and the left tube show the expression of BbaK1314001 in DH5a. The result show that nifH promoter is not working in DH5a. <img src="" width="300px">

Characterization of Bba_K1314001, Bba_K1314013, Bba_K1314014 in Azotobacter vinelandii

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Ammonium repleted condition(left) versus nitrogen depleted condition(right) for constitutive RFP expression and nifH regulated RFP expression in A. vinelandii Under high Ammonium level, there is strong expression of RFP under constitutive promoter. Under nitrogen depleted condition, there is no growth of A.vinelandii. This proves that the stable genome integration is successful. The mutated A.vinelandii has already lost nitrogenase gene, and thus no longer be diazotrophic.