Fig. 5.

Regulation of the complete refactored gene cluster. (A) Nitrogenase activity for the three controllers are shown: IPTG-inducible, aTc-inducible, and IPTG ANDN aTc logic. The gas chromatography trace is shown for each, as well as the calculated percent of WT activity (7.4% ± 2.4%, 7.2% ± 1.7%, and 6.6% ± 1.7% respectively). SD is calculated by using data from at least two experiments performed on different days. (B) Incorporation of ¹⁵N into cell biomass is shown. Nitrogen fixation from N₂ gas by the refactored gene cluster was traced by using ¹⁵N₂ and measured by using isotope ratio mass spectrometry. Data are represented as the fraction of cellular nitrogen that is ¹⁵N. The SD represents two experiments performed on different days. (C) The effect of ammonia on regulation of nitrogenase expression is shown. Acetylene reduction traces shown with (red) and without (blue) addition of 17.5 mM ammonium acetate for WT cells (Left) and cells bearing synthetic nif system (Right). The synthetic system was induced by controller 1 using 1 mM IPTG and exhibited nitrogenase activity of 1.1% ± 0.5% and 6.1% ± 0.4% with and without ammonium acetate, respectively. (D) T7* RNAP expression of controller 1 corresponding to C is shown. Strains carrying controller 1 and an RFP reporter plasmid were characterized under 1 mM IPTG induction with or without addition of ammonium acetate.