Part:BBa_K3747138:Experience

Testing Nap in Pseudomonas putida

In this experiment the Naps originating from Pseudomonas stutzeri JM300 (BBa_K3747137), Cupriavidus necator H16 and Paracoccus denitrificans DSM 413 (BBa_K3747139) were tested in P. putida. To quantify the activity of Nap, P. putida EM42 containing the construct was cultured on M9 medium with 30 mM acetate as carbon source and 2 g/L (NH₄)₂SO₄ as nitrogen source supplemented with 5 mM NaNO₃ as substrate for Nap. After 24 hours, the supernatant was taken and the NO₂^- concentration was quantified by performing a Griess Assay.

Figure 1. NO₂^- production by P. putida EM42 containing plasmid with nap operon originating from C. necator, P. stutzeri or P. denitrificans. The NO₂^- production was corrected for the change in OD over the time span of 24 hours. As control, P. putida EM42 containing pSEVAb22 without cargo was used. Three biological replicates were used for the control and Nap P. stutzeri and five biological replicates for C. necator and P. denitrificans Nap.

Both the Nap originating from C. necator and P. denitrificans worked in P. putida, but P. stutzeri Nap showed little activity. P. denitrificans Nap showed the highest activity.

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