Part:BBa_K3747139:Experience

Testing Nap in Pseudomonas putida

In this experiment the Naps originating from Pseudomonas stutzeri JM300 (BBa_K3747137), Cupriavidus necator H16 (BBa_K3747138) and Paracoccus denitrificans DSM 413 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 (Figure 1). P. denitrificans Nap showed the highest activity.

Integrating napEDABCGH from P. denitrificans in the genome of P. putida EM42

To be able to combine Nap with plasmids encoding the other denitrification enzymes, we integrated the nap operon from P. denitrificans in the genome of P. putida EM42 at the PP5322 site [57]. We compared the activity of this Nap with Nap expressed from the plasmid and we showed that the genomic Nap produced more than ten times more NO₂^- (Figure 2).

Figure 2.NO₂^- production by P. putida EM42 ∆nasT containing nap operon originating from P. denitrificans on a plasmid (Nap plasmid) and integrated in the genome (Nap genomic). 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 five biological replicates for Nap.

Nap combined with Nir

Nap was also combined with the second enzyme in the denitrification pathway, nitrite reductase (Nir). This Nir originates from Pseudomonas stutzeri JM300 (BBa_K3747142). The Nap and Nir plasmids were cloned into P. putida EM42 to create P. putida EM42 with Nap + Nir. This strain was grown with NO₃^- (5 mM) for 24 hours. Figure 3 shows the results of the growth experiment. Clearly, more NO₂^- is produced with Nap alone in comparison to Nap combined with Nir. This indicates that the produced NO₂^- from Nap is consecutively consumed by Nir.

Figure 3.NO₂^- production by P. putida EM42 containing the nap operon originating from P. denitrificans on plasmid (Nap) or containing the nir operon origination from P. stutzeri on plasmid as well (Nap + Nir). 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 five biological replicates for Nap and Nap + Nir.

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