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Part:BBa_K3747142:Experience

Designed by: Anemoon Drinkwaard   Group: iGEM21_Wageningen_UR   (2021-10-21)


Applications of BBa_K3747142

Testing Nir in Pseudomonas putida

The Nir containing strains were cultured with NO2- and the amount of NO2- consumed was estimated. The change in optical density (OD600) was also determined. While the OD of the control group (P. putida EM42) increased normally in 24 hours, the OD of the Nir strains decreased. However, we did see signs of growth: a clear pellet was found in the samples after centrifugation at the 24-hour point, while non was visible at the start of the experiment. Moreover, the medium was cloudier after 24 hours. Figure 1 shows the consumption of NO2- over the 24-hour timespan. A notable difference between the control and the Nir strains can be seen. Approximately 20% of the added NO2- is converted in 24 hours. Unfortunately, the NO-responsive biosensor could not be used to prove the production of NO. Nevertheless, these results are a clear indication Nir is active in P. putida.

Figure 2.NO2- consumption by P. putida EM42 containing the plasmid with the copper dependent nir operon originating from P. stutzeri. As control, P. putida EM42. Three biological replicates were used for the control and test strain.

Nap combined with Nir

Nir was also combined with the first enzyme in the denitrification pathway, periplasmic nitrate reductase (Nap). This Nap originates from Paracoccus denitrificans DSM 413 (BBa_K3747139). 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 2 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 2.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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