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Growth experiments

P. putida EM42 strains with the pSEVA231-CRISPR plasmid were grown at 30°C overnight in 10 mL LB supplemented with 50 μ/ml kanamycin. 15 different targeting spacers were used to downregulate the different terminal oxidases and a non-targeting spacer was used as control.

The next day, the cultures were diluted to an OD₆₀₀ of 0.2 in M9 supplemented with kanamycin and 30 mM acetate as carbon source to a total volume of 200 μl in 96-well plates. The plate was directly incubated at 30 °C in the Synergy MX plate reader for 48 hours, with OD600 measurements every 5 minutes. For these experiments both biological and technical triplicates were included.

Effect of the spacer on growht of P. putida EM42

For the project designed 15 different spacers, three for each terminal oxidase complex. The spacers were designed to target the non-template strand, right at the 3’ end of a Protospacer Adjacent Motive (PAM) sequence. For every terminal oxidase a spacer was designed for: (1) the promoter sequence, (2) sequence between the promoter and start codon and (3) start codon (Figure 1).

Figure 1. Spacer numbers corresponding to target

In Figure 2 it can be seen that targeting the promoter of Cyo oxidase (Sp₄)can impair growth for P. putida EM42. Furthermore, we computed the time at which the exponential phase started. We found that for Sp₄, the exponential phase was delayed most. Overall, the values for exponential delay were quite variable for the different spacers and therefore difficult to be conclusive alone. From these analyses, we can conclude that targeting the promoter for the Cyo oxidase is most impactful in impairing growth.

Figure 2. 16-hour growth curves of P. putida EM42 with the terminal oxidases downregulated. Each line represents the downregulation effect of spacers targeting different terminal oxidases. Values represent the mean and standard deviation of (1) doubling time, (2) start of exponential phase, (3) biomass yield for three biological and two technical replicates. Note that for Sp.4, 11, 15, only two biological replicates were tested.

This spacer most likely affects growth as it targets the Cyo terminal oxidase which has a significant effect on the cell transcriptome [1]. Moreover, this oxidase is the only oxidase active during the exponential phase, which explains why we see the growth defect only during this growth stage (Figure 2). The other oxidases, i.e. the CIO oxidase, the Aa₃ oxidase, the Cbb₃-1 oxidase and Cbb₃-2 oxidase are active during the stationary phase (Figure 2). When other oxidases take over, which has been shown upon knocking out the Cyo oxidase, the effect of downregulation could have been lost.

References

[1] Ugidos, F. Rojo, G. Morales, A. Ugidos, and F. Rojo, “Inactivation of Pseudomonas putida cyo terminal oxidaseG Inactivation of the Pseudomonas putida cytochrome o ubiquinol oxidase leads to a significant change in the transcriptome and to increased expression of the CIO and cbb3-1 terminal oxidases,” Environ. Microbiol., vol. 8, no. 10, pp. 1764–1774, 2006, doi: 10.1111/j.1462-2920.2006.01061.x.