Oxygen Control System!

PrrA/PrrB two component system

This system remains inactive under high oxygen tension, when oxygen concentration decreases, it is possible the GFP transcription. (See Rhodofactory section for a complete explanation).

We made two BioBricks (BBa_K776019 y BBa_K776021) to test the Oxygen Control System, each one has GFP as a reporter gene and the functionality was related to the fluorescence detection.

Figure 1. This BioBrick will show if our dependent promoter is functional, using the constitutive (or natural) system from R. sphaeroides or the orthologue system from R. palustris.

Figure 2. This BioBrick will show if our complete system is functional because probably we need a synthetic system to promote GFP expression by binding its target sequence (dependent promoter) in R. palustris.

Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur Photosynthetic Bacteria, the plasmids were introduced in R. sphaeroides and R. palustris, by biparental and triparental conjugation.

The measurement approach we used was:

We used 3 environmental growing conditions:

For all data results, we considered a negative control: Rhodobacter sphaeroides or Rhodopseudomonas palustris, conjugated bacteria with pRK415 vector without BioBrick.

Figure 3. Percentage of bacterial population expressing GFP.

Figure 4. Representative images obtained by fluorescence microscopy, where our systems were functional in the expected conditions.

Discussion

In R. sphaeroides, as we can see in figure 3 and 4, there was low GFP expression, probably because growing conditions were microaerophilic instead of extrictly anaerobic. In anaerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this activated PrrA binds its promoter sequence to start GFP expression. Furthermore, when we introduced the complete system (BBa_K776021), actually we are overexpressing the regulatory proteins and the signaling could not be fully controlled.

In R. palustris, we had GFP expression in PrrA dependent promoter (BBa_K776019), maybe because orthologous proteins activated it. The complete system (BBa_K776021) also was functional but in a lower level, assumably due to the interference of other proteins that regulate photosynthetic genes.

The GFP expression that we did not expected was in aerobic condition in the complete system, probably is due to the complexity of the regulatory network where this system is involved.

Conclusion

Our two BioBricks (K776019 and BBa_K776021) are functional in two photosynthetic bacteria R. palustris and R. sphaeroides, both in anaerobic/light expected condition. This is a functional system for controlling genetic expression with Oxygen tension.