"
Team/CINVESTAV-IPN-UNAM MX/oxigenresponse.htm
From 2012.igem.org
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 PrraA 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 and we have to we have to consider that our system is synthetic and the behavior can be different than expected.
Conclusion
Our two BioBricks (K776019 and BBa_K776021) are functional in two photosynthetic bacteria R. palustris and R. sphaeroides. This is a functional system for controlling genetic expression with Oxygen tension.
Rhodofactory 2012
