Team/CINVESTAV-IPN-UNAM MX/oxigenresponse.htm

In Rhodopseudomonas palustris, we can see in Figure 1A a perfect behavior of AppA/PpsR, the PpsR promoter do not show GFP expression, it indicates that other regulation systems do not bind this promoter, but when we introduce the complete system, we see a GFP expression of 31.89%, because the non natural system we designed is functional. In the case of PrrA/PrrB, the PrrA promoter do not show GFP expression, probably because the RegA/B system (the homologs system in this bacteria) do not have affinity for this sequence, but the complete construction is functional, we have to consider that it is an artificial system and the behavior can be different than expected, the functionality at these conditions is interesting because PrrB is supposed to be inactivated at aerobic conditions, or if the PrrA protein need to be phosphorylated, how it is being inactivated in this chassis. R. palustris has a different PrrA/PrrB system, and its mechanism is quite different, in fact, the main regulator to start photosynthetic metabolism is not PrrA/PrrB system, actually it is FixK enzyme (Rey, 2010). The Anaerobic/Light conditions in R. Palustris (figure 2A) made possible the functionality of our Synthetic AppA/PpsR system, the PpsR promoter show a low (0.169%) GFP expression, but the complete construction also show a little GFP expression.

In figure 3, the AppA/PpsR system is not functional, because in this condition, reduced PpsR has not affinity by its target sequence and the transcription is possible, AppA is is forming the complex with PpsR, but we can not see GFP expression. PrrA/PrrB show a low response, probably due to the independence of R. palustris for PrrA/PrrB mechanism, it functions with FixJ-FixK to regulate the change of metabolism aerobic to anaerobic (Metz, 2012).

In R. palustris, the Median Fluorescence intensity (MIF) between complete systems and promoter is quite different. Figure 8 shows that PrrA promoter works better in Anaerobic/light conditions, than in others, it is the expected result considering the participation of homolog proteins, but the complete system is different because we saw a big change of fluorescence in aerobic/light conditions, introducing a synthetic system could affect the functionality of our constructions. AppA/PpsR system is functional because our promoter is being activated by other proteins, but the signal increase with our Synthetic Construction in the complete system, and also, as it is expected, the complete system works in aerobic/light conditions.