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

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       <p>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).
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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.
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      <p><strong>Systems and Synthetic Biology Lab </strong></p>
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        As we can see, in figure 1A the PpsR promoter system, in R.  sphaeroides, shows a high signal (17.66%) of GFP expression, it implies that constitutive  proteins from this bacteria were able to activate our system. The growth  conditions were aerobic/darkness, although oxidized PpsR binds its target promoter, it is known that AppA can avoid the binding affinity of PpsR in the dark probably by the interference of an AppA-(PpsR)2  complex (Kim, 2006). In the case of AppA/PpsR complete system, we have a high GFP expression due to activity of the extra AppA and PpsR enzymes that were introduced.</p>
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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).
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      <p>The  blue columns show the low GFP expression with both PrrA  promoter and PrrA/PrrB complete system; in aerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this activated PrrA  binds to its promoter region as a transcriptional repressor (Bauer, 2003).  All the results show an equivalent result  with the images that were obtained by fluorescence microscope.</p>
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      <p>Figure 2A shows that in R. sphaeroides, the AppA/PpsR  system promoted the GFP expression, it is possible because reduced PpsR is unable to bind its promoter and AppA is a flavin with a photoreceptor, thus under light, AppA is unable to forma a complex with PpsR and we can see GFP expression in the  bacterial population. </p>
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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.
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Revision as of 03:08, 27 September 2012

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As we can see, in figure 1A the PpsR promoter system, in R. sphaeroides, shows a high signal (17.66%) of GFP expression, it implies that constitutive proteins from this bacteria were able to activate our system. The growth conditions were aerobic/darkness, although oxidized PpsR binds its target promoter, it is known that AppA can avoid the binding affinity of PpsR in the dark probably by the interference of an AppA-(PpsR)2 complex (Kim, 2006). In the case of AppA/PpsR complete system, we have a high GFP expression due to activity of the extra AppA and PpsR enzymes that were introduced.

The blue columns show the low GFP expression with both PrrA promoter and PrrA/PrrB complete system; in aerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this activated PrrA binds to its promoter region as a transcriptional repressor (Bauer, 2003).  All the results show an equivalent result with the images that were obtained by fluorescence microscope.

Figure 2A shows that in R. sphaeroides, the AppA/PpsR system promoted the GFP expression, it is possible because reduced PpsR is unable to bind its promoter and AppA is a flavin with a photoreceptor, thus under light, AppA is unable to forma a complex with PpsR and we can see GFP expression in the bacterial population.

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