Light Response.htm

Light + Redox Response: AppA/PpsR Control System

This is a repressor/antirepressor system, under high oxygen tension PpsR strongly repress GFP expression by binding a conserved regulatory sequence that avoid RNA polymerase for promoter binding. When oxygen concentration decreases AppA is activated, and bind PpsR avoiding this DNA binding activity, thus, GFP expression can begin.

How does it works?

Our genetic system consists in 2 modules, the first one is formed by appa and ppsr coupled to B0030 RBS units, J23104 Strong constitutive promoter and B0014 Double terminator to form the transcription unit. We will evaluate the functioning of J23104, B0030 and B0014 in a new chassis. The second module consists in J54103 GFP generator (B0030 RBS + E1010 GFP + B0014 Double terminator) coupled to PpsR repressible promoter.

Inspired in…

This system is inspired in AppA/PpsR repressor/antirepressor system from Rhodobacter sphaeroides, PpsR protein is a master repressor of Photosynthesis (PS) genes (Moskvin and Gomelsky 2005). Inactivation of the ppsR gene is enough to turn on PS gene expression and formation of the photosynthetic apparatus even at a high oxygen concentration, whereas ppsR overexpression is sufficient to block PS development even in the absence of oxygen. PpsR directly represses transcription of most carotenoid and pigment synthesis genes, photosystems operons, and genes involved in tetrapyrrole biosynthesis (Gomelsky and Kaplan 1995). The upstream regions of these genes contain two PpsR binding sites, TGTcN10gACA.

A second protein called AppA, which has no known homologues, plays a role in controlling gene expression in R. sphaeroides in response to both light and O2 by acting as an antirepressor of PpsR. Our parts (appa, ppsr and ppsr-promoter) were synthesized with Genescript, and are codon optimized for R. palustris.

1. Gomelsky L., Moskvin L.,  Stenzel A., Jones D., Donohue T. and Gomelsky M.(2008) Hierarchical Regulation of Photosynthesis Gene Expression by the Oxygen-Responsive PrrBA and AppA-PpsR Systems of Rhodobacter sphaeroides. J. Bacteriol. Dec. 2008, p. 8106–8114 Vol. 190, No. 24
2. Moskvin, O. V., L. Gomelsky, and M. Gomelsky. (2005). Transcriptome analysis of the Rhodobacter sphaeroides PpsR regulon: PpsR as a master regulator of photosystem development. J. Bacteriol. 187:2148–2156.
3. Gomelsky, M., and S. Kaplan. (1995). Genetic evidence that PpsR from Rhodobacter sphaeroides 2.4.1 functions as a repressor of puc and bchF expression. J. Bacteriol. 177:1634–1637.
4. Gomelsky, M., and S. Kaplan. (1995). appA, a novel gene encoding a transacting factor involved in the regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. J. Bacteriol. 177:4609–4618.