Light & Oxygen Response: AppA/PpsR

Regulation System!

This is a repressor/antirepressor system, which under high oxygen tension; PpsR represses GFP expression by avoiding RNA polymerase binding the promoter sequence. When oxygen concentration decreases and in presence of light AppA has a conformational change and can bind with PpsR, this complex prevents the union of PpsR to its target sequence, thus GFP expression can begin.
(See the next video for a visual explanation).

Our biobricks

The first biobrick consists in the complete light-oxygen dependent system, AppA and PpsR, each one with Ribosome Binding Site, under a Medium strength promoter (J23104), this first biobrick also it has the PpsR dependent promoter and GFP as a reporter gene.

The second circuit is just the PpsR dependent promoter and GFP as a reporter gene.

We were inspired in:

This system is inspired in AppA/PpsR repressor/antirepressor system from Rhodobacter sphaeroides. The 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 by Genescript, and are codon optimized for R. sphaeroides.

References
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.