Construction of a light and oxygen sensor to control protein expression regulation

Rhodobacter sphaeroides (Rhb. sphaeroides) is a purple non-sulphur bacterium that belongs to the alpha-proteobacteria, the most metabolically diverse group of organisms on the planet. Part of this versatility is attributed to regulatory proteins which coordinate different metabolic states such as anaerobic photosynthesis and chemi-heterotrophy.

Specifically, our project is concerned with two regulatory protein systems. The first one, is the two-component global activator under anaerobiosis, PrrB/PrrA. The second system is the light and oxygen mediated repressor/anti-repressor PpsR/AppA. These act in conjunction, along with a slew of other proteins in Rhb. sphaeroides, to tightly control and balance the metabolic demands of the cell. Our team will take these two regulatory systems to express them into a Rhodopseudomonas palustris chassis. The goal is to achieve a better comprehension of the R. palustris regulatory networks through the study of their properties of genetic switches in expressed in a relative isolation within another organism, considering the interference caused by other proteins could be minimal. This will allow us to study in a more precise way how the regulatory systems sense external conditions and transduce them into alternative expression levels via signaling pathways.

For us to achieve this goal a cassette in which GFP expression is light-dependent by the antirepression of PpsR and oxygen dependent by the activation of PrrA/B system has been designed. All this lab work is accompanied by a computational model which, based on our experimental data, will provide a way of testing our knowledge of these systems. This in turn allows us to enhance the functionality of the device as it expresses heterologous genes in R. palustris.

Once we have characterized the functionality of these regulatory networks we aim to take advantage of R. palustri’s metabolic versatility, and use this functional bacteria as a microbial factory that will allow the production of products of economic value from byproducts otherwise considered pollutants such as CO2 and other industrial derivatives.