Quantification of RuBisCO Expression and Photosynthetic Oxygen Evolution in Cyanobacteria

Abstract

Phototrophic microorganisms are frequently engineered to regulate the expression and the activity of targeted enzymes of interest for specific biotechnological and agricultural applications. This protocol describes a method to evaluate the expression of RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) in the model cyanobacterium Synechococcus elongatus PCC 7942, at both the transcript and protein levels by quantitative PCR and Western blot, respectively. We further describe an experimental method to determine photosynthetic activity using an oxygen electrode that measures the rate of molecular oxygen production by cyanobacterial cultures. Our protocol can be utilized to assess the effects of RuBisCO engineering at the metabolic and physiological levels.