微生物学

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.

Chlorophyll fluorescence measurements have been widely used to monitor the condition of photosynthesis. Furthermore, chlorophyll fluorescence from cyanobacteria reflects the condition of respiration, since cyanobacterial photosynthesis shares several components of electron transport chain with respiration. This protocol presents the method to monitor the condition of both photosynthesis and respiration in cyanobacteria simply by measuring chlorophyll fluorescence in the dark and in the light with pulse amplitude modulation (PAM) chlorophyll fluorometer.

Cytochrome (Cyt) b₅₅₉, an important and essential core component of photosystem II in the photosynthetic electron transport system, is a heme-bridged heterodimer protein composed of an alpha subunit (PsbE) and a beta subunit (PsbE), and its reduced form has an absorption maximum in the α-band at 559 nm. The amounts of Cyt b₅₅₉ can be determined by spectrophotometrical measurement of reduced minus oxidized difference spectra that are normalized with absorbance of isosbestic point at 580 nm. The authors use differential extinction coefficients of Cyt b₅₅₉ [Δε_{(559-580 nm)} = 15.5 mM^-1·cm^-1], which have been reported by Garewal and Wasserman (1974). In addition to the Cyt b559, this procedure can be used for quantitation of Cyt b₆ and Cyt f, the subunits of the Cyt b₆/f complex, and P₇₀₀, one of the core components of photosystem I. This protocol, which is adapted from Fujita and Murakami (1987), is used in a cyanobacterium, Synechococcus elongatus PCC 7942, and also in other cyanobacterial strains including Synechocystis sp. PCC 6803.