Kędzior Mateusz, Kacar Betul
Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ, USA.
Department of Bacteriology, University of Wisconsin-Madison, USA.
Bio Protoc. 2021 Oct 20;11(20):e4199. doi: 10.21769/BioProtoc.4199.
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 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.
光合微生物经常被改造，以调节目标感兴趣酶的表达和活性，用于特定的生物技术和农业应用。本方案描述了一种通过定量PCR和蛋白质印迹分别在转录水平和蛋白质水平评估模型蓝细菌集胞藻PCC 7942中1,5-二磷酸核酮糖羧化酶/加氧酶（RuBisCO）表达的方法。我们还描述了一种使用氧电极测定光合活性的实验方法，该电极可测量蓝细菌培养物产生分子氧的速率。我们的方案可用于评估RuBisCO工程在代谢和生理水平上的影响。
