| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | His-tagged (N-terminal) Arabidopsis thaliana plastidic and cytosolic PGI, and C-terminal Strep-tagged (Wakao and Benning, 2005; Wendt et al, 2000) plastidic G6PDH1, 2, and 3 genes were commercially synthesized by GenScript (https://www.genscript.com). All of the plasmid constructs were overexpressed in E. coli strain BL21. Cells were grown at 37°C to an OD600 of 0.6 to1 and induced with 0.5 mM isopropyl β-D-1 thiogalactopyranoside at room temperature. Cells were centrifuged and resuspended in lysis buffer (5 ml lysis buffer/g of pellet; 50 mM sodium phosphate, pH 8.0, 300 mM NaCl) containing 1 mg ml−1 lysozyme, 1 μg ml−1of DNAseI, and 1x protease inhibitor cocktail (Sigma, www.sigmaaldrich.com). Cells were then lysed by sonication (Branson Sonifier 250, us.vwr.com). | Get A Quote |
Fructose 6-phosphate is an intermediate in the Calvin-Benson cycle and can be acted on by phosphoglucoisomerase to make glucose 6-phosphate (G6P) for starch synthesis. A high concentration of G6P is favorable for starch synthesis but can also stimulate G6P dehydrogenase initiating the glucose-6-phosphate shunt an alternative pathway around the Calvin-Benson cycle. A low concentration of glucose 6-phosphate will limit this futile cycle. In order to understand the biochemical regulation of plastidic glucose 6-phosphate supply and consumption, we characterized biochemical parameters of two key enzymes, phosphoglucoisomerase (PGI) and G6P dehydrogenase (G6PDH). We have found that the plastidic PGI in has a higher ... More
