Materials and Reagents

1. 1.5 ml Eppendorf tubes (Eppendorf, catalog number: 022363204 )
   
2. Micropipette tips (200 μl) (Sigma-Aldrich, catalog number: CLS4866 )
   
3. Micropipette tips (1,000 μl) (Sigma-Aldrich, catalog number: CLS4868 )
   
4. Glass beads (unwashed) (212-300 μm) (Sigma-Aldrich, catalog number: G9143 )
   
5. 1.5 ml polystyrene spectrophotometer cuvettes with 10 mm path length (Sigma-Aldrich, catalog number: C5416 )
   
6. Parafilm (Sigma-Aldrich, catalog number: P7793 )
   
7. Bacterial cells
   Note: Amount of the cell depends on how many assays will be carried out. Supernatant yielded from a cell pellet of 50 ml well-grown cyanobacterial culture (OD₇₅₀ ≥ 1.0) and 10 ml overnight grown Escherichia coli will be sufficient for about 20 assays depending on what volume is used for each assay.
   
8. Trizma^® base (Sigma-Aldrich, catalog number: T1503 )
   
9. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
   
10. Glucose-6-phosphate disodium salt (G6P) (Sigma-Aldrich, catalog number: G7250 )
    
11. Potassium phosphate dibasic trihydrate (K₂HPO₄·3H₂O) (Sigma-Aldrich, catalog number: P5504 )
    
12. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P0662 )
    
13. Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M7506 )
    
14. Hydrochloric acid (HCl) (36.5-38.0%) (Sigma-Aldrich, catalog number: H1758 )
    
15. Maleic acid (Sigma-Aldrich, catalog number: M0375 )
    
16. β-nicotinamide adenine dinucleotide phosphate hydrate (NADP⁺) (Sigma-Aldrich, catalog number: N5755 )
    
17. Paraffin
    
18. 50 mM Tris-maleate solution (see Recipes)
    
19. Extraction buffers
    
    1. Tris-maleate buffer, pH 6.8 for Synechocystis sp. PCC6803 (see Recipes)
       
    2. Potassium phosphate buffer, pH 6.8 for E.coli (see Recipes)
       
20. Assay buffers
    a.Tris-maleate buffer, pH 7.4 for Synechocystis sp. PCC6803 (see Recipes)
    b.Tris-HCl buffer, pH 8.0 for E.coli (see Recipes)
    
21. 500 mM Tris-HCl stock solution (pH 8.0) (see Recipes)
    
22. 500 mM G6P solution (see Recipes)
    
23. 100 mM NADP⁺ solution (see Recipes)

Equipment

1. Micropipettes (20-200 μl capacity) (Nichiryo, catalog number: 00-NPX2-200 )
   
2. Micropipettes (100-1,000 μl capacity) (Nichiryo, catalog number: 00-NPX2-1000 )
   
3. Microcentrifuge, 1.5 ml Eppendorf tube rotor and at least 10,000 x g force (Sigma-Aldrich, Hettich^®, model: MIKRO120 )
   
4. FastPrep FP120 (BioSurplus, Thermo-Savant, model: FP120 )
   
5. Light microscope
   
6. Vortex mixer (Bibby Scientific, Stuart, model: SA8 )
   
7. Balance (Precision Weighing Balances, Salter Brecknell, model: ESA-150 )
   
8. pH meter (Hanna Instruments, model: HI5221 )
   
9. Vis-Spectrophotometer with 1.5 ml cuvette holder (Shimadzu Scientific Instruments, model: UV-1800 )
   

Procedure