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From 2011.igem.org

Notebook

E. Coli

Cyano

Enzymology

Media
Week 1 - June 1-4
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E. Coli
Blah

Cyano

Hexokinase coupled assay used to quantitate amounts of cyanobacteria glucose secretion. Rxn: 1. glucose + ATP --Hx-> G-6-P + ADP 2. G-6-P + NAD+ --G-6-PDeH-> G-6-P + NADH Reaction used Glucose assay kit (Genzyme) containing 2000U/L Hexokinase reagent and 4000U/L G-6-P DeH and measured glucose concentrations ranging from 0.05mM-0.25mM and absorbances were measured at 340.0 nm. Results: Final NADH concentrations were determined using the molar extinction coefficient and were proportionate to intial glucose concentrations. Discussion: Focus is now to measure not only glucose, but fructose concentrations as well as both with be secreted in the form of sucrose using a D-glucose/D-fructose assay (Megaenzyme)

Media
Week 2 - June 5-11
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E. Coli

Cyano

Assay for ethanol detection. Simple one-step assay takes ethanol and NAD+ into acetylaldahyde and NADH with use of alcohol Deh (ADH) enzyme. Final NADH concentration can be determined using its molar extinction coefficient and absorbance at 340.0 nm, and is proportionate to initial ethanol concetration. Ethanol concentrations ranging from 0.05mM-0.25mM were assayed with 173U/mL ADH. Results: Final NADH concentrations were calculated to be off by a factor of ten due to unexpectedly low absorbancies. Assay was repeated with increased ethanol concentrations (0.5-10mM), but absorbancies remained lower than expected. Disscussion: It was determined that this assay was now sensitive enough for the range of ethanol concentrations that will need to be detected in our project, therefore we will use other means of ethanol detection using simple primary alcohol detection methods using oxidizing reagents.

Media
Week 3- June 12-18
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E. Coli

Cyano

Enzymology