Team:Nevada/Notebook/June/Week2

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Revision as of 09:01, 30 June 2011

                     Week 2 - June 5-11
                     -----------------------
                             E. Coli

                             Cyano
                                Blah
                             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 1 Week 3

Week 2 - June 5-11
-----------------------------

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

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+Week 2 - June 5-11
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