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Team:Nevada/Notebook/Temp
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Week 1 - June 1st-5th
E. Coli
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Cyano
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Enzymology
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 initial 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 6th-12th
E. Coli
Cyano
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Enzymology
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. and absorbances were measured at 340.0 nm.
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.
Discussion: 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
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Week 3 - June 13th-19th
E. Coli
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Cyano
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Enzymology
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Media
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Week 4 - June 20th-26th
E. Coli
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Cyano
Promoters and integration vector for Synechocystis from iGEM Utah State received. Promoters/integration vector in pSB1C3 used to transform E. Coli. Cultures streaked and grown overnight. Colonies selected from streaked plates, liquid cultures grown overnight. Liquid cultures miniprepped to isolate plasmids. Digestion using EcoR1 and Pst1 performed on each of isolated plasmids including INV ang GLF, gels run and imaged. LB with 30% glycerol stock prepared for freezing of E. Coli. An apparatus for growing Synechocystis was prepared using a flask and glass tubing connected to an aquarium pump to aerate the culture. Synechocystis was grown in a photobioreactor for four days, and the OD730 was measured once a day to determine growth. Synechocystis was grown in BG-11 medium with either 0% glucose or 50 mM glucose. Over four days, the OD730 did not change for either type of culture. This indicates that the Synechocystis did not grow, and that modifications to the growth apparatus are necessary. A larger volume of culture is necessary, as well as a humidifying device.
Enzymology
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
