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Team:Nevada/Notebook/June/Week4
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<p><font color="red">E. Coli | <p><font color="red">E. Coli | ||
<br>Blah</font> | <br>Blah</font> | ||
| - | <p><font color="blue">Cyano</font> | + | <p><font color="blue">Cyano</font> 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. |
<p><font color="green">Hexokinase coupled assay used to quantitate amounts of cyanobacteria glucose secretion. | <p><font color="green">Hexokinase coupled assay used to quantitate amounts of cyanobacteria glucose secretion. | ||
Rxn: 1. glucose + ATP --Hx-> G-6-P + ADP | Rxn: 1. glucose + ATP --Hx-> G-6-P + ADP | ||
Revision as of 19:13, 27 June 2011
E. Coli
Cyano
Enzymology
Media
Week 4 - June 19-25
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E. Coli
Blah
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.
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
