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From 2011.igem.org

Notebook

E. Coli

Cyano

Enzymology

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Week 4 - June 19-25
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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.

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)

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