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Team:Nevada/Notebook/June/Week2
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(Created page with "<html> <title> </title> <body> <a href="https://2011.igem.org/Team:Nevada/Notebook">Notebook</a> <p><font color="red">E. Coli</font> <p><font color="blue">Cyano</font> <p><font co...") |
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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> Media preparation: from 50x BG11 stock, 1.0 L stock solution prepared, 20 mL + 930 mL nano pure H20 + 50 mL purified 1.0 M glucose. |
<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 | ||
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</font> | </font> | ||
<p><font color="red">E. Coli</font> | <p><font color="red">E. Coli</font> | ||
| - | <p><font color="blue">Cyano</font> | + | <p><font color="blue">Cyano</font> Ligation buffers prepared from 1000x stock, cloning workshop Wednesday of this week. Nevada Genomis Ceneter submission protocol reviewed. AGP knock out designed. Primers ordered to isolate genomic AGP DNA. ThiE knock out designed. E. Coli transformed with pUC57 plasmids containing, individually, GLF and INV genes. E. Coli plated on LB with ampicillin. First transformation was unsuccessful, procedure repeated successfully, five colonies for each gene were selected and grown in liquid media. Liquid cultures miniprepped using QIAgen kit and sequenced. Plasmids were successfully isolated. |
<p><font color="green">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. | <p><font color="green">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. | 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. | ||
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<p><font color="red">E. Coli</font> | <p><font color="red">E. Coli</font> | ||
| - | <p><font color="blue">Cyano</font> | + | <p><font color="blue">Cyano</font> INV and GLF genes isolated from pUC57 plasmids by digestion with EcoR1 and Pst1. Fragments run on agarose gel containing cyber safe and imaged. pSB1C3 plasmids digested with EcoR1 and Pst1. INV and GLF genes ligated into pSB1C3. E. Coli transformed with pSB1C3 vectors containing INV and GLF genes. Transformed E. Coli streaked and incubated overnight. Colonies from streaked plates isolated and used to prepare liquid cultures. Liquid cultures miniprepped, genes in iGEM vectors isolated, stored. |
| - | <p><font color="green">Enzymology</font> | + | <p><font color="green">Enzymology</font> Enzymatic assay of invertase activity, quantification of sucrose. Sucrose + H20 > invertase > glucose + fructose. Step 1) Sodium acetate buffer added to sucrose along with 1 unit/mL invertase. Step 2) Hexokinase reagent added, absorption of NADH measured at 340 nm. Absorption not measured effectively. |
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Revision as of 19:05, 27 June 2011
E. Coli
Cyano
Enzymology
Media
Week 1 - June 1-4
----------------------------------------------
E. Coli
Blah
Cyano Media preparation: from 50x BG11 stock, 1.0 L stock solution prepared, 20 mL + 930 mL nano pure H20 + 50 mL purified 1.0 M glucose.
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
----------------------------------------------
E. Coli
Cyano Ligation buffers prepared from 1000x stock, cloning workshop Wednesday of this week. Nevada Genomis Ceneter submission protocol reviewed. AGP knock out designed. Primers ordered to isolate genomic AGP DNA. ThiE knock out designed. E. Coli transformed with pUC57 plasmids containing, individually, GLF and INV genes. E. Coli plated on LB with ampicillin. First transformation was unsuccessful, procedure repeated successfully, five colonies for each gene were selected and grown in liquid media. Liquid cultures miniprepped using QIAgen kit and sequenced. Plasmids were successfully isolated.
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
----------------------------------------------
E. Coli
Cyano INV and GLF genes isolated from pUC57 plasmids by digestion with EcoR1 and Pst1. Fragments run on agarose gel containing cyber safe and imaged. pSB1C3 plasmids digested with EcoR1 and Pst1. INV and GLF genes ligated into pSB1C3. E. Coli transformed with pSB1C3 vectors containing INV and GLF genes. Transformed E. Coli streaked and incubated overnight. Colonies from streaked plates isolated and used to prepare liquid cultures. Liquid cultures miniprepped, genes in iGEM vectors isolated, stored.
Enzymology Enzymatic assay of invertase activity, quantification of sucrose. Sucrose + H20 > invertase > glucose + fructose. Step 1) Sodium acetate buffer added to sucrose along with 1 unit/mL invertase. Step 2) Hexokinase reagent added, absorption of NADH measured at 340 nm. Absorption not measured effectively.
