Team:Nevada/Notebook/June/Week2

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Revision as of 08:55, 30 June 2011

                     Week 1 - June 1-4
                     -----------------------
                             E. Coli
                                Blah
                             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 2

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-<html>
+{{Nevada__Notebook_CSS}}
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 <a href="https://2011.igem.org/Team:Nevada/Notebook">Notebook</a>
-<p><font color="red">E. Coli</font>
+<a href="https://2011.igem.org/Team:Nevada/Parts">Parts</a>
-<p><font color="blue">Cyano</font>
+<a href="https://2011.igem.org/Team:Nevada/Media">Media</a>
-<p><font color="green">Enzymology</font>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<p><font color="black">Media</font>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+<a href="https://2011.igem.org/Team:Nevada/Foundraiser">Fundraisers</a>
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+<a href="http://www.igemteamnevada.webs.com">Official Site</a>
+<a href="https://2011.igem.org/Team:Nevada/Reference">Reference</a><br><br>
+<font color="red">E. Coli</font> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+<font color="blue">Cyano</font> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+<font color="green">Enzymology</font> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+<font color="black">Media</font>
+</div>
+</div>
+<br><br><p><br><p><br>
+<div id="weekly">
+<font size="5">
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+Week 1 - June 1-4
+<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+-----------------------
+</font>
+<br><font color="red">
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+E. Coli
 <br>
-<font size="5">Week 1 - June 1-4
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+Blah
+</font>
+<br><font color="blue">
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+Cyano
 <br>
-----------------------------------------------
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+Blah
 </font>
-<p><font color="red">E. Coli
+<br><font color="green">
-<br>Blah</font>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<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.
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
-<p><font color="green">Hexokinase coupled assay used to quantitate amounts of cyanobacteria glucose secretion.
+Hexokinase coupled assay used to quantitate
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+amounts of cyanobacteria glucose secretion.
+<br>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 Rxn: 1. glucose + ATP --Hx-> G-6-P + ADP
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 . 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.
+<br><br>
-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)</font>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+Reaction used Glucose assay kit (Genzyme)
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+containing 2000U/L Hexokinase reagent and
+<br>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+U/L G-6-P DeH and measured glucose
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+concentrations ranging from 0.05mM-0.25mM
+<br>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+and absorbances were measured at 340.0 nm.
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+Results: Final NADH concentrations were
+<br>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+determined using the molar extinction
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+coefficient and were proportionate to intial
+<br>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+glucose concentrations.
+<br><br>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+Discussion: Focus is now to measure not only
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+glucose, but fructose concentrations as well as
+<br>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+both with be secreted in the form of sucrose
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+using a D-glucose/D-fructose assay (Megaenzyme)
+</font>
+<br><p>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+<font color="black">
+Media
+</font><p>
+<br>
+</div>
+<div id="bottom" align="center">
+<div id="bottomright">
+<a href="https://2011.igem.org/Team:Nevada/Notebook">Week 1</a>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+<a href="https://2011.igem.org/Team:Nevada/Notebook/June/Week2">Week 2</a>
+</div>
+</div>
+<!--
+<font size="5">Week 2 - June 5-11
+<br>
+-----------------------------
+</font>
+<p><font color="red">E. Coli
+<br>
+<img src="https://static.igem.org/mediawiki/2011/a/a2/Growth_Curve_6_10_11.png" class="shadow" style="float:center"
+height=400px width=400px>
+<br>
+<br>
+<img src="https://static.igem.org/mediawiki/2011/b/bc/Log_Growth_Curve_6_10_11.png" class="shadow" style="float:center"
+height=400px width=400px>
+</font>
+<p><font color="blue">Cyano</font>
+<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.
+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.</font>
 <p><font color="black">Media</font>
 <br>
+<font size="5">Week 3- June 12-18
+<br>
+----------------------------------------------
+</font>
+<p><font color="red">E. Coli</font>
+<p><font color="blue">Cyano</font>
+<p><font color="green">Enzymology</font>
 <font size="5">Week 2 - June 5-11
 <br>
@@ Line 44: / Line 261: @@
 <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> 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.
+-->
+</div>
+</body>
 </html>