Team:Yale/Our Project/Methods/cu growth assay

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Cu<sup>2+</sup> Growth Assay<br /> <br /> The Cu growth assay tests the correlation between the growth of the bacteria and copper disappearance in solution In our experimental system, the formation of covellite should be directly related to the disappearance of Cu2+ with time because covellite is a complex of copper and sulfur. To test this hypothesis, three growing conditions were tested. In the first condition, the E. coli grew for 6hrs in Luria Broth medium with no IPTG. In the second condition, the E. coli were grown for the same amount of time but with IPTG added at time 0. In the last condition, IPTG was added after three hours of growth. The E. coli need IPTG because it triggers the production of H2S which reacts with the copper in the solution to form covellite. As a result, over time, as covellite is formed, the concentration of copper should decrease in the media.<br />  <br />
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<B> Cu<sup>2+</sup> Growth Assay Design</B> <br /> <br /> The Copper growth assay tests the correlation between the growth of the bacteria and copper disappearance in solution. In our experimental system, the formation of covellite should be directly related to the disappearance of Cu<sup>2+</sup> with time because covellite is a complex of copper and sulfur. To test this hypothesis, three growing conditions were tested. In the first condition, the E. coli grew for 6hrs in Luria Broth medium with no IPTG. In the second condition, the E. coli were grown for the same amount of time but with IPTG added at time 0. In the last condition, IPTG was added after three hours of growth. The E. coli need IPTG because it induces the production of H<sub>2</sub>S which reacts with the copper in the solution to form covellite. As a result, over time, as covellite is formed, the concentration of copper should decrease in the media.<br />  <br />
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We hypothesize that in the first condition, the copper concentration should remain constant because H2S produce was never activated. In the third condition, copper levels should level until IPTG is added spurring H2S production and copper disappearance. Lastly, we hypothesize that in the second condition copper would decrease slowly at first because of the low density of bacteria producing H2S. However, as the density of bacteria increases, the rate of decrease of copper should increase.<br />   
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We hypothesize that in the first condition, the copper concentration should remain constant because H<sub>2</sub>S produce was never activated. In the third condition, copper levels should level until IPTG is added spurring H<sub>2</sub>S production and copper disappearance. Lastly, we hypothesize that in the second condition copper would decrease slowly at first because of the low density of bacteria producing H<sub>2</sub>S. However, as the density of bacteria increases, the rate of decrease of copper should increase.<br />   
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<img src="https://static.igem.org/mediawiki/2010/b/b5/Yale-cu-growth-assay.png" />
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We measure the concentration of copper using bathocuproinedisulfonic acid (BCS) which turns orange when it reacts with Cu+. First, a standard curve of copper concentration of Cu+ from the 10mM to 1uM range. Afterwards, the concentration of copper was determined in the stored supernatants of the three growth conditions.   
We measure the concentration of copper using bathocuproinedisulfonic acid (BCS) which turns orange when it reacts with Cu+. First, a standard curve of copper concentration of Cu+ from the 10mM to 1uM range. Afterwards, the concentration of copper was determined in the stored supernatants of the three growth conditions.   
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<br />  <br />
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<b> Assay Results </b>
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<br /> <br /> Standard Curve <br /> <br />
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<img src="https://static.igem.org/mediawiki/2010/c/c3/Yale-standard-curve.png" />
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<br /> Standard curve shows a linear relationship between absorbance and copper concentration from 1µm to 0.4mM. To measure copper concentration from Conditions 1,2&3 samples have to be diluted 10 to 100x time so that they are in the right sensitivity range of BCS.
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<br /> <br /> Copper Growth Assay
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<br /> The bacteria were grown for 6 hours with 2mM copper. Samples were taken every 30 minutes and stored at -20<sup>o</sup>C. The copper concentrations will be measured next week.
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Latest revision as of 03:39, 28 October 2010

iGEM Yale

experimental methods

Cu2+ Growth Assay Design

The Copper growth assay tests the correlation between the growth of the bacteria and copper disappearance in solution. In our experimental system, the formation of covellite should be directly related to the disappearance of Cu2+ with time because covellite is a complex of copper and sulfur. To test this hypothesis, three growing conditions were tested. In the first condition, the E. coli grew for 6hrs in Luria Broth medium with no IPTG. In the second condition, the E. coli were grown for the same amount of time but with IPTG added at time 0. In the last condition, IPTG was added after three hours of growth. The E. coli need IPTG because it induces the production of H2S which reacts with the copper in the solution to form covellite. As a result, over time, as covellite is formed, the concentration of copper should decrease in the media.

We hypothesize that in the first condition, the copper concentration should remain constant because H2S produce was never activated. In the third condition, copper levels should level until IPTG is added spurring H2S production and copper disappearance. Lastly, we hypothesize that in the second condition copper would decrease slowly at first because of the low density of bacteria producing H2S. However, as the density of bacteria increases, the rate of decrease of copper should increase.


We measure the concentration of copper using bathocuproinedisulfonic acid (BCS) which turns orange when it reacts with Cu+. First, a standard curve of copper concentration of Cu+ from the 10mM to 1uM range. Afterwards, the concentration of copper was determined in the stored supernatants of the three growth conditions.

Assay Results

Standard Curve


Standard curve shows a linear relationship between absorbance and copper concentration from 1µm to 0.4mM. To measure copper concentration from Conditions 1,2&3 samples have to be diluted 10 to 100x time so that they are in the right sensitivity range of BCS.

Copper Growth Assay
The bacteria were grown for 6 hours with 2mM copper. Samples were taken every 30 minutes and stored at -20oC. The copper concentrations will be measured next week.

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