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Team:Yale/Our Project/Methods/cu growth assay
From 2010.igem.org
our project
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.
