Team:LCG-UNAM-Mexico/Experiments/Experiments

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     <td height="123" colspan="3" id="tagline" valign="top" align="center">iGEM 2008 TEAM</td>
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           <td class="pageName"><div align="center">Experiments</div>            </td>
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      <p align="center" class="bodyText"><a href="#Devices">Devices</a> | <a href="#sensing">Sensing Dispositive </a></p>
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          <p align="justify"><span class="calHeader"><a name="Devices"></a>Devices</span></p>
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      <p align="justify" class="calHeader style17"><a name="Devices"></a>Devices</p>    
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       <p align="justify"><span class="calHeader"><a name="Sensing"></a>Sensing dispositive</span><br>
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      <a href="https://static.igem.org/mediawiki/2008/a/a0/123_exp.pdf">
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            <a href="https://static.igem.org/mediawiki/2008/4/40/Rcna_exp.pdf"><img src="https://static.igem.org/mediawiki/2008/2/21/Rcna_exp.png" width="500" border="0" /></a>
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      <p class="calHeader style17"><a name="sensing"></a>Sensing dispositive</p>
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      <p align="justify" class="bodyText">As planned, we started measuring at 0.5 OD in LB medium with gold  electrodes (gold was chosen because of its high conductivity). By this  initial approach satisfactory results were obtained the first day of  measurements. However, the second measurement day we detected there was  not a constant behaviour after each measurement replicate. The cause  seemed to be the LB usage. This medium contains yeast extract which may  be full of divalent ions. We expected to solve this problem by using a  buffer as TE pH 8 or  isotonic saline solution, but anyone of them would cause an stress to  the cell that could modify or measurements in an unpredicted way.<br><br>
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      At the same time, we detected that one of the electrodes was stained(oxidation was performed), so we changed the electrodes  metal, avoiding at the same time heating of the electrodes or medium  and the noise that this could imply. Instead of gold we used chrome  electrodes recovered by platinum.<br><br>
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        The second approach was performing measurements in LB with chrome  electrodes recovered by platinum. In this way obtained results were  more consistent after each measurement.      </p>
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      <p align="left">Full sensing dispositive explanation <a href="https://static.igem.org/mediawiki/2008/2/26/ResistivityMeasurmentsDetails.pdf">here</a>.<br>
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          <p>Once the electronic  device was built, We investigated the range where it was able to measure nickel  concentrations on LB medium. We found that the device has maximum efficiency in  the range (1e-7,4e-4).</p>
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          <p align="justify"><a href="https://static.igem.org/mediawiki/2008/b/bf/Ni_Res_loglogLR.jpg"><img src="https://static.igem.org/mediawiki/2008/b/bf/Ni_Res_loglogLR.jpg" width="595" height="350"></a></p>
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          <p align="justify">The graph shows that increasing the nickel level decreases resistivity which is in accordance with the ionic nature of nickel. Points represent the mean of three independent replicates and the line represents the linear regression model:</p>
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          <p align="justify"><img align="left" src="https://static.igem.org/mediawiki/2008/a/a7/LR_Ni_re.png" width="230" height="30"><br><br></p>
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          <p align="justify">Where Ω is the resistivity measured in ohms and [Ni] is the nickel concentration.This shows that even in a complex medium (LB) we are able to detect changes in nickel concentration in a wide range that spans several order of magnitude.</p>
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          <p align="justify">Then we investigated the time dynamics of resistivity in the presence of cells in the previously defined range of nickel concentrations. For this purpose we used cells lacking the <em>rcnA</em> gene, to reduce the effect of cells on our measurements. We have to take into account that even these cells add a variable to our measurements because they continuously import nickel to their cytoplasm.</p>
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          <p align="justify">All our measurements lasted three minutes and we have one data point each 10ms, which means that we have 18000 data points for each experiment. The plot below shows means of each time point for two or three replicates at the indicated nickel concentration. The blue line represents experiments where nickel was not added.</p>
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          <p align="justify"><a href="https://static.igem.org/mediawiki/2008/a/a9/NoNickelVSNickel.jpg"><img src="https://static.igem.org/mediawiki/2008/a/a9/NoNickelVSNickel.jpg" width="595" height="350"></a></p>
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          <p align="justify">The rapid increase that we observe in all the lines was observed in all our experiments independently of the conditions. We think that it represents a phase in which our electronic device adapts to the conditions; however, it is clear from the plot that the presence of cells does not disturb the change in resistivity that we observe</p>
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          <p align="justify">We tried to estimate the rate at which nickel enters the cell by calculating the derivative of the previous curves on each point. The logic is that by substracting the derivatives of the blue curve to the other ones we would get the change in resistivity that is caused by nickel importing to the cytoplasm, i.e. the rate (flux) at which nickel enters the cells. However, we couldn’t find any significant relation despite reducing the interval to that with the most consistent replicates.</p>
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          <p align="justify">In overall, we’ve shown that resistivity allow us to distinguish a wide range of nickel concentrations in a complex medium (LB and cells). More work is needed to standardize the protocols and to investigate the effect of other variables such as temperature. the fact that such good results were achieved with a home-made non-specific electronic device suggests that performance can be greatly enhanced by using specific electrodes.</p>
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Latest revision as of 04:09, 30 October 2008

LCG-UNAM-Mexico:Experiments

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Experiments

Devices | Sensing Dispositive

Devices

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Sensing dispositive

As planned, we started measuring at 0.5 OD in LB medium with gold electrodes (gold was chosen because of its high conductivity). By this initial approach satisfactory results were obtained the first day of measurements. However, the second measurement day we detected there was not a constant behaviour after each measurement replicate. The cause seemed to be the LB usage. This medium contains yeast extract which may be full of divalent ions. We expected to solve this problem by using a buffer as TE pH 8 or isotonic saline solution, but anyone of them would cause an stress to the cell that could modify or measurements in an unpredicted way.

At the same time, we detected that one of the electrodes was stained(oxidation was performed), so we changed the electrodes metal, avoiding at the same time heating of the electrodes or medium and the noise that this could imply. Instead of gold we used chrome electrodes recovered by platinum.

The second approach was performing measurements in LB with chrome electrodes recovered by platinum. In this way obtained results were more consistent after each measurement.

Full sensing dispositive explanation here.

 
             

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