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Team:Cornell/testing/project/wetlab/4/1
From 2012.igem.org
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<h3>Overview of Characterization in Bioelectrochemical Systems</h3> | <h3>Overview of Characterization in Bioelectrochemical Systems</h3> | ||
| - | As described in the <a href="https://2012.igem.org/Team:Cornell/testing/project/wetlab/2">Chassis</a> section, <i>S. oneidensis</i> MR-1 is capable of shuttling electrons through the Mtr pathway to reduce extracellular metals because of the negative free energy change associated with these redox reactions. Thus, to encourage <i>Shewanella</i> to transfer electrons to an electrode, we poise the potential of an electrode in a three electrode system, controlled by a potentiostat, so that electron transfer is energetically favorable to the organism. [[LINK TO THE PAGE ON THE POTENTIOSTAT]]. In short, a potentiostat works by setting the potential of a working electrode (WE) with respect to a Ag/AgCl reference electrode (RE) by injecting current through a counter electrode (CE). These electrodes can be seen in the schematic representation of our single-compartment bioelectrochemical reactors shown | + | As described in the <a href="https://2012.igem.org/Team:Cornell/testing/project/wetlab/2">Chassis</a> section, <i>S. oneidensis</i> MR-1 is capable of shuttling electrons through the Mtr pathway to reduce extracellular metals because of the negative free energy change associated with these redox reactions. Thus, to encourage <i>Shewanella</i> to transfer electrons to an electrode, we poise the potential of an electrode in a three electrode system, controlled by a potentiostat, so that electron transfer is energetically favorable to the organism. [[LINK TO THE PAGE ON THE POTENTIOSTAT]]. In short, a potentiostat works by setting the potential of a working electrode (WE) with respect to a Ag/AgCl reference electrode (RE) by injecting current through a counter electrode (CE). These electrodes can be seen in the schematic representation of our single-compartment bioelectrochemical reactors shown to the right. |
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<h3>First Lessons Learned: Control Reactors</h3> | <h3>First Lessons Learned: Control Reactors</h3> | ||
| - | + | In order to enhance the field-deployability of our final device, we initially decided to feed our reactors with LB, since a very concentrated LB source fed at a low flow rate could sustain our field reactors for extended periods of time without taking up much physical space. However, upon setting up control reactors—both in batch and continuous flow operation—we discovered that wild type <i>S. oneidensis</i> MR-1 produced significantly less current when fed with LB than M4—a commonly used media for <i>Shewanella</i>-inoculated bioelectrochemical systems, as illustrated for batch operation by the figure below.<br> | |
<img class="inline" src="https://static.igem.org/mediawiki/2012/1/1c/LBvM4_small.png"> | <img class="inline" src="https://static.igem.org/mediawiki/2012/1/1c/LBvM4_small.png"> | ||
<b> Fig. 1. Current production over time is plotted for batch reactors inoculated with wildtype <i>S. oneidensis</i> MR-1 growing on M4 media (blue) and LB media (green). Maximum current production from M4-fed <i>Shewanella</i> is much greater than that from LB-fed.</b><br><br> | <b> Fig. 1. Current production over time is plotted for batch reactors inoculated with wildtype <i>S. oneidensis</i> MR-1 growing on M4 media (blue) and LB media (green). Maximum current production from M4-fed <i>Shewanella</i> is much greater than that from LB-fed.</b><br><br> | ||
| - | + | When operated in a continuous flow setup, we observed the steady state current production from an LB fed reactor to be within the background noise of the setupM4—<i>i.e.</i>, an un-incoluated reactor was indistinguishable from a reactor inoculated with wildtype <i>S. oneidensis</i> MR-1. Because of this, we chose to use M4 media for all future characterization experiments, since optimization of signal-to-noise ratio is essential in the development of any sensing system. | |
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Revision as of 01:27, 4 October 2012
-
Wet Lab
- Overview
- Chassis
- DNA Assembly
- Testing & Results
- Future Work
- Animation
Reactor Testing
Overview of Characterization in Bioelectrochemical Systems
As described in the Chassis section, S. oneidensis MR-1 is capable of shuttling electrons through the Mtr pathway to reduce extracellular metals because of the negative free energy change associated with these redox reactions. Thus, to encourage Shewanella to transfer electrons to an electrode, we poise the potential of an electrode in a three electrode system, controlled by a potentiostat, so that electron transfer is energetically favorable to the organism. [[LINK TO THE PAGE ON THE POTENTIOSTAT]]. In short, a potentiostat works by setting the potential of a working electrode (WE) with respect to a Ag/AgCl reference electrode (RE) by injecting current through a counter electrode (CE). These electrodes can be seen in the schematic representation of our single-compartment bioelectrochemical reactors shown to the right.
Because we are interested in continuous monitoring of contaminants, characterization focused on the operation of reactors in continuous flow setup, wherein reactors approached steady state current outputs at each level of analyte. In general, all experiments were set up in bench-scale reactors provided by the Angenent Lab, with a constant fluid volume of 120mL and a consistent electrode-surface area. All characterization experiments began at an analyte concentration of zero, as media was fed to the system at a constant rate of 18 mL/min. Once a system reached steady state—for a period of greater than three system retention times—the analyte concentration in the feed tank was increased. By repeating this process after new steady state current outputs were reached, we were able to characterize the current response of our reporter and control strains to either arsenic-containing compounds or naphthalene, as appropriate.
First Lessons Learned: Control Reactors
In order to enhance the field-deployability of our final device, we initially decided to feed our reactors with LB, since a very concentrated LB source fed at a low flow rate could sustain our field reactors for extended periods of time without taking up much physical space. However, upon setting up control reactors—both in batch and continuous flow operation—we discovered that wild type S. oneidensis MR-1 produced significantly less current when fed with LB than M4—a commonly used media for Shewanella-inoculated bioelectrochemical systems, as illustrated for batch operation by the figure below.
Fig. 1. Current production over time is plotted for batch reactors inoculated with wildtype S. oneidensis MR-1 growing on M4 media (blue) and LB media (green). Maximum current production from M4-fed Shewanella is much greater than that from LB-fed.When operated in a continuous flow setup, we observed the steady state current production from an LB fed reactor to be within the background noise of the setupM4—i.e., an un-incoluated reactor was indistinguishable from a reactor inoculated with wildtype S. oneidensis MR-1. Because of this, we chose to use M4 media for all future characterization experiments, since optimization of signal-to-noise ratio is essential in the development of any sensing system.
Naphthalene & Salicylate Sensing
First characterized naphthalene sensors with salicylate, since our system indirectly senses naphthalene via salicylate.Uninduced salicylate reporter produces same current as MR-1 in rich M4 media.
First attempt to diagnose problem: Switch to minimal M4... Producing higher current, but we're still not getting a salicylate response.
Next attempt: Poise the working electrode at a higher potential in attempt to get more MtrC and MtrA for MtrB to associate with. Didn't quite do the trick either.
Fig. 2. Current production over time is plotted for continuous flow reactors inoculated with our salicylate reporter strain (blue) and wildtype S. oneidensis MR-1 (green). Both duration of transient period and value of saturating current are approximately equal for reactors corresponding to both strains.
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Arsenic Sensing
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Fig. 2. Current production over time is plotted for continuous flow reactors inoculated with our salicylate reporter strain (blue) and wildtype S. oneidensis MR-1 (green). Both duration of transient period and value of saturating current are approximately equal for reactors corresponding to both strains. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean rutrum aliquam ipsum, quis lobortis ante vestibulum eu. Nullam eget est justo. Fusce commodo arcu a dui bibendum aliquet. Sed justo eros, dictum quis dictum a, laoreet ut urna. Duis in felis at felis tempor rutrum et sed metus. In sollicitudin adipiscing nibh, eu euismod lectus faucibus eget. Cras ut nulla non velit consequat venenatis in ac velit. Etiam a elit justo. Etiam gravida nulla sit amet eros suscipit at auctor orci porta.
