http://2008.igem.org/wiki/index.php?title=Special:Contributions/Linchinlee&feed=atom&limit=50&target=Linchinlee&year=&month=2008.igem.org - User contributions [en]2024-03-29T01:43:40ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:Harvard/RecipesTeam:Harvard/Recipes2008-10-30T04:08:59Z<p>Linchinlee: /* Lactate solution */</p>
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=Recipes=<br />
We experimented with several types of growth media for Shewanella growth, both aerobically and anaerobically. The most effective growth media recipes are listed as follows.<BR><BR><br />
<br />
__TOC__<br />
==Orianna's growth media==<br />
* 50 mM K-PIPES '''18.925 g/L'''<br />
* 7.5 mM NaOH '''7.5 mL/L'''<br />
* 28 mM NH<sub>4</sub>Cl '''1.5 g/L'''<br />
* 1.3 mM KCl '''433 uL/L'''<br />
* 4.3 mM NaH<sub>2</sub>PO<sub>4</sub> '''0.59 g/L'''<br />
* 100 mM NaCl '''5.84 g/L'''<br />
* 18 mM Lactate '''1.62 g/L'''<br />
* 10 mL Vitamin solution<br />
* 10 mL Amino acid solution<br />
* 10 mL Trace mineral solution<br />
<br />
==LML medium (for anaerobic Shewanella growth)==<br />
* 0.02% yeast extract<br />
* 0.01% peptone<br />
* 20 mM lactate<br />
* 10 mM HEPES [pH 7.4]<br />
<br />
==LML agar (for Shewanella reduction test)==<br />
* LML medium<br />
* 0.8% agar<br />
* 2 mM MnO2- Mn(IV) oxide (turns from brown to clear upon reduction to Mn(II))<br />
<br />
(LML recipes from [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=9829939| Beliaev and Saffarini 1998])<br />
<br />
==Chamber media==<br />
* 5.844 g/L 100mM NaCl<br />
* 15.1185 g/L 50mM PIPES (hydrogen) <br />
''7.0 pH''<br />
<br />
==Phosphate buffer==<br />
* 2.918 g/L Monosodium phosphate, monohydrate<br />
* 4.095 g/L Disodium phosphate, anhydrous <br />
* 5.844 g/L 100mM NaCl<br />
''7.0 pH''<br />
<br />
==Lactate solution==<br />
* 13.51 g/L Lactic acid<br />
* neutralize to pH 7 with NaOH<br />
<br />
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|<br />
=Recipes=<br />
We experimented with several types of growth media for Shewanella growth, both aerobically and anaerobically. The most effective growth media recipes are listed as follows.<BR><BR><br />
<br />
__TOC__<br />
==Orianna's growth media==<br />
* 50 mM K-PIPES '''18.925 g/L'''<br />
* 7.5 mM NaOH '''7.5 mL/L'''<br />
* 28 mM NH<sub>4</sub>Cl '''1.5 g/L'''<br />
* 1.3 mM KCl '''433 uL/L'''<br />
* 4.3 mM NaH<sub>2</sub>PO<sub>4</sub> '''0.59 g/L'''<br />
* 100 mM NaCl '''5.84 g/L'''<br />
* 18 mM Lactate '''1.62 g/L'''<br />
* 10 mL Vitamin solution<br />
* 10 mL Amino acid solution<br />
* 10 mL Trace mineral solution<br />
<br />
==LML medium (for anaerobic Shewanella growth)==<br />
* 0.02% yeast extract<br />
* 0.01% peptone<br />
* 20 mM lactate<br />
* 10 mM HEPES [pH 7.4]<br />
<br />
==LML agar (for Shewanella reduction test)==<br />
* LML medium<br />
* 0.8% agar<br />
* 2 mM MnO2- Mn(IV) oxide (turns from brown to clear upon reduction to Mn(II))<br />
<br />
(LML recipes from [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=9829939| Beliaev and Saffarini 1998])<br />
<br />
==Chamber media==<br />
* 5.844 g/L 100mM NaCl<br />
* 15.1185 g/L 50mM PIPES (hydrogen) <br />
''7.0 pH''<br />
<br />
==Phosphate buffer==<br />
* 2.918 g/L Monosodium phosphate, monohydrate<br />
* 4.095 g/L Disodium phosphate, anhydrous <br />
* 5.844 g/L 100mM NaCl<br />
''7.0 pH''<br />
<br />
==Lactate solution==<br />
* 13.51 g/L lactic acid<br />
* neutralize to pH 7 with NaOH<br />
<br />
|}<br />
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<!--- end body ---><br />
|}</div>Linchinleehttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:57:56Z<p>Linchinlee: /* Wildtype vs. mtrB deficient S. oneidensis */</p>
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==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<br />
The metabolically versatile bacterium ''Shewanella oneidensis'' adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, ''S. oneidensis'' transfers electrons to an electrode of a microbial fuel cell. We sought to engineer ''S. oneidensis'' to report variations in environmental conditions through changes in current production. A previous study has shown that ''S. oneidensis'' mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.<br />
<br />
==Experimental overview==<br />
<br />
We attempted to develop three inducible systems for electrical current production in ''S. oneidensis''. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression in mtrB knock-out (KO) ''S. oneidensis'' can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach uses temperature-senstive cI which would allow for an increase in electrical output in response to heat. The third is a light-inducible system based on the 2005 UT Austin biological camera.<br />
<br />
Using the LacI system, we attempted to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we built, measuring the effects of IPTG on current production in wild type and mtrB KO ''S. oneidensis'' containing plasmids expressing inducible or constitutive mtrB.<br />
<br />
==Results==<br />
<br />
===Wildtype vs. mtrB deficient ''S. oneidensis''===<br />
<br />
''Overview: By genetically manipulating S. oneidensis, it has been found that noticeable differences of current production can be detected. We wish to control current production by introducing inducible systems in these mutant strains of S. oneidensis. These differences of current production can be detected by a computer, allowing biological systems and electrical devices to be integrated.''<br />
<br />
The basis of our project lies in the discovery that by knocking out the mtrB gene, a current production gene of S. oneidensis, noticeable differences of current production can be detected. Therefore, this allows the current production of S. oneidensis to act more than just an on/off switch.<br />
<br />
The first step of our project was then to test this hypothesis and also to understand the current production behavior of wildtype S. oneidensis and the mutant S. oneidensis (annotated as mtrB). Therefore, we ran a test with microbial fuel cells, one strain in each cell, and allowed the current production to be measured for approximately one day. In literature, mtrB produces approximately 20 to 25 percent of the current or wildtype strains.<br />
<br />
<br />
[[Image: wt_mtrB.jpg|700px]]<br />
<br />
<br />
As shown in the graph above, the results of our experiment matched those in literature. We also observed that wildtype and mtrB strains have very different current production behavior, with wildtype producing current immediately after lactate injection, and mtrB producing current much more gradually. This difference in behavior can allow for more accurate differentiation of current production between wildtype and mtrB strains when integrated with electrical devices. We wish then build on these results and build inducible systems that will allow us to control the current production.<br />
<br />
===Co-Culture Experiment===<br />
<br />
''Overview: One possible system for achieving inducible current could be to couple a inducible-lacZ system in E. coli to current production by wildtype S. oneidensis MR1 through the conversion of lactose to lactate. This leaves open the possibility of using a pre-existing E. coli lacZ reporter or creating our own.<br />
''<br />
<br />
In addition to genetically engineering S. oneidensis MR-1 to respond to chemicals and heat, we also sought to take advantage of its natural metabolic pathway of breaking down lactate to produce current. From our wildtype versus mtrB knockout experiments, we found that feeding S. oneidensis MR-1 lactate led to significant current production almost instantly. In wildtype S. oneidensis MR-1, the current production would increase and stay elevated for a period of twelve hours. This result occurred consistently, thus we sought to use controlled lactate release as a way to control current output. <br />
<br />
We also knew that wildtype E. coli takes lactose and breaks down lactate. Thus, we hypothesized that it might be possible to couple E. coli’s lactate production with S. oneidensis MR-1’s lactate breakdown to produce current. In addition, a great deal of genetic engineering has already been done on E. coli and the up-regulation or down-regulation of its Lac operon. This means that instead of genetically modifying S. oneidensis MR-1 to respond to chemicals or heat, we can instead take advantage of the vast library of E. coli genetics and couple genetically engineered E. coli with wildtype S. oneidensis MR-1. <br />
<br />
In this experiment, we tested different combinations of wildtype E. coli MG1655, S. oneidensis MR-1, and Lac-operon knockout E. coli MC4100 as follows:<br />
<br />
1. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactose <br />
<br />
2. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
3. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactose<br />
<br />
4. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
5. wt S. oneidensis MR-1 + lactose neg. control<br />
<br />
6. wt E. coli (MG1655) + lactose neg. control<br />
<br />
7. Lac-operon knockout E. coli (MC4100) + lactose neg. control<br />
<br />
Based on previous experiments, we would expect current production in combinations 2 and 4 as they both have wildtype S. oneidensis MR-1 and receive lactate. In addition, however, we would expect combination 1 to also produce current. As described above, wt E. coli would break down lactose into lactate, and S. oneidensis MR-1 would break down lactate to produce current.<br />
<br />
[[ Image: picture 5.png | 800px ]]<br />
<br />
From the data above, we found that combination 1 did indeed produce current with a delay relative to the positive control. The delay can be attributed to the time it takes for E. coli to break down lactose into lactate, thus adding an extra step in the carbon source to current production pathway compared to our positive controls. These results are exciting in that they show a possibility of taking advantage of this cooperative effort to achieve inducible current.<br />
<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''S. oneidensis'' has laid the foundations for many different exciting avenues of further inquiry which look to take advantage of a bacteria-computer interface that combines the amazing sensitivity and adaptability of bacteria with the speed and analytical abilities of electricity and computers.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006 arsenic biosensor] developed by the University of Edinburgh iGEM 2006 team could be introduced into ''S. oneidensis'', allowing for the coupling of arsenic sensing to an electrical output, a form of a data which is easier to automate and transmit. This could be further extended to other chemical sensing systems, such as the [http://parts.mit.edu/igem07/index.php/Brown lead sensor] created by the Brown iGEM 2007 team and the [http://parts.mit.edu/igem07/index.php/MIT mercury sensor] made by the MIT iGEM 2007 team, resulting ultimately in an array of different strains of ''S. oneidensis'' which all respond to the presence of different chemicals with an electrical output that can be monitored by a computer. This could theoretically allow for the remote sensing and analysis of the chemical composition of an environment over time in a cost-effective manner, making it a tool with powerful public health applications, such as monitoring water quality.<br />
<br />
Another interesting direction would be the linking of the [http://parts.mit.edu/wiki/index.php/UT_Austin_2005 light-sensing system] developed by the UT Austin iGEM team with electrical output in ''S. oneidensis''. In response to variations in light, the amount of electricity produced by ''S. oneidensis'' would change. This would allow for the intriguing possibility of not only ''S. oneidensis'' conveying information to the computer, but also the computer responding to the ''S. oneidensis''. A simple example would be that in response to a chemical input, ''S. oneidensis'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''S. oneidensis'', modifying ''S. oneidensis'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers. We tried constructing this system over summer, but as the process requires making an EnvZ knockout strain of ''S. oneidensis'', we could not finish it. We did, however, make a few parts to facilitate future attempts.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''S. oneidensis''. Either of the systems described above could be pursued through an alternative alternative strategy of co-cultures. For instance, an array of ''E. coli'' which respond to different chemicals by breaking down lactose into lactate could be cultured with ''S. oneidensis''. In response to an increase in lactate, ''S. oneidensis'' would begin to produce higher levels of electricity. Co-cultures could also allow for more complex bacteria-computer interactions. This strategy could enable the coupling of almost any ''E. coli'' ability to electrical output.<br />
<br />
These future directions in which our research can be taken demonstrate some of the exciting possibilities of BACTRICITY!<br />
<br />
|}<br />
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|}</div>Linchinleehttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:57:00Z<p>Linchinlee: /* Wildtype vs. mtrB deficient S. oneidensis */</p>
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==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<br />
The metabolically versatile bacterium ''Shewanella oneidensis'' adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, ''S. oneidensis'' transfers electrons to an electrode of a microbial fuel cell. We sought to engineer ''S. oneidensis'' to report variations in environmental conditions through changes in current production. A previous study has shown that ''S. oneidensis'' mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.<br />
<br />
==Experimental overview==<br />
<br />
We attempted to develop three inducible systems for electrical current production in ''S. oneidensis''. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression in mtrB knock-out (KO) ''S. oneidensis'' can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach uses temperature-senstive cI which would allow for an increase in electrical output in response to heat. The third is a light-inducible system based on the 2005 UT Austin biological camera.<br />
<br />
Using the LacI system, we attempted to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we built, measuring the effects of IPTG on current production in wild type and mtrB KO ''S. oneidensis'' containing plasmids expressing inducible or constitutive mtrB.<br />
<br />
==Results==<br />
<br />
===Wildtype vs. mtrB deficient ''S. oneidensis''===<br />
<br />
''Overview: By genetically manipulating S. oneidensis, it has been found that noticeable differences of current production can be detected. We wish to control current production by introducing inducible systems in these mutant strains of S. oneidensis. These differences of current production can be detected by a computer, allowing biological systems and electrical devices to be integrated.''<br />
<br />
The basis of our project lies in the discovery that by knocking out the mtrB gene, a current production gene of S. oneidensis, noticeable differences of current production can be detected. Therefore, this allows the current production of S. oneidensis to act more than just an on/off switch.<br />
<br />
The first step of our project was then to test this hypothesis and also to understand the current production behavior of wildtype S. oneidensis and the mutant S. oneidensis (annotated as mtrB). Therefore, we ran a test with microbial fuel cells, one strain in each cell, and allowed the current production to be measured for approximately one day. In literature, mtrB produces approximately 20 to 25 percent of the current or wildtype strains.<br />
<br />
<br />
[[Image: wt_mtrB.jpg|700px]]<br />
<br />
<br />
As shown in the graph above, the results of our experiment matched those in literature. We also observed that wildtype and mtrB strains have very different current production behavior, with wildtype producing current immediately after lactate injection, and mtrB producing current much more gradually. This difference in behavior can allow for more accurate differentiation of current production between wildtype and mtrB strains when we integrate it with electrical devices. We wish then build on these results and build inducible systems that will allow us to control the current production.<br />
<br />
===Co-Culture Experiment===<br />
<br />
''Overview: One possible system for achieving inducible current could be to couple a inducible-lacZ system in E. coli to current production by wildtype S. oneidensis MR1 through the conversion of lactose to lactate. This leaves open the possibility of using a pre-existing E. coli lacZ reporter or creating our own.<br />
''<br />
<br />
In addition to genetically engineering S. oneidensis MR-1 to respond to chemicals and heat, we also sought to take advantage of its natural metabolic pathway of breaking down lactate to produce current. From our wildtype versus mtrB knockout experiments, we found that feeding S. oneidensis MR-1 lactate led to significant current production almost instantly. In wildtype S. oneidensis MR-1, the current production would increase and stay elevated for a period of twelve hours. This result occurred consistently, thus we sought to use controlled lactate release as a way to control current output. <br />
<br />
We also knew that wildtype E. coli takes lactose and breaks down lactate. Thus, we hypothesized that it might be possible to couple E. coli’s lactate production with S. oneidensis MR-1’s lactate breakdown to produce current. In addition, a great deal of genetic engineering has already been done on E. coli and the up-regulation or down-regulation of its Lac operon. This means that instead of genetically modifying S. oneidensis MR-1 to respond to chemicals or heat, we can instead take advantage of the vast library of E. coli genetics and couple genetically engineered E. coli with wildtype S. oneidensis MR-1. <br />
<br />
In this experiment, we tested different combinations of wildtype E. coli MG1655, S. oneidensis MR-1, and Lac-operon knockout E. coli MC4100 as follows:<br />
<br />
1. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactose <br />
<br />
2. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
3. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactose<br />
<br />
4. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
5. wt S. oneidensis MR-1 + lactose neg. control<br />
<br />
6. wt E. coli (MG1655) + lactose neg. control<br />
<br />
7. Lac-operon knockout E. coli (MC4100) + lactose neg. control<br />
<br />
Based on previous experiments, we would expect current production in combinations 2 and 4 as they both have wildtype S. oneidensis MR-1 and receive lactate. In addition, however, we would expect combination 1 to also produce current. As described above, wt E. coli would break down lactose into lactate, and S. oneidensis MR-1 would break down lactate to produce current.<br />
<br />
[[ Image: picture 5.png | 800px ]]<br />
<br />
From the data above, we found that combination 1 did indeed produce current with a delay relative to the positive control. The delay can be attributed to the time it takes for E. coli to break down lactose into lactate, thus adding an extra step in the carbon source to current production pathway compared to our positive controls. These results are exciting in that they show a possibility of taking advantage of this cooperative effort to achieve inducible current.<br />
<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''S. oneidensis'' has laid the foundations for many different exciting avenues of further inquiry which look to take advantage of a bacteria-computer interface that combines the amazing sensitivity and adaptability of bacteria with the speed and analytical abilities of electricity and computers.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006 arsenic biosensor] developed by the University of Edinburgh iGEM 2006 team could be introduced into ''S. oneidensis'', allowing for the coupling of arsenic sensing to an electrical output, a form of a data which is easier to automate and transmit. This could be further extended to other chemical sensing systems, such as the [http://parts.mit.edu/igem07/index.php/Brown lead sensor] created by the Brown iGEM 2007 team and the [http://parts.mit.edu/igem07/index.php/MIT mercury sensor] made by the MIT iGEM 2007 team, resulting ultimately in an array of different strains of ''S. oneidensis'' which all respond to the presence of different chemicals with an electrical output that can be monitored by a computer. This could theoretically allow for the remote sensing and analysis of the chemical composition of an environment over time in a cost-effective manner, making it a tool with powerful public health applications, such as monitoring water quality.<br />
<br />
Another interesting direction would be the linking of the [http://parts.mit.edu/wiki/index.php/UT_Austin_2005 light-sensing system] developed by the UT Austin iGEM team with electrical output in ''S. oneidensis''. In response to variations in light, the amount of electricity produced by ''S. oneidensis'' would change. This would allow for the intriguing possibility of not only ''S. oneidensis'' conveying information to the computer, but also the computer responding to the ''S. oneidensis''. A simple example would be that in response to a chemical input, ''S. oneidensis'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''S. oneidensis'', modifying ''S. oneidensis'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers. We tried constructing this system over summer, but as the process requires making an EnvZ knockout strain of ''S. oneidensis'', we could not finish it. We did, however, make a few parts to facilitate future attempts.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''S. oneidensis''. Either of the systems described above could be pursued through an alternative alternative strategy of co-cultures. For instance, an array of ''E. coli'' which respond to different chemicals by breaking down lactose into lactate could be cultured with ''S. oneidensis''. In response to an increase in lactate, ''S. oneidensis'' would begin to produce higher levels of electricity. Co-cultures could also allow for more complex bacteria-computer interactions. This strategy could enable the coupling of almost any ''E. coli'' ability to electrical output.<br />
<br />
These future directions in which our research can be taken demonstrate some of the exciting possibilities of BACTRICITY!<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Linchinleehttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:56:50Z<p>Linchinlee: /* Wildtype vs. mtrB deficient S. oneidensis */</p>
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==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<br />
The metabolically versatile bacterium ''Shewanella oneidensis'' adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, ''S. oneidensis'' transfers electrons to an electrode of a microbial fuel cell. We sought to engineer ''S. oneidensis'' to report variations in environmental conditions through changes in current production. A previous study has shown that ''S. oneidensis'' mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.<br />
<br />
==Experimental overview==<br />
<br />
We attempted to develop three inducible systems for electrical current production in ''S. oneidensis''. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression in mtrB knock-out (KO) ''S. oneidensis'' can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach uses temperature-senstive cI which would allow for an increase in electrical output in response to heat. The third is a light-inducible system based on the 2005 UT Austin biological camera.<br />
<br />
Using the LacI system, we attempted to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we built, measuring the effects of IPTG on current production in wild type and mtrB KO ''S. oneidensis'' containing plasmids expressing inducible or constitutive mtrB.<br />
<br />
==Results==<br />
<br />
===Wildtype vs. mtrB deficient ''S. oneidensis''===<br />
''Overview: By genetically manipulating S. oneidensis, it has been found that noticeable differences of current production can be detected. We wish to control current production by introducing inducible systems in these mutant strains of S. oneidensis. These differences of current production can be detected by a computer, allowing biological systems and electrical devices to be integrated.''<br />
<br />
The basis of our project lies in the discovery that by knocking out the mtrB gene, a current production gene of S. oneidensis, noticeable differences of current production can be detected. Therefore, this allows the current production of S. oneidensis to act more than just an on/off switch.<br />
<br />
The first step of our project was then to test this hypothesis and also to understand the current production behavior of wildtype S. oneidensis and the mutant S. oneidensis (annotated as mtrB). Therefore, we ran a test with microbial fuel cells, one strain in each cell, and allowed the current production to be measured for approximately one day. In literature, mtrB produces approximately 20 to 25 percent of the current or wildtype strains.<br />
<br />
<br />
[[Image: wt_mtrB.jpg|700px]]<br />
<br />
<br />
As shown in the graph above, the results of our experiment matched those in literature. We also observed that wildtype and mtrB strains have very different current production behavior, with wildtype producing current immediately after lactate injection, and mtrB producing current much more gradually. This difference in behavior can allow for more accurate differentiation of current production between wildtype and mtrB strains when we integrate it with electrical devices. We wish then build on these results and build inducible systems that will allow us to control the current production.<br />
<br />
===Co-Culture Experiment===<br />
<br />
''Overview: One possible system for achieving inducible current could be to couple a inducible-lacZ system in E. coli to current production by wildtype S. oneidensis MR1 through the conversion of lactose to lactate. This leaves open the possibility of using a pre-existing E. coli lacZ reporter or creating our own.<br />
''<br />
<br />
In addition to genetically engineering S. oneidensis MR-1 to respond to chemicals and heat, we also sought to take advantage of its natural metabolic pathway of breaking down lactate to produce current. From our wildtype versus mtrB knockout experiments, we found that feeding S. oneidensis MR-1 lactate led to significant current production almost instantly. In wildtype S. oneidensis MR-1, the current production would increase and stay elevated for a period of twelve hours. This result occurred consistently, thus we sought to use controlled lactate release as a way to control current output. <br />
<br />
We also knew that wildtype E. coli takes lactose and breaks down lactate. Thus, we hypothesized that it might be possible to couple E. coli’s lactate production with S. oneidensis MR-1’s lactate breakdown to produce current. In addition, a great deal of genetic engineering has already been done on E. coli and the up-regulation or down-regulation of its Lac operon. This means that instead of genetically modifying S. oneidensis MR-1 to respond to chemicals or heat, we can instead take advantage of the vast library of E. coli genetics and couple genetically engineered E. coli with wildtype S. oneidensis MR-1. <br />
<br />
In this experiment, we tested different combinations of wildtype E. coli MG1655, S. oneidensis MR-1, and Lac-operon knockout E. coli MC4100 as follows:<br />
<br />
1. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactose <br />
<br />
2. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
3. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactose<br />
<br />
4. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
5. wt S. oneidensis MR-1 + lactose neg. control<br />
<br />
6. wt E. coli (MG1655) + lactose neg. control<br />
<br />
7. Lac-operon knockout E. coli (MC4100) + lactose neg. control<br />
<br />
Based on previous experiments, we would expect current production in combinations 2 and 4 as they both have wildtype S. oneidensis MR-1 and receive lactate. In addition, however, we would expect combination 1 to also produce current. As described above, wt E. coli would break down lactose into lactate, and S. oneidensis MR-1 would break down lactate to produce current.<br />
<br />
[[ Image: picture 5.png | 800px ]]<br />
<br />
From the data above, we found that combination 1 did indeed produce current with a delay relative to the positive control. The delay can be attributed to the time it takes for E. coli to break down lactose into lactate, thus adding an extra step in the carbon source to current production pathway compared to our positive controls. These results are exciting in that they show a possibility of taking advantage of this cooperative effort to achieve inducible current.<br />
<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''S. oneidensis'' has laid the foundations for many different exciting avenues of further inquiry which look to take advantage of a bacteria-computer interface that combines the amazing sensitivity and adaptability of bacteria with the speed and analytical abilities of electricity and computers.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006 arsenic biosensor] developed by the University of Edinburgh iGEM 2006 team could be introduced into ''S. oneidensis'', allowing for the coupling of arsenic sensing to an electrical output, a form of a data which is easier to automate and transmit. This could be further extended to other chemical sensing systems, such as the [http://parts.mit.edu/igem07/index.php/Brown lead sensor] created by the Brown iGEM 2007 team and the [http://parts.mit.edu/igem07/index.php/MIT mercury sensor] made by the MIT iGEM 2007 team, resulting ultimately in an array of different strains of ''S. oneidensis'' which all respond to the presence of different chemicals with an electrical output that can be monitored by a computer. This could theoretically allow for the remote sensing and analysis of the chemical composition of an environment over time in a cost-effective manner, making it a tool with powerful public health applications, such as monitoring water quality.<br />
<br />
Another interesting direction would be the linking of the [http://parts.mit.edu/wiki/index.php/UT_Austin_2005 light-sensing system] developed by the UT Austin iGEM team with electrical output in ''S. oneidensis''. In response to variations in light, the amount of electricity produced by ''S. oneidensis'' would change. This would allow for the intriguing possibility of not only ''S. oneidensis'' conveying information to the computer, but also the computer responding to the ''S. oneidensis''. A simple example would be that in response to a chemical input, ''S. oneidensis'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''S. oneidensis'', modifying ''S. oneidensis'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers. We tried constructing this system over summer, but as the process requires making an EnvZ knockout strain of ''S. oneidensis'', we could not finish it. We did, however, make a few parts to facilitate future attempts.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''S. oneidensis''. Either of the systems described above could be pursued through an alternative alternative strategy of co-cultures. For instance, an array of ''E. coli'' which respond to different chemicals by breaking down lactose into lactate could be cultured with ''S. oneidensis''. In response to an increase in lactate, ''S. oneidensis'' would begin to produce higher levels of electricity. Co-cultures could also allow for more complex bacteria-computer interactions. This strategy could enable the coupling of almost any ''E. coli'' ability to electrical output.<br />
<br />
These future directions in which our research can be taken demonstrate some of the exciting possibilities of BACTRICITY!<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Linchinleehttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:55:17Z<p>Linchinlee: /* Wildtype vs. mtrB deficient S. oneidensis */</p>
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|<br />
==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<br />
The metabolically versatile bacterium ''Shewanella oneidensis'' adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, ''S. oneidensis'' transfers electrons to an electrode of a microbial fuel cell. We sought to engineer ''S. oneidensis'' to report variations in environmental conditions through changes in current production. A previous study has shown that ''S. oneidensis'' mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.<br />
<br />
==Experimental overview==<br />
<br />
We attempted to develop three inducible systems for electrical current production in ''S. oneidensis''. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression in mtrB knock-out (KO) ''S. oneidensis'' can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach uses temperature-senstive cI which would allow for an increase in electrical output in response to heat. The third is a light-inducible system based on the 2005 UT Austin biological camera.<br />
<br />
Using the LacI system, we attempted to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we built, measuring the effects of IPTG on current production in wild type and mtrB KO ''S. oneidensis'' containing plasmids expressing inducible or constitutive mtrB.<br />
<br />
==Results==<br />
<br />
===Wildtype vs. mtrB deficient ''S. oneidensis''===<br />
''Overview: By genetically manipulating S. oneidensis, it has been found that noticeable differences of current production can be detected. We wish to control current production by introducing inducible systems in these mutant strains of S. oneidensis. These differences of current production can be detected by a computer, allowing biological systems and electrical devices to be integrated.''<br />
<br />
One important basis of our project lies in the discovery that by knocking out the mtrB gene, a current production gene of S. oneidensis, noticeable differences of current production can be detected. Therefore, this allows the current production of S. oneidensis to act more than just an on/off switch.<br />
<br />
The first step of our project was then to test this hypothesis and also to understand the current production behavior of wildtype S. oneidensis and the mutant S. oneidensis (annotated as mtrB). Therefore, we ran a test with microbial fuel cells, one strain in each cell, and allowed the current production to be measured for approximately one day. In literature, mtrB produces approximately 20 to 25 percent of the current or wildtype strains.<br />
<br />
<br />
[[Image: wt_mtrB.jpg|700px]]<br />
<br />
<br />
As shown in the graph above, the results of our experiment matched those in literature. We also observed that wildtype and mtrB strains have very different current production behavior, with wildtype producing current immediately after lactate injection, and mtrB producing current much more gradually. This difference in behavior can allow for more accurate differentiation of current production between wildtype and mtrB strains when we integrate it with electrical devices. We wish then build on these results and build inducible systems that will allow us to control the current production.<br />
<br />
===Co-Culture Experiment===<br />
<br />
''Overview: One possible system for achieving inducible current could be to couple a inducible-lacZ system in E. coli to current production by wildtype S. oneidensis MR1 through the conversion of lactose to lactate. This leaves open the possibility of using a pre-existing E. coli lacZ reporter or creating our own.<br />
''<br />
<br />
In addition to genetically engineering S. oneidensis MR-1 to respond to chemicals and heat, we also sought to take advantage of its natural metabolic pathway of breaking down lactate to produce current. From our wildtype versus mtrB knockout experiments, we found that feeding S. oneidensis MR-1 lactate led to significant current production almost instantly. In wildtype S. oneidensis MR-1, the current production would increase and stay elevated for a period of twelve hours. This result occurred consistently, thus we sought to use controlled lactate release as a way to control current output. <br />
<br />
We also knew that wildtype E. coli takes lactose and breaks down lactate. Thus, we hypothesized that it might be possible to couple E. coli’s lactate production with S. oneidensis MR-1’s lactate breakdown to produce current. In addition, a great deal of genetic engineering has already been done on E. coli and the up-regulation or down-regulation of its Lac operon. This means that instead of genetically modifying S. oneidensis MR-1 to respond to chemicals or heat, we can instead take advantage of the vast library of E. coli genetics and couple genetically engineered E. coli with wildtype S. oneidensis MR-1. <br />
<br />
In this experiment, we tested different combinations of wildtype E. coli MG1655, S. oneidensis MR-1, and Lac-operon knockout E. coli MC4100 as follows:<br />
<br />
1. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactose <br />
<br />
2. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
3. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactose<br />
<br />
4. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
5. wt S. oneidensis MR-1 + lactose neg. control<br />
<br />
6. wt E. coli (MG1655) + lactose neg. control<br />
<br />
7. Lac-operon knockout E. coli (MC4100) + lactose neg. control<br />
<br />
Based on previous experiments, we would expect current production in combinations 2 and 4 as they both have wildtype S. oneidensis MR-1 and receive lactate. In addition, however, we would expect combination 1 to also produce current. As described above, wt E. coli would break down lactose into lactate, and S. oneidensis MR-1 would break down lactate to produce current.<br />
<br />
[[ Image: picture 5.png | 800px ]]<br />
<br />
From the data above, we found that combination 1 did indeed produce current with a delay relative to the positive control. The delay can be attributed to the time it takes for E. coli to break down lactose into lactate, thus adding an extra step in the carbon source to current production pathway compared to our positive controls. These results are exciting in that they show a possibility of taking advantage of this cooperative effort to achieve inducible current.<br />
<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''S. oneidensis'' has laid the foundations for many different exciting avenues of further inquiry which look to take advantage of a bacteria-computer interface that combines the amazing sensitivity and adaptability of bacteria with the speed and analytical abilities of electricity and computers.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006 arsenic biosensor] developed by the University of Edinburgh iGEM 2006 team could be introduced into ''S. oneidensis'', allowing for the coupling of arsenic sensing to an electrical output, a form of a data which is easier to automate and transmit. This could be further extended to other chemical sensing systems, such as the [http://parts.mit.edu/igem07/index.php/Brown lead sensor] created by the Brown iGEM 2007 team and the [http://parts.mit.edu/igem07/index.php/MIT mercury sensor] made by the MIT iGEM 2007 team, resulting ultimately in an array of different strains of ''S. oneidensis'' which all respond to the presence of different chemicals with an electrical output that can be monitored by a computer. This could theoretically allow for the remote sensing and analysis of the chemical composition of an environment over time in a cost-effective manner, making it a tool with powerful public health applications, such as monitoring water quality.<br />
<br />
Another interesting direction would be the linking of the [http://parts.mit.edu/wiki/index.php/UT_Austin_2005 light-sensing system] developed by the UT Austin iGEM team with electrical output in ''S. oneidensis''. In response to variations in light, the amount of electricity produced by ''S. oneidensis'' would change. This would allow for the intriguing possibility of not only ''S. oneidensis'' conveying information to the computer, but also the computer responding to the ''S. oneidensis''. A simple example would be that in response to a chemical input, ''S. oneidensis'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''S. oneidensis'', modifying ''S. oneidensis'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers. We tried constructing this system over summer, but as the process requires making an EnvZ knockout strain of ''S. oneidensis'', we could not finish it. We did, however, make a few parts to facilitate future attempts.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''S. oneidensis''. Either of the systems described above could be pursued through an alternative alternative strategy of co-cultures. For instance, an array of ''E. coli'' which respond to different chemicals by breaking down lactose into lactate could be cultured with ''S. oneidensis''. In response to an increase in lactate, ''S. oneidensis'' would begin to produce higher levels of electricity. Co-cultures could also allow for more complex bacteria-computer interactions. This strategy could enable the coupling of almost any ''E. coli'' ability to electrical output.<br />
<br />
These future directions in which our research can be taken demonstrate some of the exciting possibilities of BACTRICITY!<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Linchinleehttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:53:15Z<p>Linchinlee: /* Wildtype vs. mtrB deficient S. oneidensis */</p>
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{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<br />
The metabolically versatile bacterium ''Shewanella oneidensis'' adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, ''S. oneidensis'' transfers electrons to an electrode of a microbial fuel cell. We sought to engineer ''S. oneidensis'' to report variations in environmental conditions through changes in current production. A previous study has shown that ''S. oneidensis'' mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.<br />
<br />
==Experimental overview==<br />
<br />
We attempted to develop three inducible systems for electrical current production in ''S. oneidensis''. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression in mtrB knock-out (KO) ''S. oneidensis'' can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach uses temperature-senstive cI which would allow for an increase in electrical output in response to heat. The third is a light-inducible system based on the 2005 UT Austin biological camera.<br />
<br />
Using the LacI system, we attempted to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we built, measuring the effects of IPTG on current production in wild type and mtrB KO ''S. oneidensis'' containing plasmids expressing inducible or constitutive mtrB.<br />
<br />
==Results==<br />
<br />
===Wildtype vs. mtrB deficient ''S. oneidensis''===<br />
''Overview: By genetically manipulating S. oneidensis, it has been found that noticeable differences of current production can be detected. We wish to control current production by introducing inducible systems in these mutant strains of S. oneidensis. These differences of current production can be detected by a computer, allowing biological systems and electrical devices to be integrated.''<br />
<br />
One important basis of our project lies in the discovery that by knocking out the mtrB gene, a current production gene, of S. oneidensis, noticeable differences of current production can be detected. Therefore, this allows the current production of S. oneidensis to act more than just an on/off switch.<br />
<br />
The first step of our project was then to test this hypothesis and also to understand the current production behavior of wildtype S. oneidensis and the mutant S. oneidensis (annotated as mtrB). Therefore, we ran a test with two chambers, one strain in each chamber, and allowed the current production to be measured for approximately one day. In literature, mtrB produces approximately 20 to 25 percent of the current or wildtype strains.<br />
<br />
<br />
[[Image: wt_mtrB.jpg|700px]]<br />
<br />
<br />
As shown in the graph above, the results of our experiment matched those in literature. We also observed that wildtype and mtrB strains have very different current production behavior, with wildtype producing current immediately after lactate injection, and mtrB producing current much more gradually. We wish then build on these results and build inducible systems that will allow us to control the current production.<br />
<br />
===Co-Culture Experiment===<br />
<br />
''Overview: One possible system for achieving inducible current could be to couple a inducible-lacZ system in E. coli to current production by wildtype S. oneidensis MR1 through the conversion of lactose to lactate. This leaves open the possibility of using a pre-existing E. coli lacZ reporter or creating our own.<br />
''<br />
<br />
In addition to genetically engineering S. oneidensis MR-1 to respond to chemicals and heat, we also sought to take advantage of its natural metabolic pathway of breaking down lactate to produce current. From our wildtype versus mtrB knockout experiments, we found that feeding S. oneidensis MR-1 lactate led to significant current production almost instantly. In wildtype S. oneidensis MR-1, the current production would increase and stay elevated for a period of twelve hours. This result occurred consistently, thus we sought to use controlled lactate release as a way to control current output. <br />
<br />
We also knew that wildtype E. coli takes lactose and breaks down lactate. Thus, we hypothesized that it might be possible to couple E. coli’s lactate production with S. oneidensis MR-1’s lactate breakdown to produce current. In addition, a great deal of genetic engineering has already been done on E. coli and the up-regulation or down-regulation of its Lac operon. This means that instead of genetically modifying S. oneidensis MR-1 to respond to chemicals or heat, we can instead take advantage of the vast library of E. coli genetics and couple genetically engineered E. coli with wildtype S. oneidensis MR-1. <br />
<br />
In this experiment, we tested different combinations of wildtype E. coli MG1655, S. oneidensis MR-1, and Lac-operon knockout E. coli MC4100 as follows:<br />
<br />
1. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactose <br />
<br />
2. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
3. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactose<br />
<br />
4. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
5. wt S. oneidensis MR-1 + lactose neg. control<br />
<br />
6. wt E. coli (MG1655) + lactose neg. control<br />
<br />
7. Lac-operon knockout E. coli (MC4100) + lactose neg. control<br />
<br />
Based on previous experiments, we would expect current production in combinations 2 and 4 as they both have wildtype S. oneidensis MR-1 and receive lactate. In addition, however, we would expect combination 1 to also produce current. As described above, wt E. coli would break down lactose into lactate, and S. oneidensis MR-1 would break down lactate to produce current.<br />
<br />
[[ Image: picture 5.png | 800px ]]<br />
<br />
From the data above, we found that combination 1 did indeed produce current with a delay relative to the positive control. The delay can be attributed to the time it takes for E. coli to break down lactose into lactate, thus adding an extra step in the carbon source to current production pathway compared to our positive controls. These results are exciting in that they show a possibility of taking advantage of this cooperative effort to achieve inducible current.<br />
<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''S. oneidensis'' has laid the foundations for many different exciting avenues of further inquiry which look to take advantage of a bacteria-computer interface that combines the amazing sensitivity and adaptability of bacteria with the speed and analytical abilities of electricity and computers.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006 arsenic biosensor] developed by the University of Edinburgh iGEM 2006 team could be introduced into ''S. oneidensis'', allowing for the coupling of arsenic sensing to an electrical output, a form of a data which is easier to automate and transmit. This could be further extended to other chemical sensing systems, such as the [http://parts.mit.edu/igem07/index.php/Brown| lead sensor] created by the Brown iGEM 2007 team and the [http://parts.mit.edu/igem07/index.php/MIT| mercury sensor] made by the MIT iGEM 2007 team, resulting ultimately in an array of different strains of ''S. oneidensis'' which all respond to the presence of different chemicals with an electrical output that can be monitored by a computer. This could theoretically allow for the remote sensing and analysis of the chemical composition of an environment over time in a cost-effective manner, making it a tool with powerful public health applications, such as monitoring water quality.<br />
<br />
Another interesting direction would be the linking of the [http://parts.mit.edu/wiki/index.php/UT_Austin_2005| light-sensing system] developed by the UT Austin iGEM team with electrical output in ''S. oneidensis''. In response to variations in light, the amount of electricity produced by ''S. oneidensis'' would change. This would allow for the intriguing possibility of not only ''S. oneidensis'' conveying information to the computer, but also the computer responding to the ''S. oneidensis''. A simple example would be that in response to a chemical input, ''S. oneidensis'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''S. oneidensis'', modifying ''S. oneidensis'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers. We tried constructing this system over summer, but as the process requires making an EnvZ knockout strain of ''S. oneidensis'', we could not finish it. We did, however, make a few parts to facilitate future attempts.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''S. oneidensis''. Either of the systems described above could be pursued through an alternative alternative strategy of co-cultures. For instance, an array of ''E. coli'' which respond to different chemicals by breaking down lactose into lactate could be cultured with ''S. oneidensis''. In response to an increase in lactate, ''S. oneidensis'' would begin to produce higher levels of electricity. Co-cultures could also allow for more complex bacteria-computer interactions. This strategy could enable the coupling of almost any ''E. coli'' ability to electrical output.<br />
<br />
These future directions in which our research can be taken demonstrate some of the exciting possibilities of BACTRICITY!<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Linchinleehttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:52:51Z<p>Linchinlee: /* Wildtype vs. mtrB deficient S. oneidensis */</p>
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==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<br />
The metabolically versatile bacterium ''Shewanella oneidensis'' adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, ''S. oneidensis'' transfers electrons to an electrode of a microbial fuel cell. We sought to engineer ''S. oneidensis'' to report variations in environmental conditions through changes in current production. A previous study has shown that ''S. oneidensis'' mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.<br />
<br />
==Experimental overview==<br />
<br />
We attempted to develop three inducible systems for electrical current production in ''S. oneidensis''. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression in mtrB knock-out (KO) ''S. oneidensis'' can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach uses temperature-senstive cI which would allow for an increase in electrical output in response to heat. The third is a light-inducible system based on the 2005 UT Austin biological camera.<br />
<br />
Using the LacI system, we attempted to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we built, measuring the effects of IPTG on current production in wild type and mtrB KO ''S. oneidensis'' containing plasmids expressing inducible or constitutive mtrB.<br />
<br />
==Results==<br />
<br />
===Wildtype vs. mtrB deficient ''S. oneidensis''===<br />
[[Image: wt_mtrB.jpg|700px]]<br />
''<br />
Overview: By genetically manipulating S. oneidensis, it has been found that noticeable differences of current production can be detected. We wish to control current production by introducing inducible systems in these mutant strains of S. oneidensis. These differences of current production can be detected by a computer, allowing biological systems and electrical devices to be integrated.''<br />
<br />
One important basis of our project lies in the discovery that by knocking out the mtrB gene, a current production gene, of S. oneidensis, noticeable differences of current production can be detected. Therefore, this allows the current production of S. oneidensis to act more than just an on/off switch.<br />
<br />
The first step of our project was then to test this hypothesis and also to understand the current production behavior of wildtype S. oneidensis and the mutant S. oneidensis (annotated as mtrB). Therefore, we ran a test with two chambers, one strain in each chamber, and allowed the current production to be measured for approximately one day. In literature, mtrB produces approximately 20 to 25 percent of the current or wildtype strains.<br />
<br />
===Co-Culture Experiment===<br />
<br />
''Overview: One possible system for achieving inducible current could be to couple a inducible-lacZ system in E. coli to current production by wildtype S. oneidensis MR1 through the conversion of lactose to lactate. This leaves open the possibility of using a pre-existing E. coli lacZ reporter or creating our own.<br />
''<br />
<br />
In addition to genetically engineering S. oneidensis MR-1 to respond to chemicals and heat, we also sought to take advantage of its natural metabolic pathway of breaking down lactate to produce current. From our wildtype versus mtrB knockout experiments, we found that feeding S. oneidensis MR-1 lactate led to significant current production almost instantly. In wildtype S. oneidensis MR-1, the current production would increase and stay elevated for a period of twelve hours. This result occurred consistently, thus we sought to use controlled lactate release as a way to control current output. <br />
<br />
We also knew that wildtype E. coli takes lactose and breaks down lactate. Thus, we hypothesized that it might be possible to couple E. coli’s lactate production with S. oneidensis MR-1’s lactate breakdown to produce current. In addition, a great deal of genetic engineering has already been done on E. coli and the up-regulation or down-regulation of its Lac operon. This means that instead of genetically modifying S. oneidensis MR-1 to respond to chemicals or heat, we can instead take advantage of the vast library of E. coli genetics and couple genetically engineered E. coli with wildtype S. oneidensis MR-1. <br />
<br />
In this experiment, we tested different combinations of wildtype E. coli MG1655, S. oneidensis MR-1, and Lac-operon knockout E. coli MC4100 as follows:<br />
<br />
1. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactose <br />
<br />
2. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
3. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactose<br />
<br />
4. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
5. wt S. oneidensis MR-1 + lactose neg. control<br />
<br />
6. wt E. coli (MG1655) + lactose neg. control<br />
<br />
7. Lac-operon knockout E. coli (MC4100) + lactose neg. control<br />
<br />
Based on previous experiments, we would expect current production in combinations 2 and 4 as they both have wildtype S. oneidensis MR-1 and receive lactate. In addition, however, we would expect combination 1 to also produce current. As described above, wt E. coli would break down lactose into lactate, and S. oneidensis MR-1 would break down lactate to produce current.<br />
<br />
[[ Image: picture 5.png | 800px ]]<br />
<br />
From the data above, we found that combination 1 did indeed produce current with a delay relative to the positive control. The delay can be attributed to the time it takes for E. coli to break down lactose into lactate, thus adding an extra step in the carbon source to current production pathway compared to our positive controls. These results are exciting in that they show a possibility of taking advantage of this cooperative effort to achieve inducible current.<br />
<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''S. oneidensis'' has laid the foundations for many different exciting avenues of further inquiry which look to take advantage of a bacteria-computer interface that combines the amazing sensitivity and adaptability of bacteria with the speed and analytical abilities of electricity and computers.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006 arsenic biosensor] developed by the University of Edinburgh iGEM 2006 team could be introduced into ''S. oneidensis'', allowing for the coupling of arsenic sensing to an electrical output, a form of a data which is easier to automate and transmit. This could be further extended to other chemical sensing systems, such as the [http://parts.mit.edu/igem07/index.php/Brown| lead sensor] created by the Brown iGEM 2007 team and the [http://parts.mit.edu/igem07/index.php/MIT| mercury sensor] made by the MIT iGEM 2007 team, resulting ultimately in an array of different strains of ''S. oneidensis'' which all respond to the presence of different chemicals with an electrical output that can be monitored by a computer. This could theoretically allow for the remote sensing and analysis of the chemical composition of an environment over time in a cost-effective manner, making it a tool with powerful public health applications, such as monitoring water quality.<br />
<br />
Another interesting direction would be the linking of the [http://parts.mit.edu/wiki/index.php/UT_Austin_2005| light-sensing system] developed by the UT Austin iGEM team with electrical output in ''S. oneidensis''. In response to variations in light, the amount of electricity produced by ''S. oneidensis'' would change. This would allow for the intriguing possibility of not only ''S. oneidensis'' conveying information to the computer, but also the computer responding to the ''S. oneidensis''. A simple example would be that in response to a chemical input, ''S. oneidensis'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''S. oneidensis'', modifying ''S. oneidensis'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers. We tried constructing this system over summer, but as the process requires making an EnvZ knockout strain of ''S. oneidensis'', we could not finish it. We did, however, make a few parts to facilitate future attempts.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''S. oneidensis''. Either of the systems described above could be pursued through an alternative alternative strategy of co-cultures. For instance, an array of ''E. coli'' which respond to different chemicals by breaking down lactose into lactate could be cultured with ''S. oneidensis''. In response to an increase in lactate, ''S. oneidensis'' would begin to produce higher levels of electricity. Co-cultures could also allow for more complex bacteria-computer interactions. This strategy could enable the coupling of almost any ''E. coli'' ability to electrical output.<br />
<br />
These future directions in which our research can be taken demonstrate some of the exciting possibilities of BACTRICITY!<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Linchinleehttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:25:04Z<p>Linchinlee: /* Co-Culture Experiment */</p>
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|<br />
==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<br />
The metabolically versatile bacterium ''Shewanella oneidensis'' adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, ''S. oneidensis'' transfers electrons to an electrode of a microbial fuel cell. We sought to engineer ''S. oneidensis'' to report variations in environmental conditions through changes in current production. A previous study has shown that ''S. oneidensis'' mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.<br />
<br />
==Experimental overview==<br />
<br />
We attempted to develope three inducible systems for electrical current production in ''S. oneidensis''. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression in mtrB knock-out (KO) ''S. oneidensis'' can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach uses temperature-senstive cI which would allow for an increase in electrical output in response to heat. The third is a light-inducible system based on the 2005 UT Austin biological camera.<br />
<br />
Using the LacI system, we attempted to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we built, measuring the effects of IPTG on current production in wild type and mtrB KO ''S. oneidensis'' containing plasmids expressing inducible or constitutive mtrB.<br />
<br />
==Results==<br />
<br />
===Wildtype vs. mtrB===<br />
[[Image: wt_mtrB.jpg|700px]]<br />
<br />
===Co-Culture Experiment===<br />
<br />
''Overview: One possible system for achieving inducible current could be to couple a inducible-lacZ system in E. coli to current production by wildtype S. oneidensis MR1 through the conversion of lactose to lactate. This leaves open the possibility of using a pre-existing E. coli lacZ reporter or creating our own.<br />
''<br />
<br />
In addition to genetically engineering S. oneidensis MR-1 to respond to chemicals and heat, we also sought to take advantage of its natural metabolic pathway of breaking down lactate to produce current. From our wildtype versus mtrB knockout experiments, we found that feeding S. oneidensis MR-1 lactate led to significant current production almost instantly. In wildtype S. oneidensis MR-1, the current production would increase and stay elevated for a period of twelve hours. This result occurred consistently, thus we sought to use controlled lactate release as a way to control current output. <br />
<br />
We also knew that wildtype E. coli takes lactose and breaks down lactate. Thus, we hypothesized that it might be possible to couple E. coli’s lactate production with S. oneidensis MR-1’s lactate breakdown to produce current. In addition, a great deal of genetic engineering has already been done on E. coli and the up-regulation or down-regulation of its Lac operon. This means that instead of genetically modifying S. oneidensis MR-1 to respond to chemicals or heat, we can instead take advantage of the vast library of E. coli genetics and couple genetically engineered E. coli with wildtype S. oneidensis MR-1. <br />
<br />
In this experiment, we tested different combinations of wildtype E. coli MG1655, S. oneidensis MR-1, and Lac-operon knockout E. coli MC4100 as follows:<br />
<br />
1. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactose <br />
<br />
2. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
3. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactose<br />
<br />
4. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
5. wt S. oneidensis MR-1 + lactose neg. control<br />
<br />
6. wt E. coli (MG1655) + lactose neg. control<br />
<br />
7. Lac-operon knockout E. coli (MC4100) + lactose neg. control<br />
<br />
Based on previous experiments, we would expect current production in combinations 2 and 4 as they both have wildtype S. oneidensis MR-1 and receive lactate. In addition, however, we would expect combination 1 to also produce current. As described above, wt E. coli would break down lactose into lactate, and S. oneidensis MR-1 would break down lactate to produce current.<br />
<br />
[[ Image: picture 5.png | 800px ]]<br />
<br />
From the data above, we found that combination 1 did indeed produce current with a delay relative to the positive control. The delay can be attributed to the time it takes for E. coli to break down lactose into lactate, thus adding an extra step in the carbon source to current production pathway compared to our positive controls. These results are exciting in that they show a possibility of taking advantage of this cooperative effort to achieve inducible current.<br />
<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''S. oneidensis'' has laid the foundations for many different exciting avenues of further inquiry which look to take advantage of a bacteria-computer interface that combines the amazing sensitivity and adaptability of bacteria with the speed and analytical abilities of electricity and computers.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006 arsenic biosensor] developed by the University of Edinburgh iGEM 2006 team could be introduced into ''S. oneidensis'', allowing for the coupling of arsenic sensing to an electrical output, a form of a data which is easier to automate and transmit. This could be further extended to other chemical sensing systems, resulting ultimately in an array of different strains ''S. oneidensis'' which all respond to the presence of different chemicals with an electrical output that can be monitored by a computer. This could theoretically allow for the remote sensing and analysis of the chemical composition of an environment over time.<br />
<br />
Another interesting direction would be the linking of the light-sensing system developed by the UT Austin iGEM team with electrical output in ''S. oneidensis''. In response to variations in light, the amount of electricity produced by ''S. oneidensis'' would change. This would allow for the intriguing possibility of not only ''S. oneidensis'' conveying information to the computer, but also the computer responding to the ''S. oneidensis''. A simple example would be that in response to a chemical input, ''S. oneidensis'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''S. oneidensis'', modifying ''S. oneidensis'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers. We tried constructing this system over summer, but as the process requires making an EnvZ knockout strain of ''S. oneidensis'', we could not finish it. We did, however, make a few parts to facilitate future attempts.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''S. oneidensis''. Either of the systems described above could be pursued through an alternative alternative strategy of co-cultures. For instance, an array of ''E. coli'' which respond to different chemicals by breaking down lactose into lactate could be cultured with ''S. oneidensis''. In response to an increase in lactate, ''S. oneidensis'' would begin to produce higher levels of electricity. Co-cultures could also allow for more complex bacteria-computer interactions. This strategy could enable the coupling of almost any ''E. coli'' ability to electrical output.<br />
<br />
These future directions in which our research can be taken demonstrate some of the exciting possibilities of BACTRICITY!<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Linchinleehttp://2008.igem.org/File:Lacinducible.jpgFile:Lacinducible.jpg2008-10-30T03:23:23Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:Wt_mtrB.jpgFile:Wt mtrB.jpg2008-10-30T02:51:19Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week3/WidgetryTeam:Harvard/Dailybook/Week3/Widgetry2008-10-30T02:10:28Z<p>Linchinlee: /* Lab */</p>
<hr />
<div>=Goals for this week=<br />
#Make Orianna's growth media<br />
#construct more chambers to run parallel tests<br />
#set up Keithley for multiple tests<br />
#grow mtrA, mtrB,wt, E. coli in liquid cultures<br />
#negative control E. coli in LB/lactate<br />
#standardize tests (OD, bubbling, etc)<br />
<br />
=Monday: June 7, 2008=<br />
<br />
* E. Coli Negative Control in LB <br />
<br />
[[Image:EColi_LB.JPG|600px]]<br />
<br />
[[Image:EColi_LB_O2.JPG|600px]]<br />
<br />
=Tuesday: June 8, 2008=<br />
<br />
* Constructed two new MFCs<br />
* grew mtrA, mtrB, wt, and E. coli in LB+lactate liquid cultures<br />
* Ran negative control with E. coli in NaPIPES media<br />
<br />
<br />
[[Image:EColi_NaPIPES.JPG|600px]]<br />
<br />
=Wednesday: June 9, 2008=<br />
<br />
* Made Orianna's growth media<br />
:: First followed recipe exactly: pH = 3.4<br />
::* Added NaOH to neutralize media<br />
:: Missing amino acids<br />
<br />
* Grew wt Shewie, mtrA, mtrB, and mtrC/omcA in Orianna's growth media <br />
** overnight cultures<br />
** '''RESULTS''': <br />
:: '''OD readings''':<br />
:: wt: -0.001<br />
:: mtrA: 0.001<br />
:: mtrB: 0.003<br />
:: mtrC/omcA: 0.002 <br />
:: '''CONCLUSION''':<br />
:: Orianna's growth media isn't working. Go see Colleen for possible sources of error. <br />
<br />
* OD readings from liquid cultures grown in LB + lactate growth media<br />
** wt: 2.489<br />
** mtrB: 1.711<br />
<br />
* Ran MFC overnight with mtrB and wt Shewie strains.<br />
** added strains first without lactate<br />
** waited until chambers became anaerobic (color-change) to add lactate<br />
** wt cell chamber denser than mtrB cell chamber<br />
** N2 stopped in wt chamber. turned off N2 in mtrB chamber to mirror this change<br />
[[Image:Mfc_wt_mtrB.png|600px]]<br />
<br />
* Experiment continued<br />
** replenished lactate in wt chamber<br />
** fluctuations may have resulted from air/N2 flow<br />
[[Image:Mfc_wt_mtrB_cont.png|600px]]<br />
<br />
=Thursday: June 10, 2008=<br />
<br />
*Continued running experiment from Wednesday<br />
*Purchased pipe cleaners, resistors<br />
*Made OGM with potassium PIPES<br />
<br />
=Friday: June 11, 2008=<br />
<br />
==Lab==<br />
*set up three chamber system<br />
*tested wt, mtrA, negative control:<br />
** resistances:<br />
*** wt:<br />
*** mtrA:<br />
*** none:<br />
[[Image:Mfc_overnight1.png|600px]]<br />
<br />
465 s: injected Shewie<br />
<br />
3500 s: injected Lactate & stopped N<sub>2</sub><br />
<br />
[[Image:Mfc_overnight2.png|600px]]<br />
<br />
* Nitrogen stopped bubbling somewhere between 70,000 & 80,000<br />
<br />
<br />
[[Image:Mfc_overnight3.png|600px]]<br />
<br />
'''Conclusion'''<br />
* Day 1, current production of wt and mtrA Shewie match that in literature<br />
** When wt peaks at around 275 microamps, mtrA Shewie is producing 25-50 microamps</div>Linchinleehttp://2008.igem.org/Team:Harvard/HardwareTeam:Harvard/Hardware2008-10-30T01:44:10Z<p>Linchinlee: /* Motivation */</p>
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=Fuel Cell Development=<br />
==Motivation==<br />
<br />
The broad goal of our project was to engineer s. Oneidensis to produce a detectable change in electric current in response to some environmental stimulus. In order to observe such a reaction, our first task was to design an environment capable of housing bacteria and measuring current production.<br />
<br />
==Solution - Microbial Fuel Cells==<br />
<br />
===Background===<br />
<br />
Microbial fuel cells (MFCs) are devices that use bacteria as the catalysts to oxidize organic and inorganic matter and generate current <ref></ref>. The principle behind these devices is to physically separate an oxidation reaction from a reduction reaction while providing a path for electrons to travel between them.<br />
<br />
===Context===<br />
[[Image:Chambers.jpg|600px]]<br />
<br />
==Design Goal==<br />
<br />
===Functional description===<br />
<br />
The final product is a complete system capable of introducing separate strains of bacteria to multiple different environments while simultaneously measuring and recording current readings from each. The experimenter specifies the number of bacteria/environment combinations to be run, as well as the initial conditions for each. Data collection and storage is automated, with a computer displaying live current readings and graphing historical current levels. The experimenter can change the conditions of any fuel cell throughout the course of the experiment without affecting other fuel cells. The fuel cells themselves are stand-alone, capable of being treated as individual circuit components.<br />
<br />
===Specifications===<br />
<br />
* automated<br />
Some experiments can last several days. Measurements must be automated to allow for overnight observation. <br />
<br />
* anaerobic/aerobic<br />
s. Odenisis only oxidizes substrates in anaerobic environments. The chambers housing the bacteria must be oxygen free and airtight. <br />
<br />
* sterile<br />
Fuel cells must be capable of being sterilized to prevent contamination<br />
<br />
* reproducible<br />
Individual fuel cells must be similar enough to produce consistent results.<br />
<br />
* accessible<br />
Experimenters must have access to the bacterial environment. <br />
<br />
<br />
==Approach==<br />
<br />
===Decomposition into components===<br />
<br />
Construction of the system was broken down into three distinct parts. The most important components were the fuel cells themselves. Once these devices were built, a measurement system was constructed to sample current readings from each simultaneously. Software was developed to orchestrate these readings, recording and displaying them in real time.<br />
<br />
===Component descriptions and approaches===<br />
<br />
====Fuel Cells====<br />
<br />
Fuel cells were constructed to provide an environment in which bacteria could live and produce electric current. After researching various microbial fuel cell designs, we concluded that a two chamber system would best suit our goals. <br />
<br />
The simple picture of our design was a hollow tube, sealed at both ends with endplates, and cut in half horizontally to allow for insertion of a semi-permeable membrane. The two halves would then be clamped back together to close the system. Ports needed to be drilled into the walls of the tube to allow for insertion of the anode and cathode, as well as to initially fill the sealed chambers with media and inject bacteria and food. Previous research suggested bubbling gasses into the chambers could improve performance. In the anode chamber, nitrogen was typically bubbled to purge the chamber of oxygen, keeping the bacteria in a controlled anaerobic environment. In the cathode chamber, air was typically bubbled to provide a continuous source of oxygen, ensuring an efficient reduction reaction. We decided to include ports for gas inlet and outlet as well.<br />
<br />
Materials for the chamber were then selected. The central tube and endplates were made from polycarbonate. This is a clear, hard plastic that is easily bonded to itself with silicone glue. It has the added benefit of allowing visual monitoring of the chambers, it is easily milled, and it can be autoclaved. Silicone was used in glue form to bond the tubes to their endplates, and it was used in solidified from to construct gaskets which sealed the two chambers together when clamped. The clamping mechanism involved drilling holes through the overlap of the endplates, inserting iron threaded rods down the length of the chambers, and fastening the ends of the rods with wing nuts. <br />
<br />
====Measurement====<br />
<br />
We required that our measurement system be automated and capable of monitoring multiple fuel cells at once. Our original goal was to measure and compare output current levels between separate fuel cell environments. We choose to use current as the measured variable since previous studies in s. Onedisis and mutants gave us reasonable expectation values of the current magnitudes we would observe. As these reported current levels were on the order of microamps for similarly sized systems, we decided to purchase a high throughput commercial digital multimeter (DMM) to achieve the desired resolution. Finding a multiplexed system with large quantities of current channels proved difficult, and we adapted our schematic to measure voltage and resistance and then calculate current.<br />
<br />
Our final product choice was the Keithley 2700 with a Keithley 7700 multiplexer accessory. This configuration provided 100 nA current resolution while allowing up to 20 devices to be sampled from at once. <br />
<br />
We choose to use 470 Ohms as our resistance connecting the anode and cathode of each fuel cell. This choice was based on several considerations. We measured the resistance path from the surface of an anode to the surface of a cathode and found that it ranged from 5-10 Ohms. Our resistance had to be large enough that voltage drops across this internal resistance did not significantly affect the measured voltage drop across our external resistor. We also measured the resistance of the path from anode to cathode through the fuel cells media and membrane. This resistance was highly variable but typically ranged from 15K to 25K. Our external resistance had to be low enough that the vast majority of freed electrons traveled externally rather than through this variable media/membrane path.<br />
<br />
====Software====<br />
<br />
The task of our software program was to coordinate measurements through the DMM, record data, and display live data in graphical format. Some members of our team had prior experience with LabVIEW, which we used to develop these routines. <br />
<br />
Working with the Keithley DMM, our program first samples resistances across each fuel cell's external resistor. It then switches the DMM measurement channel to voltage and enters a measurement loop. In each loop iteration, voltage samples are taken from all connected fuel cells and are divided by their previously measured resistances. This computed current is displayed in both graphical and tabular format, as well as stored in a .xls file. Current values are compared against a user specified threshold value, and a display indicates which chambers are over threshold. To avoid spikes, this function only returns true if a chamber's current level has been above threshold for the previous ten readings. An iteration ends by waiting for the next multiple of a user specified time. This ensures synchronous measurement intervals.<br />
<br />
* In LabVIEW, a block diagram is used to visually write c code. Our block diagram is shown here.<br />
[[Image:LV_block1.png |350px|left]] [[Image:LV_block2.png | 350px|]]<br />
<br />
<br />
* In Labview, an instrument panel is used to allow users to specify inputs and to display the status of program variables. Our instrument panel is shown here.<br />
[[Image:LV_vi1.png |350px| left]][[Image:LV_vi2.png | 350px]]<br />
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<hr />
<div>=Baseline Experiment for Chemical/Heat Inducible Systems=<br />
[[Image:bline1.jpg | 600px]]<br />
<br />
'''Baseline Chemical and Heat Test'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested wt and mtrB strains of Shewie with adding IPTG, Tet, and heating to 40C<br />
<br />
<br />
Results:<br />
<br />
-Current production was very low for all chambers<br />
<br />
:-Less than 3 microAmps even for wt Shewie which is nearly a hundred-fold lower than what we normally found<br />
<br />
-Chambers were not anaerobic<br />
<br />
-Need to redo this test</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week9/WidgetryTeam:Harvard/Dailybook/Week9/Widgetry2008-10-30T01:39:10Z<p>Linchinlee: /* Thursday: August 20, 2008 */</p>
<hr />
<div>=Friday: August 21, 2008=<br />
[[Image:bline1.jpg | 600px]]<br />
<br />
'''Baseline Chemical and Heat Test'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested wt and mtrB strains of Shewie with adding IPTG, Tet, and heating to 40C<br />
<br />
<br />
Results:<br />
<br />
-Current production was very low for all chambers<br />
<br />
:-Less than 3 microAmps even for wt Shewie which is nearly a hundred-fold lower than what we normally found<br />
<br />
-Chambers were not anaerobic<br />
<br />
-Need to redo this test</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week9/WidgetryTeam:Harvard/Dailybook/Week9/Widgetry2008-10-30T01:39:05Z<p>Linchinlee: /* Wednesday: August 19, 2008 */</p>
<hr />
<div>=Thursday: August 20, 2008=<br />
<br />
=Friday: August 21, 2008=<br />
[[Image:bline1.jpg | 600px]]<br />
<br />
'''Baseline Chemical and Heat Test'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested wt and mtrB strains of Shewie with adding IPTG, Tet, and heating to 40C<br />
<br />
<br />
Results:<br />
<br />
-Current production was very low for all chambers<br />
<br />
:-Less than 3 microAmps even for wt Shewie which is nearly a hundred-fold lower than what we normally found<br />
<br />
-Chambers were not anaerobic<br />
<br />
-Need to redo this test</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week9/WidgetryTeam:Harvard/Dailybook/Week9/Widgetry2008-10-30T01:38:59Z<p>Linchinlee: /* Tuesday: August 18, 2008 */</p>
<hr />
<div>=Wednesday: August 19, 2008=<br />
<br />
=Thursday: August 20, 2008=<br />
<br />
=Friday: August 21, 2008=<br />
[[Image:bline1.jpg | 600px]]<br />
<br />
'''Baseline Chemical and Heat Test'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested wt and mtrB strains of Shewie with adding IPTG, Tet, and heating to 40C<br />
<br />
<br />
Results:<br />
<br />
-Current production was very low for all chambers<br />
<br />
:-Less than 3 microAmps even for wt Shewie which is nearly a hundred-fold lower than what we normally found<br />
<br />
-Chambers were not anaerobic<br />
<br />
-Need to redo this test</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week9/WidgetryTeam:Harvard/Dailybook/Week9/Widgetry2008-10-30T01:38:51Z<p>Linchinlee: /* Monday: August 17, 2008 */</p>
<hr />
<div>=Tuesday: August 18, 2008=<br />
<br />
=Wednesday: August 19, 2008=<br />
<br />
=Thursday: August 20, 2008=<br />
<br />
=Friday: August 21, 2008=<br />
[[Image:bline1.jpg | 600px]]<br />
<br />
'''Baseline Chemical and Heat Test'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested wt and mtrB strains of Shewie with adding IPTG, Tet, and heating to 40C<br />
<br />
<br />
Results:<br />
<br />
-Current production was very low for all chambers<br />
<br />
:-Less than 3 microAmps even for wt Shewie which is nearly a hundred-fold lower than what we normally found<br />
<br />
-Chambers were not anaerobic<br />
<br />
-Need to redo this test</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week7/WidgetryTeam:Harvard/Dailybook/Week7/Widgetry2008-10-29T21:24:33Z<p>Linchinlee: /* Wednesday: August 6, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Shewanella and E. coli co-culture experiments<br />
** If Shewanella responds rapidly to lactate, and E. coli breaks down lactose into lactate, can we couple lactose breakdown by E. coli with current production by Shewanella?<br />
** Motivation:<br />
*** An inducible system in E. coli could be used to control Shewanella current production<br />
<br />
=Monday: August 4, 2008=<br />
<br />
== Co-culture experiment setup ==<br />
<br />
* Chambers 1 & 2: wt E. coli + wt Shewie + lactose<br />
* Chambers 4 &5 : Lac-operon knockout E. coli + wt Shewie + lactose<br />
<br />
'''Positive Controls'''<br />
* Chamber 3: wt E. coli + wt Shewie + lactate<br />
* Chamber 6: Lac-operon knockout E. coli + wt Shewie + lactate<br />
<br />
'''Negative Controls'''<br />
* Chamber 7: wt Shewie + lactose<br />
* Chamber 8: wt E. coli + lactose<br />
* Chamber 9: Lac-operon knockout E. coli + lactose<br />
<br />
=Tuesday: August 5, 2008=<br />
<br />
==Co-culture experiment results, day 1==<br />
<br />
:[[Image: Co-culture_part1.JPG | 700 px]]<br />
:* Notes:<br />
:** At t = 2000 s, all cells added<br />
:** At t = 80000 s, all injection done<br />
<br />
<br />
=== Zoomed-in graph of results, part 1 ===<br />
:[[Image: Co-culture_part1_zoomed.jpg | 700 px]]<br />
:* Notes:<br />
:** Zoomed-in on time since injections<br />
:** Positive controls omitted<br />
<br />
<br />
'''Preliminary Conclusions'''<br />
:* Noticeable difference in behavior between wt E. coli + Shewie and Lac-operon knockout + Shewie co-cultures<br />
:* Positive and negative controls show most likely no other variables involved<br />
:* Could be used to create tic-tac-toe which uses the difference in behavior to determine moves<br />
<br />
=Wednesday: August 6, 2008 to Friday: August 8, 2008=<br />
<br />
==Co-culture experiment results, day 1 & 2 ==<br />
<br />
=== wt E. coli vs. Lac-operon knockout E. coli ===<br />
: [[Image: Co-culture_part2_test.jpg | 700px]]<br />
:* Notes:<br />
:** At t = 260000 s, adjusted gas for Chamber 5<br />
:** At t = 340000 s, lactose injection for Chamber 1<br />
:** Initially, very noticeable difference between wt E. coli and Lac-operon knockout E. coli, but the current production of one of the E. coli chambers rises after a day, making the difference less distinguishable<br />
<br />
=== Results w/ positive controls ===<br />
:[[Image: Co-culture_part2_wpos.jpg | 700px]]<br />
:* Notes: <br />
:* At t = 340000 s, lactose injections for Chambers 1, 3, and 6<br />
:** Wanted to see if the positive controls would exhibit the same behavior when lactose injected instead of lactate<br />
<br />
<br />
=== Results (entire) ===<br />
:[[Image: Co-culture_part2.jpg | 700px]]<br />
<br />
:'''Concerns'''<br />
:* Chambers with co-cultures contained double the amount of cells of the controls<br />
:* Current production in non-positive co-culture chambers<br />
:** wt Shewie + lactose<br />
:** lac-operon knockout E. coli + Shewie + lactose<br />
:** lac-operon knockout E. coli + lactose<br />
<br />
:'''Conclusions'''<br />
:* Although behavior of wt E. coli + Shewie and Lac-operon knockout E. coli + Shewie co-cultures are not as distinguishable after a day, our focus can only be on the first day<br />
:* Will try the same experiment with DH5(alpha) and DH5(alpha)/pUC19 strains<br />
:* Need to look for inducible lacZ systems<br />
:** Light-repressible (RU1012 with plasmid) system?</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week7/WidgetryTeam:Harvard/Dailybook/Week7/Widgetry2008-10-29T21:24:10Z<p>Linchinlee: /* Friday: August 8, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Shewanella and E. coli co-culture experiments<br />
** If Shewanella responds rapidly to lactate, and E. coli breaks down lactose into lactate, can we couple lactose breakdown by E. coli with current production by Shewanella?<br />
** Motivation:<br />
*** An inducible system in E. coli could be used to control Shewanella current production<br />
<br />
=Monday: August 4, 2008=<br />
<br />
== Co-culture experiment setup ==<br />
<br />
* Chambers 1 & 2: wt E. coli + wt Shewie + lactose<br />
* Chambers 4 &5 : Lac-operon knockout E. coli + wt Shewie + lactose<br />
<br />
'''Positive Controls'''<br />
* Chamber 3: wt E. coli + wt Shewie + lactate<br />
* Chamber 6: Lac-operon knockout E. coli + wt Shewie + lactate<br />
<br />
'''Negative Controls'''<br />
* Chamber 7: wt Shewie + lactose<br />
* Chamber 8: wt E. coli + lactose<br />
* Chamber 9: Lac-operon knockout E. coli + lactose<br />
<br />
=Tuesday: August 5, 2008=<br />
<br />
==Co-culture experiment results, day 1==<br />
<br />
:[[Image: Co-culture_part1.JPG | 700 px]]<br />
:* Notes:<br />
:** At t = 2000 s, all cells added<br />
:** At t = 80000 s, all injection done<br />
<br />
<br />
=== Zoomed-in graph of results, part 1 ===<br />
:[[Image: Co-culture_part1_zoomed.jpg | 700 px]]<br />
:* Notes:<br />
:** Zoomed-in on time since injections<br />
:** Positive controls omitted<br />
<br />
<br />
'''Preliminary Conclusions'''<br />
:* Noticeable difference in behavior between wt E. coli + Shewie and Lac-operon knockout + Shewie co-cultures<br />
:* Positive and negative controls show most likely no other variables involved<br />
:* Could be used to create tic-tac-toe which uses the difference in behavior to determine moves<br />
<br />
=Wednesday: August 6, 2008=<br />
<br />
==Co-culture experiment results, day 1 & 2 ==<br />
<br />
=== wt E. coli vs. Lac-operon knockout E. coli ===<br />
: [[Image: Co-culture_part2_test.jpg | 700px]]<br />
:* Notes:<br />
:** At t = 260000 s, adjusted gas for Chamber 5<br />
:** At t = 340000 s, lactose injection for Chamber 1<br />
:** Initially, very noticeable difference between wt E. coli and Lac-operon knockout E. coli, but the current production of one of the E. coli chambers rises after a day, making the difference less distinguishable<br />
<br />
=== Results w/ positive controls ===<br />
:[[Image: Co-culture_part2_wpos.jpg | 700px]]<br />
:* Notes: <br />
:* At t = 340000 s, lactose injections for Chambers 1, 3, and 6<br />
:** Wanted to see if the positive controls would exhibit the same behavior when lactose injected instead of lactate<br />
<br />
<br />
=== Results (entire) ===<br />
:[[Image: Co-culture_part2.jpg | 700px]]<br />
<br />
:'''Concerns'''<br />
:* Chambers with co-cultures contained double the amount of cells of the controls<br />
:* Current production in non-positive co-culture chambers<br />
:** wt Shewie + lactose<br />
:** lac-operon knockout E. coli + Shewie + lactose<br />
:** lac-operon knockout E. coli + lactose<br />
<br />
:'''Conclusions'''<br />
:* Although behavior of wt E. coli + Shewie and Lac-operon knockout E. coli + Shewie co-cultures are not as distinguishable after a day, our focus can only be on the first day<br />
:* Will try the same experiment with DH5(alpha) and DH5(alpha)/pUC19 strains<br />
:* Need to look for inducible lacZ systems<br />
:** Light-repressible (RU1012 with plasmid) system?</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week7/WidgetryTeam:Harvard/Dailybook/Week7/Widgetry2008-10-29T21:24:01Z<p>Linchinlee: /* Thursday: August 7, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Shewanella and E. coli co-culture experiments<br />
** If Shewanella responds rapidly to lactate, and E. coli breaks down lactose into lactate, can we couple lactose breakdown by E. coli with current production by Shewanella?<br />
** Motivation:<br />
*** An inducible system in E. coli could be used to control Shewanella current production<br />
<br />
=Monday: August 4, 2008=<br />
<br />
== Co-culture experiment setup ==<br />
<br />
* Chambers 1 & 2: wt E. coli + wt Shewie + lactose<br />
* Chambers 4 &5 : Lac-operon knockout E. coli + wt Shewie + lactose<br />
<br />
'''Positive Controls'''<br />
* Chamber 3: wt E. coli + wt Shewie + lactate<br />
* Chamber 6: Lac-operon knockout E. coli + wt Shewie + lactate<br />
<br />
'''Negative Controls'''<br />
* Chamber 7: wt Shewie + lactose<br />
* Chamber 8: wt E. coli + lactose<br />
* Chamber 9: Lac-operon knockout E. coli + lactose<br />
<br />
=Tuesday: August 5, 2008=<br />
<br />
==Co-culture experiment results, day 1==<br />
<br />
:[[Image: Co-culture_part1.JPG | 700 px]]<br />
:* Notes:<br />
:** At t = 2000 s, all cells added<br />
:** At t = 80000 s, all injection done<br />
<br />
<br />
=== Zoomed-in graph of results, part 1 ===<br />
:[[Image: Co-culture_part1_zoomed.jpg | 700 px]]<br />
:* Notes:<br />
:** Zoomed-in on time since injections<br />
:** Positive controls omitted<br />
<br />
<br />
'''Preliminary Conclusions'''<br />
:* Noticeable difference in behavior between wt E. coli + Shewie and Lac-operon knockout + Shewie co-cultures<br />
:* Positive and negative controls show most likely no other variables involved<br />
:* Could be used to create tic-tac-toe which uses the difference in behavior to determine moves<br />
<br />
=Wednesday: August 6, 2008=<br />
<br />
==Co-culture experiment results, day 1 & 2 ==<br />
<br />
=== wt E. coli vs. Lac-operon knockout E. coli ===<br />
: [[Image: Co-culture_part2_test.jpg | 700px]]<br />
:* Notes:<br />
:** At t = 260000 s, adjusted gas for Chamber 5<br />
:** At t = 340000 s, lactose injection for Chamber 1<br />
:** Initially, very noticeable difference between wt E. coli and Lac-operon knockout E. coli, but the current production of one of the E. coli chambers rises after a day, making the difference less distinguishable<br />
<br />
=== Results w/ positive controls ===<br />
:[[Image: Co-culture_part2_wpos.jpg | 700px]]<br />
:* Notes: <br />
:* At t = 340000 s, lactose injections for Chambers 1, 3, and 6<br />
:** Wanted to see if the positive controls would exhibit the same behavior when lactose injected instead of lactate<br />
<br />
<br />
=== Results (entire) ===<br />
:[[Image: Co-culture_part2.jpg | 700px]]<br />
<br />
:'''Concerns'''<br />
:* Chambers with co-cultures contained double the amount of cells of the controls<br />
:* Current production in non-positive co-culture chambers<br />
:** wt Shewie + lactose<br />
:** lac-operon knockout E. coli + Shewie + lactose<br />
:** lac-operon knockout E. coli + lactose<br />
<br />
:'''Conclusions'''<br />
:* Although behavior of wt E. coli + Shewie and Lac-operon knockout E. coli + Shewie co-cultures are not as distinguishable after a day, our focus can only be on the first day<br />
:* Will try the same experiment with DH5(alpha) and DH5(alpha)/pUC19 strains<br />
:* Need to look for inducible lacZ systems<br />
:** Light-repressible (RU1012 with plasmid) system?<br />
<br />
<br />
<br />
=Friday: August 8, 2008=</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-29T21:22:07Z<p>Linchinlee: /* First Nine-chamber Test */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
** Use argon instead of nitrogen on anode side<br />
<br />
=First Nine-chamber Test=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
** Reused cathodes, but replaced Nafion membrane and anode<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production<br />
<br />
:-Therefore, amount of gas bubbling directly affects current production<br />
<br />
- Possible to use this as direct test for computer interface</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-29T21:21:08Z<p>Linchinlee: /* First Nine-chamber Test */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
** Use argon instead of nitrogen on anode side<br />
<br />
=First Nine-chamber Test=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
** Reused cathodes, but replaced Nafion membrane and anode<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production<br />
<br />
- Possible to use this as direct test for computer interface</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-29T21:20:18Z<p>Linchinlee: /* Friday: August 1, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
** Use argon instead of nitrogen on anode side<br />
<br />
=First Nine-chamber Test=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
** Reused cathodes, but replaced Nafion membrane and anode<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-29T21:20:05Z<p>Linchinlee: /* Thursday: July 31, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
** Use argon instead of nitrogen on anode side<br />
<br />
=First Nine-chamber Test=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
** Reused cathodes, but replaced Nafion membrane and anode<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=Friday: August 1, 2008=<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-29T21:19:56Z<p>Linchinlee: /* Wednesday: July 30, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
** Use argon instead of nitrogen on anode side<br />
<br />
=First Nine-chamber Test=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
** Reused cathodes, but replaced Nafion membrane and anode<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
<br />
<br />
<br />
<br />
=Thursday: July 31, 2008=<br />
=Friday: August 1, 2008=<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-29T21:19:48Z<p>Linchinlee: /* Tuesday: July 29, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
** Use argon instead of nitrogen on anode side<br />
<br />
=First Nine-chamber Test=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
** Reused cathodes, but replaced Nafion membrane and anode<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
<br />
<br />
=Wednesday: July 30, 2008=<br />
=Thursday: July 31, 2008=<br />
=Friday: August 1, 2008=<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-29T21:19:39Z<p>Linchinlee: /* Monday: July 28, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
** Use argon instead of nitrogen on anode side<br />
<br />
=First Nine-chamber Test=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
** Reused cathodes, but replaced Nafion membrane and anode<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
=Tuesday: July 29, 2008=<br />
=Wednesday: July 30, 2008=<br />
=Thursday: July 31, 2008=<br />
=Friday: August 1, 2008=<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week5/WidgetryTeam:Harvard/Dailybook/Week5/Widgetry2008-10-29T19:22:27Z<p>Linchinlee: /* Monday: July 21, 2008 */</p>
<hr />
<div>= Goals for the Week =<br />
* Last week, had experiment with no current at all<br />
** Hypothesized that cells were dead?<br />
** Run test with 1 day vs. 2 day old Shewie to determine if there is an effect<br />
<br />
<br />
<br />
=Tuesday: July 22, 2008=<br />
*started mtrB and wt cultures<br />
** mtrB culture to be used as 2-day culture on Thursday<br />
*troubleshoot LabVIEW<br />
** Resistances were measured to be different as expected in the past experiment<br />
** Some as low as 160 ohms<br />
** Perhaps the resistance of the chambers are less than we expected?<br />
<br />
=Wednesday: July 23, 2008=<br />
* Started another mtrB culture<br />
** To be used as 1 day culture in experiment on Thursday<br />
* LabVIEW<br />
** Determined that resistance should be measured before connecting to the fuel cells<br />
** Resistance of chambers is the same as what we measured initially<br />
<br />
=Thursday: July 24, 2008=<br />
* mtrB 1 day vs. 2 day experiment<br />
** 6 chambers<br />
*** 3 chambers with 1-day old mtrB<br />
*** 3 chambers with 2-day old mtrB<br />
*** Injected lactate after the initial current spike came down<br />
** Hypothesis<br />
***2-day cultures will have lower (if any) current production<br />
<br />
=Friday: July 25, 2008=<br />
[[Image:mtrb_1dv2d.jpg | 600px]]<br />
<br />
'''mtrB: Overnight versus 2 Day Liquid Cultures'''<br />
<br />
<br />
What we did:<br />
<br />
-Ran 1-day old mtrB versus 2-day old mtrB<br />
<br />
<br />
Results:<br />
<br />
-The shapes of the curves were all very similar. <br />
<br />
-Overnight cultures had slightly higher current production than 2-day old cultures.</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-28T21:59:11Z<p>Linchinlee: /* Goals for this week */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
** Use argon instead of nitrogen on anode side<br />
<br />
=Monday: July 28, 2008=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
** Reused cathodes, but replaced Nafion membrane and anode<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
=Tuesday: July 29, 2008=<br />
=Wednesday: July 30, 2008=<br />
=Thursday: July 31, 2008=<br />
=Friday: August 1, 2008=<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-28T21:58:11Z<p>Linchinlee: /* Monday: July 28, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
<br />
=Monday: July 28, 2008=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
** Reused cathodes, but replaced Nafion membrane and anode<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
=Tuesday: July 29, 2008=<br />
=Wednesday: July 30, 2008=<br />
=Thursday: July 31, 2008=<br />
=Friday: August 1, 2008=<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-28T21:57:17Z<p>Linchinlee: /* Monday: July 28, 2008 */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
<br />
=Monday: July 28, 2008=<br />
* Clean and re-build fuel cells in preparation for experiment<br />
* Placed built fuel cells under UV light to prevent contamination<br />
<br />
=Tuesday: July 29, 2008=<br />
=Wednesday: July 30, 2008=<br />
=Thursday: July 31, 2008=<br />
=Friday: August 1, 2008=<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week6/WidgetryTeam:Harvard/Dailybook/Week6/Widgetry2008-10-28T21:56:19Z<p>Linchinlee: /* Goals for this week */</p>
<hr />
<div>=Goals for this week=<br />
* Finish building remainder of chambers to bring us up to a total of 9 working fuel cells<br />
* Configure Kiethley/LabVIEW to support measurement from 9 fuel cells<br />
* Run test with 9 identical wt chambers to test for reproducibility of results<br />
<br />
=Monday: July 28, 2008=<br />
=Tuesday: July 29, 2008=<br />
=Wednesday: July 30, 2008=<br />
=Thursday: July 31, 2008=<br />
=Friday: August 1, 2008=<br />
[[Image:shaking.jpg | 600px]]<br />
<br />
'''Variance Test with Shaking'''<br />
<br />
<br />
What we did:<br />
<br />
-Tested how much variance existed among chambers by running 9 parallel chambers with wt Shewie<br />
<br />
-Switched nitrogen gas with argon gas on the anode side<br />
<br />
-Shook chamber 1 manually to see the effect of shaking on current production<br />
<br />
<br />
Results:<br />
<br />
-Current production range was between 50-75 microAmps<br />
<br />
-Red line (wt w/ shaking) went up to 500 microAmps when shaking vigorously<br />
<br />
:-Shows that shaking directly affects current production</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week5/WidgetryTeam:Harvard/Dailybook/Week5/Widgetry2008-10-28T21:53:41Z<p>Linchinlee: /* Thursday: July 24, 2008 */</p>
<hr />
<div>= Goals for the Week =<br />
* Last week, had experiment with no current at all<br />
** Hypothesized that cells were dead?<br />
** Run test with 1 day vs. 2 day old Shewie to determine if there is an effect<br />
<br />
=Monday: July 21, 2008=<br />
*Lab meeting<br />
*Cleaned chambers<br />
<br />
=Tuesday: July 22, 2008=<br />
*started mtrB and wt cultures<br />
** mtrB culture to be used as 2-day culture on Thursday<br />
*troubleshoot LabVIEW<br />
** Resistances were measured to be different as expected in the past experiment<br />
** Some as low as 160 ohms<br />
** Perhaps the resistance of the chambers are less than we expected?<br />
<br />
=Wednesday: July 23, 2008=<br />
* Started another mtrB culture<br />
** To be used as 1 day culture in experiment on Thursday<br />
* LabVIEW<br />
** Determined that resistance should be measured before connecting to the fuel cells<br />
** Resistance of chambers is the same as what we measured initially<br />
<br />
=Thursday: July 24, 2008=<br />
* mtrB 1 day vs. 2 day experiment<br />
** 6 chambers<br />
*** 3 chambers with 1-day old mtrB<br />
*** 3 chambers with 2-day old mtrB<br />
*** Injected lactate after the initial current spike came down<br />
** Hypothesis<br />
***2-day cultures will have lower (if any) current production<br />
<br />
=Friday: July 25, 2008=<br />
[[Image:mtrb_1dv2d.jpg | 600px]]<br />
<br />
'''mtrB: Overnight versus 2 Day Liquid Cultures'''<br />
<br />
<br />
What we did:<br />
<br />
-Ran 1-day old mtrB versus 2-day old mtrB<br />
<br />
<br />
Results:<br />
<br />
-The shapes of the curves were all very similar. <br />
<br />
-Overnight cultures had slightly higher current production than 2-day old cultures.</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week5/WidgetryTeam:Harvard/Dailybook/Week5/Widgetry2008-10-28T21:53:29Z<p>Linchinlee: /* Thursday: July 24, 2008 */</p>
<hr />
<div>= Goals for the Week =<br />
* Last week, had experiment with no current at all<br />
** Hypothesized that cells were dead?<br />
** Run test with 1 day vs. 2 day old Shewie to determine if there is an effect<br />
<br />
=Monday: July 21, 2008=<br />
*Lab meeting<br />
*Cleaned chambers<br />
<br />
=Tuesday: July 22, 2008=<br />
*started mtrB and wt cultures<br />
** mtrB culture to be used as 2-day culture on Thursday<br />
*troubleshoot LabVIEW<br />
** Resistances were measured to be different as expected in the past experiment<br />
** Some as low as 160 ohms<br />
** Perhaps the resistance of the chambers are less than we expected?<br />
<br />
=Wednesday: July 23, 2008=<br />
* Started another mtrB culture<br />
** To be used as 1 day culture in experiment on Thursday<br />
* LabVIEW<br />
** Determined that resistance should be measured before connecting to the fuel cells<br />
** Resistance of chambers is the same as what we measured initially<br />
<br />
=Thursday: July 24, 2008=<br />
* mtrB 1 day vs. 2 day experiment<br />
** 6 chambers<br />
*** 3 chambers with 1-day old mtrB<br />
*** 3 chambers with 2-day old mtrB<br />
*** Injected lactate after the initial current spike came down<br />
** Hypothesis: 2-day cultures will have lower (if any) current production<br />
<br />
=Friday: July 25, 2008=<br />
[[Image:mtrb_1dv2d.jpg | 600px]]<br />
<br />
'''mtrB: Overnight versus 2 Day Liquid Cultures'''<br />
<br />
<br />
What we did:<br />
<br />
-Ran 1-day old mtrB versus 2-day old mtrB<br />
<br />
<br />
Results:<br />
<br />
-The shapes of the curves were all very similar. <br />
<br />
-Overnight cultures had slightly higher current production than 2-day old cultures.</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week5/WidgetryTeam:Harvard/Dailybook/Week5/Widgetry2008-10-28T21:52:49Z<p>Linchinlee: /* Thursday: July 24, 2008 */</p>
<hr />
<div>= Goals for the Week =<br />
* Last week, had experiment with no current at all<br />
** Hypothesized that cells were dead?<br />
** Run test with 1 day vs. 2 day old Shewie to determine if there is an effect<br />
<br />
=Monday: July 21, 2008=<br />
*Lab meeting<br />
*Cleaned chambers<br />
<br />
=Tuesday: July 22, 2008=<br />
*started mtrB and wt cultures<br />
** mtrB culture to be used as 2-day culture on Thursday<br />
*troubleshoot LabVIEW<br />
** Resistances were measured to be different as expected in the past experiment<br />
** Some as low as 160 ohms<br />
** Perhaps the resistance of the chambers are less than we expected?<br />
<br />
=Wednesday: July 23, 2008=<br />
* Started another mtrB culture<br />
** To be used as 1 day culture in experiment on Thursday<br />
* LabVIEW<br />
** Determined that resistance should be measured before connecting to the fuel cells<br />
** Resistance of chambers is the same as what we measured initially<br />
<br />
=Thursday: July 24, 2008=<br />
* mtrB 1 day vs. 2 day experiment<br />
** 6 chambers<br />
*** 3 chambers with 1-day old mtrB<br />
*** 3 chambers with 2-day old mtrB<br />
*** Injected lactate after the initial current spike came down<br />
<br />
=Friday: July 25, 2008=<br />
[[Image:mtrb_1dv2d.jpg | 600px]]<br />
<br />
'''mtrB: Overnight versus 2 Day Liquid Cultures'''<br />
<br />
<br />
What we did:<br />
<br />
-Ran 1-day old mtrB versus 2-day old mtrB<br />
<br />
<br />
Results:<br />
<br />
-The shapes of the curves were all very similar. <br />
<br />
-Overnight cultures had slightly higher current production than 2-day old cultures.</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week5/WidgetryTeam:Harvard/Dailybook/Week5/Widgetry2008-10-28T21:51:44Z<p>Linchinlee: /* Wednesday: July 23, 2008 */</p>
<hr />
<div>= Goals for the Week =<br />
* Last week, had experiment with no current at all<br />
** Hypothesized that cells were dead?<br />
** Run test with 1 day vs. 2 day old Shewie to determine if there is an effect<br />
<br />
=Monday: July 21, 2008=<br />
*Lab meeting<br />
*Cleaned chambers<br />
<br />
=Tuesday: July 22, 2008=<br />
*started mtrB and wt cultures<br />
** mtrB culture to be used as 2-day culture on Thursday<br />
*troubleshoot LabVIEW<br />
** Resistances were measured to be different as expected in the past experiment<br />
** Some as low as 160 ohms<br />
** Perhaps the resistance of the chambers are less than we expected?<br />
<br />
=Wednesday: July 23, 2008=<br />
* Started another mtrB culture<br />
** To be used as 1 day culture in experiment on Thursday<br />
* LabVIEW<br />
** Determined that resistance should be measured before connecting to the fuel cells<br />
** Resistance of chambers is the same as what we measured initially<br />
<br />
=Thursday: July 24, 2008=<br />
=Friday: July 25, 2008=<br />
[[Image:mtrb_1dv2d.jpg | 600px]]<br />
<br />
'''mtrB: Overnight versus 2 Day Liquid Cultures'''<br />
<br />
<br />
What we did:<br />
<br />
-Ran 1-day old mtrB versus 2-day old mtrB<br />
<br />
<br />
Results:<br />
<br />
-The shapes of the curves were all very similar. <br />
<br />
-Overnight cultures had slightly higher current production than 2-day old cultures.</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week5/WidgetryTeam:Harvard/Dailybook/Week5/Widgetry2008-10-28T21:49:59Z<p>Linchinlee: /* Tuesday: July 22, 2008 */</p>
<hr />
<div>= Goals for the Week =<br />
* Last week, had experiment with no current at all<br />
** Hypothesized that cells were dead?<br />
** Run test with 1 day vs. 2 day old Shewie to determine if there is an effect<br />
<br />
=Monday: July 21, 2008=<br />
*Lab meeting<br />
*Cleaned chambers<br />
<br />
=Tuesday: July 22, 2008=<br />
*started mtrB and wt cultures<br />
** mtrB culture to be used as 2-day culture on Thursday<br />
*troubleshoot LabVIEW<br />
** Resistances were measured to be different as expected in the past experiment<br />
** Some as low as 160 ohms<br />
** Perhaps the resistance of the chambers are less than we expected?<br />
<br />
=Wednesday: July 23, 2008=<br />
=Thursday: July 24, 2008=<br />
=Friday: July 25, 2008=<br />
[[Image:mtrb_1dv2d.jpg | 600px]]<br />
<br />
'''mtrB: Overnight versus 2 Day Liquid Cultures'''<br />
<br />
<br />
What we did:<br />
<br />
-Ran 1-day old mtrB versus 2-day old mtrB<br />
<br />
<br />
Results:<br />
<br />
-The shapes of the curves were all very similar. <br />
<br />
-Overnight cultures had slightly higher current production than 2-day old cultures.</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week5/WidgetryTeam:Harvard/Dailybook/Week5/Widgetry2008-10-28T21:48:26Z<p>Linchinlee: /* Tuesday: July 22, 2008 */</p>
<hr />
<div>= Goals for the Week =<br />
* Last week, had experiment with no current at all<br />
** Hypothesized that cells were dead?<br />
** Run test with 1 day vs. 2 day old Shewie to determine if there is an effect<br />
<br />
=Monday: July 21, 2008=<br />
*Lab meeting<br />
*Cleaned chambers<br />
<br />
=Tuesday: July 22, 2008=<br />
*started mtrB and wt cultures<br />
** mtrB culture to be used as 2-day culture on Thursday<br />
*troubleshoot LabVIEW<br />
<br />
=Wednesday: July 23, 2008=<br />
=Thursday: July 24, 2008=<br />
=Friday: July 25, 2008=<br />
[[Image:mtrb_1dv2d.jpg | 600px]]<br />
<br />
'''mtrB: Overnight versus 2 Day Liquid Cultures'''<br />
<br />
<br />
What we did:<br />
<br />
-Ran 1-day old mtrB versus 2-day old mtrB<br />
<br />
<br />
Results:<br />
<br />
-The shapes of the curves were all very similar. <br />
<br />
-Overnight cultures had slightly higher current production than 2-day old cultures.</div>Linchinleehttp://2008.igem.org/Team:Harvard/Dailybook/Week5/WidgetryTeam:Harvard/Dailybook/Week5/Widgetry2008-10-28T21:45:40Z<p>Linchinlee: /* Monday: July 21, 2008 */</p>
<hr />
<div>= Goals for the Week =<br />
* Last week, had experiment with no current at all<br />
** Hypothesized that cells were dead?<br />
** Run test with 1 day vs. 2 day old Shewie to determine if there is an effect<br />
<br />
=Monday: July 21, 2008=<br />
*Lab meeting<br />
*Cleaned chambers<br />
<br />
=Tuesday: July 22, 2008=<br />
*started mtrB and wt cultures<br />
*troubleshoot LabVIEW<br />
<br />
=Wednesday: July 23, 2008=<br />
=Thursday: July 24, 2008=<br />
=Friday: July 25, 2008=<br />
[[Image:mtrb_1dv2d.jpg | 600px]]<br />
<br />
'''mtrB: Overnight versus 2 Day Liquid Cultures'''<br />
<br />
<br />
What we did:<br />
<br />
-Ran 1-day old mtrB versus 2-day old mtrB<br />
<br />
<br />
Results:<br />
<br />
-The shapes of the curves were all very similar. <br />
<br />
-Overnight cultures had slightly higher current production than 2-day old cultures.</div>Linchinleehttp://2008.igem.org/File:Chambers.JPGFile:Chambers.JPG2008-10-28T17:56:50Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:Wt-mtrB-bubbling.pngFile:Wt-mtrB-bubbling.png2008-10-28T17:55:48Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:Mfc_3xwt.pngFile:Mfc 3xwt.png2008-10-28T17:55:41Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:7-18-08.pngFile:7-18-08.png2008-10-28T17:55:34Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:ShewieCurrentTest2.jpgFile:ShewieCurrentTest2.jpg2008-10-28T17:54:19Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:ShewieCurrentProduction2.jpgFile:ShewieCurrentProduction2.jpg2008-10-28T17:54:10Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:ShewieCurrentProduction1.jpgFile:ShewieCurrentProduction1.jpg2008-10-28T17:54:04Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:MtrA.jpgFile:MtrA.jpg2008-10-28T17:53:57Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:Mfc_resazurin.JPGFile:Mfc resazurin.JPG2008-10-28T17:53:51Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:Mfc_happykids.JPGFile:Mfc happykids.JPG2008-10-28T17:53:48Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinleehttp://2008.igem.org/File:Mfc_current4.JPGFile:Mfc current4.JPG2008-10-28T17:53:06Z<p>Linchinlee: </p>
<hr />
<div></div>Linchinlee