http://2008.igem.org/wiki/index.php?title=Special:Contributions/Lauren&feed=atom&limit=50&target=Lauren&year=&month=2008.igem.org - User contributions [en]2024-03-28T14:02:57ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:Harvard/ShewieTeam:Harvard/Shewie2008-10-30T03:55:08Z<p>Lauren: /* Molecular Biology with Shewanella oneidensis */</p>
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=Organism=<br />
Most of our work this summer is founded upon the diverse metabolism of the bacterium ''Shewanella oneidensis MR-1''. Throughout the summer, we came to better understand how to work with this organism, and we hope our findings will help establish ''S. oneidensis'' as a interesting chasis for synthetic biology.<br />
==So, who is this "Shewie"?==<br />
<br />
This summer we worked with ''Shewanella oneidensis MR-1'', a gram-negative facultative anaerobe (Myers and Myers 1997). Under anaerobic conditions, it reduces a number of electron acceptors such as MN(IV). This ability can be harnessed by microbial fuel cells (MFC) to produce an electric current (Bretschger et al. 2007). When the bacteria are grown anaerobically in the anode chamber of an MFC, they release electrons onto the electrode, creating an electrical current. These diverse respiratory capabilities require a complex electron transport systems, including 39 c-type cytochromes (Heidelberg et al. 2002). These characteristics of ''S. oneidensis MR-1'' make it an important organism for toxin-reduction based bioremediation and biotechnology applications.<br />
<br />
==Molecular Biology with <i>Shewanella oneidensis</i>==<br />
<br />
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The ''Shewanella oneidensis MR-1'' genome was sequenced in 2002, greatly increasing its usefulness as a model organism. It was found that it had a 4,969,803 base pair circular chromosome and a 161,613 base pair plasmid (Heidelberg et al. 2002). When cloning in ''S. oneidensis MR-1'', it has also been shown that plasmids with p15A origins replicate freely, whereas plasmids with a pMB1 origin of replication do not (Myers and Myers 1997). We further found that the pSC101* origin from Lutz and Bujard (2007) and the CloDF3 origin on the [http://www.emdbiosciences.com/html/NVG/DuetTable.html pCDF-Duet vector] from Novagen work in ''S. oneidensis''. However, they are not pir+, so the R6K pir+ dependent origin does not work for them. ''S. oneidensis MR-1'' grows at 30 ºC, can be electroporated (see protocol in our [[Team:Harvard/GenProtocols| Notebook]]) and forms round orange pink colonies on plates. It is resistant to ampicillin, but other resistance markers, such as gentamycin and spectomycin, can be used (Saffarini). Together, these characteristics make ''S. oneidensis MR-1'' a genetically tractable organism good for exploring the possibilities of regulated bacterial electrical output.<br />
|<div style="text-indent:0pt;color:black">[[Image:Shewanella colonies growing on plate.JPG|thumb|300px|''S. oneidensis MR-1'' colonies from a transformation]]</div><br />
|-<br />
|}<br />
<br />
==Online resources for working with ''S. oneidensis''==<br />
-[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=211586&aa=11&style=N| Codon usage table]<br />
<br />
-[http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=gsp| Annotated genome, with information on RBS, terminators, protein domains, gene ontology, etc]<br />
<br />
-[http://archaea.ucsc.edu/cgi-bin/hgPcr?org=Shewanella+oneidensis&db=shewOnei&hgsid=159191| ''In silico'' PCR of ''S. oneidensis'' genome]<br />
<br />
==Chassis and the Registry==<br />
To facilitate easy manipulation in different organisms, it may be advantageous for the Registry to standardize a chassis specification sheet. Below, we provide a quick summary of ''S. oneidensis MR-1'' following what we think may be a suitable documentation format. Since iGEM teams frequently work with species other than ''E. coli'', if only to clone some interesting gene product, a set of such sheets could be built up to facilitate synthetic biology in a more diverse set of organisms. A [https://static.igem.org/mediawiki/2008/7/7f/Shewanellachassis.pdf PDF version] with links is available.<br />
[[Image:Chassis.png|720px|center]]<br />
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|}</div>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:54:19Z<p>Lauren: /* Future directions */</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 />
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>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:53:48Z<p>Lauren: /* Future directions */</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 />
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>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:52:39Z<p>Lauren: /* Future directions */</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 />
===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>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:50:21Z<p>Lauren: /* Future directions */</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 />
[[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, 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 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>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:45:11Z<p>Lauren: /* The Genetic Circuitry */</p>
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=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
<br />
We were able to create three such inducible systems:<br />
<br />
[[Team:Harvard/Parts/LacI| Lactose inducible system]]<br />
<br />
[[Team:Harvard/Parts/Other| Tetracycline inducible system]]<br />
<br />
[[Team:Harvard/Parts/Tempsenseci| Heat inducible system]]</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:44:37Z<p>Lauren: /* The Genetic Circuitry */</p>
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| align="center" style="background:#c4dbea"|<br />
<html><a href = "https://2008.igem.org/Team:Harvard"><img src="https://static.igem.org/mediawiki/2008/b/b9/Harvard_logo.png"></a></html><br />
<br><br />
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{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
<br />
We were able to create three such inducible systems:<br />
<br />
[[Team:Harvard/GenProtocols| Notebook]]<br />
<br />
[[Harvard/Parts/LacI| Lactose inducible system]]<br />
<br />
[[Team:Harvard/Parts/Other| Tetracycline inducible system]]<br />
<br />
[https://2008.igem.org/Team:Harvard/Parts/Tempsenseci| Heat inducible system]</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:44:22Z<p>Lauren: /* The Genetic Circuitry */</p>
<hr />
<div>__NOTOC__<br />
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<br><br />
<br />
{{Template:Main}}<br />
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{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
<br />
We were able to create three such inducible systems:<br />
<br />
[[Team:Harvard/GenProtocols| Notebook]]<br />
<br />
[[Harvard/Parts/LacI| Lactose inducible system]]<br />
<br />
[[https://2008.igem.org/Team:Harvard/Parts/Other| Tetracycline inducible system]]<br />
<br />
[https://2008.igem.org/Team:Harvard/Parts/Tempsenseci| Heat inducible system]</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:43:59Z<p>Lauren: /* The Genetic Circuitry */</p>
<hr />
<div>__NOTOC__<br />
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{|<br />
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<br><br />
<br />
{{Template:Main}}<br />
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{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
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{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
<br />
We were able to create three such inducible systems:<br />
<br />
[[Team:Harvard/GenProtocols| Notebook]]<br />
<br />
[[Harvard/Parts/LacI| Lactose inducible system]]<br />
<br />
[https://2008.igem.org/Team:Harvard/Parts/Other| Tetracycline inducible system]<br />
<br />
[https://2008.igem.org/Team:Harvard/Parts/Tempsenseci| Heat inducible system]</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:40:45Z<p>Lauren: /* The Genetic Circuitry */</p>
<hr />
<div>__NOTOC__<br />
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{|<br />
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<br><br />
<br />
{{Template:Main}}<br />
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{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
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{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
<br />
We were able to create three such inducible systems:<br />
<br />
[https://2008.igem.org/Team:Harvard/Parts/LacI| Lactose inducible system]<br />
<br />
[https://2008.igem.org/Team:Harvard/Parts/Other| Tetracycline inducible system]<br />
<br />
[https://2008.igem.org/Team:Harvard/Parts/Tempsenseci| Heat inducible system]</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:39:39Z<p>Lauren: /* The Genetic Circuitry */</p>
<hr />
<div>__NOTOC__<br />
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| align="center" style="background:#c4dbea"|<br />
<html><a href = "https://2008.igem.org/Team:Harvard"><img src="https://static.igem.org/mediawiki/2008/b/b9/Harvard_logo.png"></a></html><br />
<br><br />
<br />
{{Template:Main}}<br />
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{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
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<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
<br />
We were able to create three such inducible systems:<br />
[https://2008.igem.org/Team:Harvard/Parts/LacI| Lactose inducible system]<br />
[https://2008.igem.org/Team:Harvard/Parts/Other| Tetracycline inducible system]<br />
[https://2008.igem.org/Team:Harvard/Parts/Tempsenseci| Heat inducible system]</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:36:40Z<p>Lauren: /* The Genetic Circuitry */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
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<style><br />
table {<br />
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{|<br />
| align="center" style="background:#c4dbea"|<br />
<html><a href = "https://2008.igem.org/Team:Harvard"><img src="https://static.igem.org/mediawiki/2008/b/b9/Harvard_logo.png"></a></html><br />
<br><br />
<br />
{{Template:Main}}<br />
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{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
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<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
<br />
We were able to create three such inducible systems: a lactose inducible system, a tetracycline inducible system, and a heat inducible system.<br />
<br />
<br />
////////***** PROVIDE LINKS TO THE SUBPART PAGES HERE****/////</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:36:19Z<p>Lauren: /* The Genetic Circuitry */</p>
<hr />
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=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
#<br />
We were able to create three such inducible systems: a lactose inducible system, a tetracycline inducible system, and a heat inducible system.<br />
<br />
<br />
////////***** PROVIDE LINKS TO THE SUBPART PAGES HERE****/////</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:36:04Z<p>Lauren: /* The Genetic Circuitry */</p>
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=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
We were able to create three such inducible systems: a lactose inducible system, a tetracycline inducible system, and a heat inducible system.<br />
<br />
<br />
////////***** PROVIDE LINKS TO THE SUBPART PAGES HERE****/////</div>Laurenhttp://2008.igem.org/Team:Harvard/HardwareTeam:Harvard/Hardware2008-10-30T02:34:12Z<p>Lauren: /* 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 />
To measure the electric current production in Shewanella oneidensis MR-1, we constructed microbial fuel cells (MFCs) to harvest the electricity produced. Traditional fuel cells convert chemical energy to electrical energy. MFCs are unique in that the fuel source used is microbially degradable organic matter (Lovley 2006). Such organic matter is a form of renewable energy and can come from materials in wastewater, sediments, or agricultural wastes (Cho et al. 2007). Compared to hydrogen- and methanol-driven fuel cells, MFCs are particular attractive due to their ease of operation and wide range of fuel sources available. They do not require expensive catalysts, high operating temperatures, nor explosive or toxic fuels (Lovley 2006). Indeed, we found the construction and operation of our microbial fuel cells to be relatively straightforward. <br />
<br />
MFCs operate using principles similar to normal fuel cells. MFCs require an anode and a cathode separated by a semi-permeable membrane (permeable to protons) (Lovley 2006). The anode compartment is generally anaerobic and is the site of oxidation of organic matter (Lovley 2006); the cathode transfers electrons to the electron acceptor (Lovley 2006). MFCs take advantage of the natural metabolism of microorganisms that break down organic matter by intercepting the electrons that would be donated to naturally occurring electron acceptors. Instead, these electrons are transferred from the anode to the cathode, usually along metal wires to which current readings can be taken. <br />
<br />
A figure of a microbial fuel cell is shown below (NB: this is not what we used in our experiments). <br />
<br />
[[ Image: Snapshot1.png | 350px ]]<br />
<br />
On the anode side, glucose is being metabolized by the microorganism to yield electrons which are transferred to the cathode side where oxygen is reduced to water (Lovley 2006).<br />
<br />
===Context===<br />
<br />
Shewanella oneidensis MR-1 is a suitable microorganism for a MFC. It is known to break down lactate (Bretschger et al. 2007), and in anaerobic conditions, S. oneidensis MR-1 produces nanowires that shuttle electrons to electron acceptors as its aerobic electron acceptor, oxygen, is unavailable (Gorby et al. 2006). Taking advantage of this, we designed our microbial fuel cell with an anaerobic anode where completion of the lactate reduction pathway must be completed by transferring electrons to the cathode side at which reduction of oxygen to water occurs. The anode chamber, besides housing our bacteria, was also the site of chemical injections. We provided S. oneidensis MR-1 with lactate as its sole carbon source and sought to induce the mtrB gene, thus “turning on” current production, with chemicals such as lactose and tetracycline. In addition, our heat tests were directed towards the anode chamber as well. Thus, the detection of electricity production of S. oneidensis MR-1 using MFCs serves as an innovative way to detect gene expression, combining both traditional molecular biology with renewable energy engineering. <br />
<br />
<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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==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 />
==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 />
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|}</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:30:10Z<p>Lauren: /* The Critical Gene: mtrB */</p>
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=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''S. oneidensis''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''S. oneidensis'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''S. oneidensis'', would conceivably create a strain of ''S. oneidensis'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''S. oneidensis'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
&&&We were able to create three such inducible systems: a lactose inducible system, a tetracycline inducible system, and a heat inducible system.<br />
<br />
<br />
////////***** PROVIDE LINKS TO THE SUBPART PAGES HERE****/////</div>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T02:21:05Z<p>Lauren: /* Experimental overview */</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 developed 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 have built, measuring the affects of IPTG on plasmids expressing inducible or constitutive mtrB in wild type and mtrB KO ''S. oneidensis''.<br />
<br />
==Results==<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>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T02:18:45Z<p>Lauren: /* Experimental overview */</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 developed 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 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 have built, measuring the affects of<br />
<br />
==Results==<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>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T02:12:15Z<p>Lauren: /* Future directions */</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 are developing two 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 can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach is a light-inducible system based on the 2005 UT Austin biological camera. We are also considering a third approach using cI lambda which would be dependent on temperature.<br />
<br />
Using one or both of these systems, we hope to be able to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we have built. Communication between the fuel cells and a computer then has the potential to be programmable into a game of sorts, such as Tic-Tac-Toe, in which the level of current production induced by a chemical or light input is understood by the computer as a "turn." This rudimentary game is just one of many possibilities for an inducible interaction between computers and bacteria.<br />
<br />
==Results==<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>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T02:10:26Z<p>Lauren: /* Experimental overview */</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 are developing two 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 can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach is a light-inducible system based on the 2005 UT Austin biological camera. We are also considering a third approach using cI lambda which would be dependent on temperature.<br />
<br />
Using one or both of these systems, we hope to be able to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we have built. Communication between the fuel cells and a computer then has the potential to be programmable into a game of sorts, such as Tic-Tac-Toe, in which the level of current production induced by a chemical or light input is understood by the computer as a "turn." This rudimentary game is just one of many possibilities for an inducible interaction between computers and bacteria.<br />
<br />
==Results==<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''Shewanella'' 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 ''Shewanella'', 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 ''Shewanella'' 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 ''Shewanella''. In response to variations in light, the amount of electricity produced by ''Shewanella'' would change. This would allow for the intriguing possibility of not only ''Shewanella'' conveying information to the computer, but also the computer responding to the ''Shewanella''. A simple example would be that in response to a chemical input, ''Shewanella'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''Shewanella'', modifying ''Shewanella'''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 ''Shewanella''. 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 ''Shewanella''. In response to an increase in lactate, ''Shewanella'' 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>Laurenhttp://2008.igem.org/Team:Harvard/ShewieTeam:Harvard/Shewie2008-10-30T02:09:33Z<p>Lauren: /* Organism */</p>
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=Organism=<br />
Most of our work this summer is founded upon the diverse metabolism of the bacterium ''Shewanella oneidensis MR-1''. Throughout the summer, we came to better understand how to work with this organism, and we hope our findings will help establish ''S. oneidensis'' as a interesting chasis for synthetic biology.<br />
==''Shewanella oneidensis MR-1'': an Introduction==<br />
<br />
This summer we worked with ''Shewanella oneidensis MR-1'', a gram-negative facultative anaerobe (Myers and Myers 1997). Under anaerobic conditions, it reduces a number of electron acceptors such as MN(IV). This ability can be harnessed by microbial fuel cells (MFC) to produce an electric current (Bretschger et al. 2007). When the bacteria are grown anaerobically in the anode chamber of an MFC, they release electrons onto the electrode, creating an electrical current. These diverse respiratory capabilities require a complex electron transport systems, including 39 c-type cytochromes (Heidelberg et al. 2002). These interesting characteristics of ''S. oneidensis MR-1'' make it an important model organism for both studies of bioremediation as well as biotechnology applications.<br />
<br />
==Molecular Biology with <i>Shewanella oneidensis</i>==<br />
<br />
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The ''Shewanella oneidensis MR-1'' genome was sequenced in 2002, greatly increasing its usefulness as a model organism. It was found that it had a 4,969,803 base pair circular chromosome and a 161,613 base pair plasmid (Heidelberg et al. 2002). When cloning in ''S. oneidensis MR-1'', it has also been shown that plasmids with p15A origins replicate freely, whereas plasmids with a pMB1 origin of replication do not (Myers and Myers 1997). We further found that the pSC101* origin from Lutz and Bujard (2007) and the CloDF3 origin on the [http://www.emdbiosciences.com/html/NVG/DuetTable.html| pCDF-Duet vector] from Novagen work in ''S. oneidensis''. However, they are not pir+, so the R6K pir+ dependent origin does not work for them. ''S. oneidensis MR-1'' grows at 30 ºC, can be electroporated (see protocol in our [[Team:Harvard/GenProtocols| Notebook]]) and forms round orange pink colonies on plates. It is resistant to ampicillin, but other resistance markers, such as gentamycin and spectomycin, can be used (Saffarini). Together, these characteristics make ''S. oneidensis MR-1'' a genetically tractable organism good for exploring the possibilities of regulated bacterial electrical output.<br />
|<div style="text-indent:0pt;color:black">[[Image:Shewanella colonies growing on plate.JPG|thumb|300px|''S. oneidensis MR-1'' colonies from a transformation]]</div><br />
|-<br />
|}<br />
<br />
==Online resources for working with ''S. oneidensis''==<br />
-[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=211586&aa=11&style=N| Codon usage table]<br />
<br />
-[http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=gsp| Annotated genome, with information on RBS, terminators, protein domains, gene ontology, etc]<br />
<br />
-[http://archaea.ucsc.edu/cgi-bin/hgPcr?org=Shewanella+oneidensis&db=shewOnei&hgsid=159191| ''In silico'' PCR of ''S. oneidensis'' genome]<br />
<br />
==Chassis and the Registry==<br />
To facilitate easy manipulation in different organisms, it may be advantageous for the Registry to standardize a chassis specification sheet. Below, we provide a quick summary of ''S. oneidensis MR-1'' following what we think may be a suitable documentation format. Since iGEM teams frequently work with species other than ''E. coli'', if only to clone some interesting gene product, a set of such sheets could be built up to facilitate synthetic biology in a more diverse set of organisms. A [https://static.igem.org/mediawiki/2008/7/7f/Shewanellachassis.pdf PDF version] with links is available.<br />
[[Image:Chassis.png|720px|center]]<br />
|}<br />
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==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<b>B</b>acteria <b>A</b>s <b>C</b>urrent <b>T</b>ransmitters <b>R</b>eport <b>I</b>nduced <b>C</b>hanges <b>I</b>mportant <b>T</b>o <b>Y</b>ou<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 are developing two inducible systems for electrical current production in Shewanella. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach is a light-inducible system based on the 2005 UT Austin biological camera. We are also considering a third approach using cI lambda which would be dependent on temperature.<br />
<br />
Using one or both of these systems, we hope to be able to interact with engineered Shewanella in multiple anaerobic microbial fuel cells which we have built. Communication between the fuel cells and a computer then has the potential to be programmable into a game of sorts, such as Tic-Tac-Toe, in which the level of current production induced by a chemical or light input is understood by the computer as a "turn." This rudimentary game is just one of many possibilities for an inducible interaction between computers and bacteria.<br />
<br />
==Results==<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''Shewanella'' 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 ''Shewanella'', 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 ''Shewanella'' 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 ''Shewanella''. In response to variations in light, the amount of electricity produced by ''Shewanella'' would change. This would allow for the intriguing possibility of not only ''Shewanella'' conveying information to the computer, but also the computer responding to the ''Shewanella''. A simple example would be that in response to a chemical input, ''Shewanella'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''Shewanella'', modifying ''Shewanella'''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 ''Shewanella''. 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 ''Shewanella''. In response to an increase in lactate, ''Shewanella'' 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>Laurenhttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T01:35:28Z<p>Lauren: /* lacI Inducible System */</p>
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=LacI Inducible System=<br />
<br />
In this system, the lac repressor (LacI) is controlled by a strong constitutive promoter, and is upstream of mtrB under the control of pLac, a LacI regulated promoter. In the default state, LacI is expressed, and inhibits transcription at pLac. Thus, in the default state, mtrB is not expressed. IPTG (an analog of allolactose) induces mtrB expression by binding to LacI, thereby preventing it from inhibiting transcription at pLac.<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:BBa_K098984.png|thumb|650px|center|BBa_K098984 with BioBrick Prefix and Suffix]]</div><br />
<br />
==Inducibility Test for Lac System with GFP==<br />
An induction test was designed to test the inducibility of the lac system by IPTG when the repressor (LacI) is driven by a weak promoter.<br />
===Method===<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|150px|Click for diagram of induction experimental method]]</div><br><br />
<br />
===Results===<br />
Induction of GFP expression was observed at both 2 and 4 hours after adding IPTG. Levels of GFP expression in uninduced samples, however, remained relatively the same throughout the 4 hours. Meanwhile, IPTG induction was not observed in either the negative control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]) or the constitutive GFP control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]).<br />
<br />
<br />
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<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|720px|thumb|center|Lac Inducibility Results]]</div><br />
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=Parts Submitted to Registry=<br />
You can find the complete list of parts we submitted to the registry [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard here].<br />
==The Critical Gene: ''mtrB''==<br />
Many genes are involved in ''Shewanella''’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in ''E. coli'' (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of ''S. oneidensis'' by looking at the effects of knock-out and complementation of mtrB on the electrical output of ''Shewanella''. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in ''Shewanella'', it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out ''Shewanella'', would conceivably create a strain of ''Shewanella'' which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of ''Shewanella'' to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
==The Genetic Circuitry==<br />
In order to control the expression of exogenous mtrB we sought to create several different inducible systems. As depicted below, these systems consist of a repressor under the control of a constitutive promoter (blue). In the default state, the repressor will bind to the downstream promoter (red), preventing RNA polymerase from attaching to the DNA strand to start transcription. Thus, in this state, mtrB is not expressed.<br />
<br />
In the presence of an inducer, mtrB expression does occur. In this case, the inducer binds the repressor protein, preventing it from attaching to the DNA sequence of the promoter (green). RNA polymerase is therefore able to bind to the DNA at the promoter, allowing for expression of mtrB.<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|Induction results in mtrB expression]]<br />
</div><br />
<br />
&&&We were able to create three such inducible systems: a lactose inducible system, a tetracycline inducible system, and a heat inducible system.<br />
<br />
<br />
////////***** PROVIDE LINKS TO THE SUBPART PAGES HERE****/////</div>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T01:30:16Z<p>Lauren: /* Project Overview */</p>
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=Project Overview=<br />
<br />
<h4><b>BACTRICITY: Bacterial Biosensors with Electrical Output</b></h4><br />
<br />
<b>B</b>acteria <b>A</b>s <b>C</b>urrent <b>T</b>ransmitters <b>R</b>eport <b>I</b>nduced <b>C</b>hanges <b>I</b>mportant <b>T</b>o <b>Y</b>ou<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 />
==Results==<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''Shewanella'' 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 ''Shewanella'', 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 ''Shewanella'' 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 ''Shewanella''. In response to variations in light, the amount of electricity produced by ''Shewanella'' would change. This would allow for the intriguing possibility of not only ''Shewanella'' conveying information to the computer, but also the computer responding to the ''Shewanella''. A simple example would be that in response to a chemical input, ''Shewanella'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''Shewanella'', modifying ''Shewanella'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''Shewanella''. 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 ''Shewanella''. In response to an increase in lactate, ''Shewanella'' 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>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T01:28:56Z<p>Lauren: /* Project Overview */</p>
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=Project Overview=<br />
<br />
<h4><b>BACTRICITY: Bacterial Biosensors with Electrical Output</b></h4><br />
<br />
<b>B</b>acteria <b>A</b>s <b>C</b>urrent <b>T</b>ransmitters <b>R</b>eport <b>I</b>nduced <b>C</b>hanges <b>I</b>mportant <b>T</b>o <b>Y</b>ou<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 />
==Results==<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''Shewanella'' 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 ''Shewanella'', 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 ''Shewanella'' 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 ''Shewanella''. In response to variations in light, the amount of electricity produced by ''Shewanella'' would change. This would allow for the intriguing possibility of not only ''Shewanella'' conveying information to the computer, but also the computer responding to the ''Shewanella''. A simple example would be that in response to a chemical input, ''Shewanella'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''Shewanella'', modifying ''Shewanella'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''Shewanella''. 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 ''Shewanella''. In response to an increase in lactate, ''Shewanella'' 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>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T01:26:07Z<p>Lauren: /* Future directions */</p>
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|<br />
=Project Overview=<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 />
==Results==<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''Shewanella'' 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 ''Shewanella'', 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 ''Shewanella'' 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 ''Shewanella''. In response to variations in light, the amount of electricity produced by ''Shewanella'' would change. This would allow for the intriguing possibility of not only ''Shewanella'' conveying information to the computer, but also the computer responding to the ''Shewanella''. A simple example would be that in response to a chemical input, ''Shewanella'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''Shewanella'', modifying ''Shewanella'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''Shewanella''. 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 ''Shewanella''. In response to an increase in lactate, ''Shewanella'' 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>Laurenhttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T01:18:44Z<p>Lauren: /* Future directions */</p>
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<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Project Overview=<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 />
==Results==<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in Shewanella 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 Shewanella, 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 Shewanella 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 Shewanella. In response, to changes of light the amount of electricity produced by Shewanella could change. This would allow for the intriguing possibility of not only Shewanella conveying information to the computer, but also the computer responding to the Shewanella. A simple example would be that in response to a chemical input, Shewanella may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the Shewanella, modifying Shewanella's output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of E. coli and Shewanella. 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 Shewanella. In response to an increase in lactate, Shewanella 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>Laurenhttp://2008.igem.org/Team:Harvard/FutureTeam:Harvard/Future2008-10-30T01:09:03Z<p>Lauren: /* Future Directions */</p>
<hr />
<div>=Future Directions=<br />
<br />
Our work with creating a system of inducible electrical output in Shewanella 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 Shewanella, 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 Shewanella 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 />
<br />
Another interesting direction would be the linking of the light-sensing system developed by the UT Austin iGEM team with electrical output in Shewanella. In response, to changes of light the amount of electricity produced by Shewanella could change. This would allow for the intriguing possibility of not only Shewanella conveying information to the computer, but also the computer responding to the Shewanella. A simple example would be that in response to a chemical input, Shewanella may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the Shewanella, modifying Shewanella's output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of E. coli and Shewanella. 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 Shewanella. In response to an increase in lactate, Shewanella 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!</div>Laurenhttp://2008.igem.org/Team:Harvard/FutureTeam:Harvard/Future2008-10-30T01:05:00Z<p>Lauren: /* Future Directions */</p>
<hr />
<div>=Future Directions=<br />
<br />
Our work with creating a system of inducible electrical output in Shewanella 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 Shewanella, 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 Shewanella 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 />
<br />
Another interesting direction would be the linking of the light-sensing system developed by the UT Austin iGEM team with electrical output in Shewanella. In response, to changes of light the amount of electricity produced by Shewanella could change. This would allow for the intriguing possibility of not only Shewanella conveying information to the computer, but also the computer responding to the Shewanella. A simple example would be that in response to a chemical input, Shewanella may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the Shewanella, modifying Shewanella's output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of E. coli and Shewanella. 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 Shewanella. In response to an increase in lactate, Shewanella would begin to produce higher levels of electricity. This strategy could allow for 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!</div>Laurenhttp://2008.igem.org/Team:Harvard/FutureTeam:Harvard/Future2008-10-30T00:50:21Z<p>Lauren: /* Future Directions */</p>
<hr />
<div>=Future Directions=<br />
<br />
Our work with creating a system of inducible electrical output in Shewanella has laid the foundations for many different exciting avenues of further inquiry.<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 Shewanella, 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 Shewanella 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 />
<br />
Another interesting direction would be the linking of the light-sensing system developed by the UT Austin iGEM team with electrical output in Shewanella. In response, to changes of light the amount of electricity produced by Shewanella could change. This would allow for the intriguing possibility of not only Shewanella conveying information to the computer, but also the computer responding to the Shewanella. A simple example would be that in response to a chemical input, Shewanella may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the Shewanella, modifying Shewanella's output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers.</div>Laurenhttp://2008.igem.org/Team:Harvard/FutureTeam:Harvard/Future2008-10-30T00:48:53Z<p>Lauren: /* Future Directions */</p>
<hr />
<div>=Future Directions=<br />
<br />
Our work with creating a system of inducible electrical output in Shewanella has laid the foundations for many different exciting avenues of further inquiry.<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 Shewanella, 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 Shewanella 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 />
<br />
Another interesting direction would be the linking of the light-sensing system developed by the UT Austin iGEM team with electrical output in Shewanella. In response, to changes of light the amount of electricity produced by Shewanella could change. This would allow for the intriguing possibility of not only Shewanella conveying information to the computer, but also the computer responding to the Shewanella. A simple example would be that in response to a chemical input, Shewanella may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the Shewanella, modifying Shewanella's output, creating interesting feedback loops.</div>Laurenhttp://2008.igem.org/Team:Harvard/FutureTeam:Harvard/Future2008-10-30T00:26:59Z<p>Lauren: /* Future Directions */</p>
<hr />
<div>=Future Directions=<br />
<br />
Our work with creating a system of inducible electrical output in Shewanella has laid the foundations for many different exciting avenues of further inquiry.<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 Shewanella, 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 Shewanella 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.</div>Laurenhttp://2008.igem.org/Team:Harvard/FutureTeam:Harvard/Future2008-10-30T00:26:12Z<p>Lauren: /* Future Directions */</p>
<hr />
<div>=Future Directions=<br />
<br />
Our work with creating a system of inducible electrical output in Shewanella has laid the foundations for many different exciting avenues of further inquiry.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006| arsenic system] developed by the University of Edinburgh iGEM 2006 team could be introduced into Shewanella, 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 Shewanella 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.</div>Laurenhttp://2008.igem.org/Team:Harvard/FutureTeam:Harvard/Future2008-10-30T00:25:55Z<p>Lauren: New page: =Future Directions= Our work with creating a system of inducible electrical output in Shewanella has laid the foundations for many different exciting avenues of further inquiry. Using th...</p>
<hr />
<div>=Future Directions=<br />
<br />
Our work with creating a system of inducible electrical output in Shewanella has laid the foundations for many different exciting avenues of further inquiry.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006|arsenic system] developed by the University of Edinburgh iGEM 2006 team could be introduced into Shewanella, 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 Shewanella 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.</div>Laurenhttp://2008.igem.org/Team:Harvard/ShewieTeam:Harvard/Shewie2008-10-29T05:11:29Z<p>Lauren: /* Molecular Biology with Shewanella oneidensis */</p>
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=Organism=<br />
Most of our work this summer is founded upon the diverse metabolism of the bacterium ''Shewanella oneidensis MR-1''. Throughout the summer, we came to better understand how to work with this organism, and we hope our findings will help establish ''S. oneidensis'' as a interesting chasis for synthetic biology.<br />
==''Shewanella oneidensis MR-1'': an Introduction==<br />
<br />
This summer we worked with ''Shewanella oneidensis MR-1'', a gram-negative facultative anaerobe (Myers and Myers 1997). Under anaerobic conditions, it reduces a number of electron acceptors such as MN(IV). This ability can be harnessed by microbial fuel cells (MFC) to produce an electric current (Bretschger et al. 2007). When the bacteria are grown anaerobically in the anode chamber of an MFC, they release electrons onto the electrode, creating an electrical current. These diverse respiratory capabilities require a complex electron transport systems, including 39 c-type cytochromes (Heidelberg et al. 2002). These interesting characteristics of ''S. oneidensis MR-1'' make it an important model organism for both studies of bioremediation as well as biotechnology applications.<br />
<br />
==Molecular Biology with <i>Shewanella oneidensis</i>==<br />
<br />
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|<br />
The ''Shewanella oneidensis MR-1'' genome was sequenced in 2002, greatly increasing its usefulness as a model organism. It was found that it had a 4,969,803 base pair circular chromosome and a 161,613 base pair plasmid (Heidelberg et al. 2002). When cloning in ''S. oneidensis MR-1'', it has also been shown that plasmids with p15A origins replicate freely, whereas plasmids with a pMB1 origin of replication do not (Myers and Myers 1997). We further found that the pSC101* origin from Lutz and Bujard (2007) and the CloDF3 origin on the [http://www.emdbiosciences.com/html/NVG/DuetTable.html| pCDF-Duet vector] from Novagen work in ''Shewanella''. However, they are not pir+, so the R6K pir+ dependent origin does not work for them. ''S. oneidensis MR-1'' grows at 30 ºC, can be electroporated (see protocol in our [[Team:Harvard/GenProtocols| Notebook]]) and forms round orange pink colonies on plates. It is resistant to ampicillin, but other resistance markers, such as gentamycin and spectomycin, can be used (Saffarini). Together, these characteristics make ''S. oneidensis MR-1'' a genetically tractable organism good for exploring the possibilities of regulated bacterial electrical output.<br />
|<div style="text-indent:0pt;color:black">[[Image:Shewanella colonies growing on plate.JPG|thumb|300px|''S. oneidensis MR-1'' colonies from a transformation]]</div><br />
|-<br />
|}<br />
<br />
==Online resources for working with ''S. oneidensis MR-1''==<br />
-[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=211586&aa=11&style=N| Codon usage table]<br />
<br />
-[http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=gsp| Annotated genome, with information on RBS, terminators, protein domains, gene ontology, etc]<br />
<br />
-[http://archaea.ucsc.edu/cgi-bin/hgPcr?org=Shewanella+oneidensis&db=shewOnei&hgsid=159191| ''In silico'' PCR of ''S. oneidensis'' genome]<br />
<br />
==Chassis and the Registry==<br />
To facilitate easy manipulation in different organisms, it may be advantageous for the Registry to standardize a chassis specification sheet. Below, we provide a quick summary of ''S. oneidensis MR-1'' following what we think may be a suitable documentation format. Since iGEM teams frequently work with species other than ''E. coli'', if only to clone some interesting gene product, a set of such sheets could be built up to facilitate synthetic biology in a more diverse set of organisms. A [https://static.igem.org/mediawiki/2008/7/7f/Shewanellachassis.pdf PDF version] with links is available.<br />
[[Image:Chassis.png|720px|center]]<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Laurenhttp://2008.igem.org/Team:Harvard/ReferencesTeam:Harvard/References2008-10-29T05:06:38Z<p>Lauren: </p>
<hr />
<div>Bretschger, O., Obraztsove, A., Sturm, C., Chang, I., Gorby, Y., Reed, S., Culley, D., Reardon, C., Barua, S., Romine, M., Zhou, J., Beliaev, A., Bouhenni, R., Saffrini, D., Mansfeld, F., Kim, B., Fredrickson, J., and Nealson, K. (2007). Current Production and Metal Oxide Reduction by ''Shewanella oneidensis'' MR01 Wild Type and Mutants. ''Appl. Environ. Microbiol''. 73, 7003-7012.<br />
<br />
Heidelberg, J. et al. (2002). Genome sequence of the dissimilatory metal ion-reducing bacterium Sewanella oneidensis. ''Nature Biotechnology''. 20, 1118-1123.<br />
<br />
Leipold, R., Krewson, C., and Dhurjati, P. (1994). Mathematical Model of Temperature-Senstive Plasmid Replication. ''Plasmid'' 32, 131-167.<br />
<br />
Lutz, R. and Bujard, H. (1997). Independent and tight regulation of transcriptional units in ''Escherichia coli'' via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. ''Nucleic Acids Research'' 25, 1203-1210.<br />
<br />
Myers, C., and Myers, J. (1997). Replication of plasmids with the p15A origin in ''Shewanela putrfaciens'' MR-1. ''Letters in Applied Microbiology''. 24, 221-225.<br />
<br />
Saffrini, Daad. (2008) Personal correspondence.</div>Laurenhttp://2008.igem.org/Team:Harvard/ReferencesTeam:Harvard/References2008-10-29T05:02:18Z<p>Lauren: </p>
<hr />
<div>Bretschger, O., Obraztsove, A., Sturm, C., Chang, I., Gorby, Y., Reed, S., Culley, D., Reardon, C., Barua, S., Romine, M., Zhou, J., Beliaev, A., Bouhenni, R., Saffrini, D., Mansfeld, F., Kim, B., Fredrickson, J., and Nealson, K. (2007). Current Production and Metal Oxide Reduction by ''Shewanella oneidensis'' MR01 Wild Type and Mutants. ''Appl. Environ. Microbiol''. 73, 7003-7012.<br />
<br />
Leipold, R., Krewson, C., and Dhurjati, P. (1994). Mathematical Model of Temperature-Senstive Plasmid Replication. ''Plasmid'' 32, 131-167.<br />
<br />
Lutz, R. and Bujard, H. (1997). Independent and tight regulation of transcriptional units in ''Escherichia coli'' via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. ''Nucleic Acids Research'' 25, 1203-1210.<br />
<br />
Myers, C., and Myers, J. (1997). Replication of plasmids with the p15A origin in ''Shewanela putrfaciens'' MR-1. ''Letters in Applied Microbiology''. 24, 221-225.<br />
<br />
Saffrini, Daad. (2008) Personal correspondence.</div>Laurenhttp://2008.igem.org/Team:Harvard/ReferencesTeam:Harvard/References2008-10-29T04:59:33Z<p>Lauren: </p>
<hr />
<div>Bretschger, O., Obraztsove, A., Sturm, C., Chang, I., Gorby, Y., Reed, S., Culley, D., Reardon, C., Barua, S., Romine, M., Zhou, J., Beliaev, A., Bouhenni, R., Saffrini, D., Mansfeld, F., Kim, B., Fredrickson, J., and Nealson, K. (2007). Current Production and Metal Oxide Reduction by ''Shewanella oneidensis'' MR01 Wild Type and Mutants. ''Appl. Environ. Microbiol''. 73, 7003-7012.<br />
<br />
Leipold, R., Krewson, C., and Dhurjati, P. (1994). Mathematical Model of Temperature-Senstive Plasmid Replication. ''Plasmid'' 32, 131-167.<br />
<br />
Lutz, R. and Bujard, H. (1997). Independent and tight regulation of transcriptional units in ''Escherichia coli'' via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. ''Nucleic Acids Research'' 25, 1203-1210.<br />
<br />
Saffrini, Daad. (2008) Personal correspondence.</div>Laurenhttp://2008.igem.org/Team:Harvard/ReferencesTeam:Harvard/References2008-10-29T04:53:34Z<p>Lauren: </p>
<hr />
<div>Leipold, R., Krewson, C., and Dhurjati, P. (1994). Mathematical Model of Temperature-Senstive Plasmid Replication. ''Plasmid'' 32, 131-167.<br />
<br />
Lutz, R. and Bujard, H. (1997). Independent and tight regulation of transcriptional units in ''Escherichia coli'' via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. ''Nucleic Acids Research'' 25, 1203-1210.<br />
<br />
Saffrini, Daad. (2008) Personal correspondence.</div>Laurenhttp://2008.igem.org/Team:Harvard/ReferencesTeam:Harvard/References2008-10-29T04:51:34Z<p>Lauren: </p>
<hr />
<div>Leipold, R., Krewson, C., and Dhurjati, P. (1994). Mathematical Model of Temperature-Senstive Plasmid Replication. ''Plasmid'' 32, 131-167.<br />
<br />
Lutz, R. and Bujard, H. (1997). Independent and tight regulation of transcriptional units in ''Escherichia coli'' via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. ''Nucleic Acids Research'' 25, 1203-1210.</div>Laurenhttp://2008.igem.org/Team:Harvard/ReferencesTeam:Harvard/References2008-10-29T04:47:58Z<p>Lauren: New page: Leipold, R., Krewson, C., and Dhurjati, P. (1994). Mathematical Model of Temperature-Senstive Plasmid Replication. ''Plasmid'' 32, 131-167.</p>
<hr />
<div>Leipold, R., Krewson, C., and Dhurjati, P. (1994). Mathematical Model of Temperature-Senstive Plasmid Replication. ''Plasmid'' 32, 131-167.</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-29T04:43:58Z<p>Lauren: /* Thermoinducible cI System */</p>
<hr />
<div>__NOTOC__<br />
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{{Template:Main}}<br />
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<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
<br />
<br />
==Short intro==<br />
See [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard list] of all parts we submitted.<br />
__TOC__<br />
\\******change subsection headers*******\\<br />
<br />
==General overview of QPIs==<br />
<br />
<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|//EDIT CAPTION//]]<br />
</div><br />
<br />
==General overview of mtrB==<br />
Many genes are involved in Shewanella’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in E. coli (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of Shewanella by looking at the effects of knock-out and complementation of mtrB on the electrical output of Shewanella. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in Shewanella, it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out Shewanella, would conceivably create a strain of Shewanella which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of Shewanella to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
===lac system (will be moved to separate page)===<br />
description of complete system<br />
Amy's induction data<br />
use sublevels, as entire section will become new page<br />
<br />
<br />
<div style="text-indent:0pt">[[Image:BBa_K098984.png|thumb|650px|center|BBa_K098984 with BioBrick Prefix and Suffix]]</div><br />
<br />
===QPIs that we did not use with mtrB===<br />
<br />
<br />
<br />
===tet===<br />
===Thermoinducible cI System===<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<br />
<br />
The thermoinducible cI lambda system uses cI857 (a mutant form of cI from [http://www.addgene.org/pgvec1?f=c&vectorid=5079&cmd=genvecmap&dim=800&format=html&mtime=1188314819| pGW7] purchased from [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx#40554| ATCC]) to regulate expression of genes under the control of the lambda promoter. The cI857 repressor is repressed by thermal denaturation. Activity of cI857 begins to decrease around 30 ºC and is fully denatured by around 42 ºC (Leipold et al., 1994). Thus transcription of the gene under the control of the lambda promoter can be induced by increasing the temperature from 30 ºC to 37 ºC-40 ºC. <br />
<br />
Edit stuff here; we'll move entire section to new page.<br />
|}<br />
<br><br><br />
<br />
<!--- end body ---><br />
|}</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-29T04:39:49Z<p>Lauren: /* heat sensitive cI */</p>
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{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
<br />
<br />
==Short intro==<br />
See [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard list] of all parts we submitted.<br />
__TOC__<br />
\\******change subsection headers*******\\<br />
<br />
==General overview of QPIs==<br />
<br />
<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|//EDIT CAPTION//]]<br />
</div><br />
<br />
==General overview of mtrB==<br />
Many genes are involved in Shewanella’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in E. coli (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of Shewanella by looking at the effects of knock-out and complementation of mtrB on the electrical output of Shewanella. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in Shewanella, it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out Shewanella, would conceivably create a strain of Shewanella which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of Shewanella to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
===lac system (will be moved to separate page)===<br />
description of complete system<br />
Amy's induction data<br />
use sublevels, as entire section will become new page<br />
<br />
<br />
<div style="text-indent:0pt">[[Image:BBa_K098984.png|thumb|650px|center|BBa_K098984 with BioBrick Prefix and Suffix]]</div><br />
<br />
===QPIs that we did not use with mtrB===<br />
<br />
<br />
<br />
===tet===<br />
===Thermoinducible cI System===<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<br />
<br />
The thermoinducible cI lambda system uses cI857 (a mutant form of cI from pGW7 purchased from ATCC) to regulate expression of genes under the control of the lambda promoter. The cI857 repressor is repressed by thermal denaturation. Activity of cI857 begins to decrease around 30 ºC and is fully denatured by around 42 ºC (Leipold et al., 1994). Thus transcription of the gene under the control of the lambda promoter can be induced by increasing the temperature from 30 ºC to 37 ºC-40 ºC. <br />
<br />
Edit stuff here; we'll move entire section to new page.<br />
|}<br />
<br><br><br />
<br />
<!--- end body ---><br />
|}</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-29T03:58:35Z<p>Lauren: /* heat sensitive ci */</p>
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<br><br />
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{{Template:Main}}<br />
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<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
<br />
<br />
==Short intro==<br />
See [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard list] of all parts we submitted.<br />
__TOC__<br />
\\******change subsection headers*******\\<br />
<br />
==General overview of QPIs==<br />
<br />
<br />
<div style="text-indent:0pt">[[Image:Harvsystem.png|thumb|650px|center|//EDIT CAPTION//]]<br />
</div><br />
<br />
==General overview of mtrB==<br />
Many genes are involved in Shewanella’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in E. coli (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of Shewanella by looking at the effects of knock-out and complementation of mtrB on the electrical output of Shewanella. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in Shewanella, it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out Shewanella, would conceivably create a strain of Shewanella which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of Shewanella to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
===lac complete system (will be moved to separate page)===<br />
complete description<br />
<br />
<br />
<div style="text-indent:0pt">[[Image:BBa_K098984.png|thumb|650px|center|BBa_K098984]]</div><br />
<br />
===QPIs that we did not use with mtrB===<br />
<br />
===lac===<br />
short description, Amy's data on separate page: put most details here:<br />
[[Team:Harvard/lacsys]]<br />
<br />
===tet===<br />
===heat sensitive cI===<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<br />
<br />
short description, Amy's data on separate page: put most details here:<br />
[[Team:Harvard/citssys]]<br />
|}<br />
<br><br><br />
<br />
<!--- end body ---><br />
|}</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-29T03:24:05Z<p>Lauren: /* heat sensitive ci */</p>
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{{Template:Main}}<br />
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{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
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<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<br />
=Parts Submitted to Registry=<br />
__TOC__<br />
<br />
==Short intro==<br />
See [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard list] of all parts we submitted.<br />
<br />
\\******change subsection headers*******\\<br />
<br />
==General overview of QPIs==<br />
<br />
{|align="center" cellpadding="0" width="98%"|<br />
|[[Image:Harvsystem.png|thumb|650px|center|//EDIT CAPTION OR DELETE//]]<br />
|-<br />
|}<br />
<br />
==General overview of mtrB==<br />
Many genes are involved in Shewanella’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in E. coli (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of Shewanella by looking at the effects of knock-out and complementation of mtrB on the electrical output of Shewanella. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in Shewanella, it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out Shewanella, would conceivably create a strain of Shewanella which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of Shewanella to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
===lac complete system===<br />
complete description<br />
<br />
{|align="center" cellpadding="0" width="98%"|<br />
|[[Image:BBa_K098984.png|thumb|650px|center|BBa_K098984]]<br />
|-<br />
|}<br />
<br />
===QPIs that we did not use with mtrB===<br />
<br />
===lac===<br />
short description, Amy's data on separate page: put most details here:<br />
[[Team:Harvard/lacsys]]<br />
<br />
===tet===<br />
===heat sensitive ci===<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<br />
<br />
short description, Amy's data on separate page: put most details here:<br />
[[Team:Harvard/citssys]]<br />
|}<br />
<br><br><br />
<br />
<!--- end body ---><br />
|}</div>Laurenhttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-29T03:22:05Z<p>Lauren: /* General overview of mtrB */</p>
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<br><br />
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=Parts Submitted to Registry=<br />
__TOC__<br />
<br />
==Short intro==<br />
See [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Harvard list] of all parts we submitted.<br />
<br />
\\******change subsection headers*******\\<br />
<br />
==General overview of QPIs==<br />
<br />
{|align="center" cellpadding="0" width="98%"|<br />
|[[Image:Harvsystem.png|thumb|650px|center|//EDIT CAPTION OR DELETE//]]<br />
|-<br />
|}<br />
<br />
==General overview of mtrB==<br />
Many genes are involved in Shewanella’s complex respiratory system (Heidelberg et al. 2002). We focused on mtrB, a 679-amino-acid-long outer membrane protein thought to be involved in the binding of metals and the localization of outer membrane cytochromes during reduction (Bretschger et al. 2007). It is unfortunately toxic in E. coli (Saffarini). Bretschger et al. recently characterized the role of mtrB in anaerobic respiration of Shewanella by looking at the effects of knock-out and complementation of mtrB on the electrical output of Shewanella. It was found that the strain which lacked mtrB produced less than 20% of the current generated by the wild type strain. In complemented strains, where mtrB is expressed constitutively under the control of the lacZ promoter in the knock-out strain, the phenotype was rescued with a similar amount of current being produced to that of the wild type (Bretschger et al. 2007). Not only does this experiment demonstrate the importance of mtrB in reduction in Shewanella, it also suggests a mechanism by which this electrical output could be controlled. Transforming plasmids containing mtrB under the control of an inducible promoter into mtrB knock out Shewanella, would conceivably create a strain of Shewanella which could increase its electrical output in response to the turning-on of the promoter controlling mtrB expression. The creation of a strain with an inducible electrical output could have important applications in biotechnology by creating a system which couples the ability of Shewanella to respond to a diverse array of stimuli with the speed and ubiquity of electricity.<br />
<br />
===lac complete system===<br />
complete description<br />
<br />
{|align="center" cellpadding="0" width="98%"|<br />
|[[Image:BBa_K098984.png|thumb|650px|center|BBa_K098984]]<br />
|-<br />
|}<br />
<br />
===QPIs that we did not use with mtrB===<br />
<br />
===lac===<br />
short description, Amy's data on separate page: put most details here:<br />
[[Team:Harvard/lacsys]]<br />
<br />
===tet===<br />
===heat sensitive ci===<br />
short description, Amy's data on separate page: put most details here:<br />
[[Team:Harvard/citssys]]<br />
|}<br />
<br><br><br />
<br />
<!--- end body ---><br />
|}</div>Laurenhttp://2008.igem.org/Team:Harvard/ShewieTeam:Harvard/Shewie2008-10-29T03:21:05Z<p>Lauren: /* Molecular Biology with Shewanella oneidensis */</p>
<hr />
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<html><a href = "https://2008.igem.org/Team:Harvard"><img src="https://static.igem.org/mediawiki/2008/b/b9/Harvard_logo.png"></a></html><br />
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<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Organism=<br />
Most of our work this summer is founded upon the diverse metabolism of the bacterium ''Shewanella oneidensis MR-1''. Throughout the summer, we came to better understand how to work with this organism, and we hope our findings will help establish ''S. oneidensis'' as a interesting chasis for synthetic biology.<br />
==''Shewanella oneidensis MR-1'': an Introduction==<br />
<br />
This summer we worked with ''Shewanella oneidensis MR-1'', a gram-negative facultative anaerobe (Myers and Myers 1997). Under anaerobic conditions, it reduces a number of electron acceptors such as MN(IV). This ability can be harnessed by microbial fuel cells (MFC) to produce an electric current (Bretschger et al. 2007). When the bacteria are grown anaerobically in the anode chamber of an MFC, they release electrons onto the electrode, creating an electrical current. These diverse respiratory capabilities require a complex electron transport systems, including 39 c-type cytochromes (Heidelberg et al. 2002). These interesting characteristics of ''S. oneidensis MR-1'' make it an important model organism for both studies of bioremediation as well as biotechnology applications.<br />
<br />
==Molecular Biology with <i>Shewanella oneidensis</i>==<br />
<br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; text-align:justify" width="100%"<br />
|<br />
The ''Shewanella oneidensis MR-1'' genome was sequenced in 2002, greatly increasing its usefulness as a model organism. It was found that it had a 4,969,803 base pair circular chromosome and a 161,613 base pair plasmid (Heidelberg et al. 2002). When cloning in ''S. oneidensis MR-1'', it has also been shown that plasmids with p15A origins replicate freely, whereas plasmids with a pMB1 origin of replication do not (Myers and Myers 1997). We further found that the pSC101* origin from Lutz and Bujard (2007) and the CloDF3 origin on the [http://www.emdbiosciences.com/html/NVG/DuetTable.html| pCDF-Duet vector] from Novagen work in ''Shewanella''. ''S. oneidensis MR-1'' grows at 30 ºC, can be electroporated (see protocol in our [[Team:Harvard/GenProtocols| Notebook]]) and forms round orange pink colonies on plates. It is resistant to ampicillin, but other resistance markers work (Saffarini). Together, these characteristics make ''S. oneidensis MR-1'' a genetically tractable organism good for exploring the possibilities of regulated bacterial electrical output.</p><br />
|[[Image:Shewanella colonies growing on plate.JPG|thumb|300px|''S. oneidensis MR-1'' colonies from a transformation]]<br />
|-<br />
|}<br />
<br />
==Online resources for working with ''S. oneidensis MR-1''==<br />
*[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=211586&aa=11&style=N| Codon usage table]<br />
*[http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=gsp| Annotated genome, with information on RBS, terminators, protein domains, gene ontology, etc]<br />
*[http://archaea.ucsc.edu/cgi-bin/hgPcr?org=Shewanella+oneidensis&db=shewOnei&hgsid=159191| ''In silico'' PCR of ''S. oneidensis'' genome]<br />
<br />
==Chassis and the Registry==<br />
To facilitate easy manipulation in different organisms, it may be advantageous to standardize a specification sheet. Below, we provide a quick summary of ''S. oneidensis MR-1'' following what we think may be a suitable format. Since iGEM teams frequently work with species other than ''E. coli'', if only to clone some interesting gene product, a set of such sheets could be built up to facilitate synthetic biology in a more diverse set of organisms.<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Laurenhttp://2008.igem.org/Team:Harvard/ShewieTeam:Harvard/Shewie2008-10-29T03:18:15Z<p>Lauren: /* Molecular Biology with Shewanella oneidensis */</p>
<hr />
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<html><a href = "https://2008.igem.org/Team:Harvard"><img src="https://static.igem.org/mediawiki/2008/b/b9/Harvard_logo.png"></a></html><br />
<br><br />
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<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Organism=<br />
Most of our work this summer is founded upon the diverse metabolism of the bacterium ''Shewanella oneidensis MR-1''. Throughout the summer, we came to better understand how to work with this organism, and we hope our findings will help establish ''S. oneidensis'' as a interesting chasis for synthetic biology.<br />
==''Shewanella oneidensis MR-1'': an Introduction==<br />
<br />
This summer we worked with ''Shewanella oneidensis MR-1'', a gram-negative facultative anaerobe (Myers and Myers 1997). Under anaerobic conditions, it reduces a number of electron acceptors such as MN(IV). This ability can be harnessed by microbial fuel cells (MFC) to produce an electric current (Bretschger et al. 2007). When the bacteria are grown anaerobically in the anode chamber of an MFC, they release electrons onto the electrode, creating an electrical current. These diverse respiratory capabilities require a complex electron transport systems, including 39 c-type cytochromes (Heidelberg et al. 2002). These interesting characteristics of ''S. oneidensis MR-1'' make it an important model organism for both studies of bioremediation as well as biotechnology applications.<br />
<br />
==Molecular Biology with <i>Shewanella oneidensis</i>==<br />
<br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; text-align:justify" width="100%"<br />
|<br />
The ''Shewanella oneidensis MR-1'' genome was sequenced in 2002, greatly increasing its usefulness as a model organism. It was found that it had a 4,969,803 base pair circular chromosome and a 161,613 base pair plasmid (Heidelberg et al. 2002). When cloning in ''S. oneidensis MR-1'', it has also been shown that plasmids with p15A origins replicate freely, whereas plasmids with a pMB1 origin of replication do not (Myers and Myers 1997). We further found that the pSC101* origin from Lutz and Bujard (2007) and the CloDF3 origin on the pCDF-Duet vector from Novagen work in ''Shewanella''. ''S. oneidensis MR-1'' grows at 30 ºC, can be electroporated (see protocol in our [[Team:Harvard/GenProtocols| Notebook]]) and forms round orange pink colonies on plates. It is resistant to ampicillin, but other resistance markers work (Saffarini). Together, these characteristics make ''S. oneidensis MR-1'' a genetically tractable organism good for exploring the possibilities of regulated bacterial electrical output.</p><br />
|[[Image:Shewanella colonies growing on plate.JPG|thumb|300px|''S. oneidensis MR-1'' colonies from a transformation]]<br />
|-<br />
|}<br />
<br />
==Online resources for working with ''S. oneidensis MR-1''==<br />
*[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=211586&aa=11&style=N| Codon usage table]<br />
*[http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=gsp| Annotated genome, with information on RBS, terminators, protein domains, gene ontology, etc]<br />
*[http://archaea.ucsc.edu/cgi-bin/hgPcr?org=Shewanella+oneidensis&db=shewOnei&hgsid=159191| ''In silico'' PCR of ''S. oneidensis'' genome]<br />
<br />
==Chassis and the Registry==<br />
To facilitate easy manipulation in different organisms, it may be advantageous to standardize a specification sheet. Below, we provide a quick summary of ''S. oneidensis MR-1'' following what we think may be a suitable format. Since iGEM teams frequently work with species other than ''E. coli'', if only to clone some interesting gene product, a set of such sheets could be built up to facilitate synthetic biology in a more diverse set of organisms.<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Laurenhttp://2008.igem.org/Team:Harvard/ShewieTeam:Harvard/Shewie2008-10-29T03:17:38Z<p>Lauren: /* Molecular Biology with Shewanella oneidensis */</p>
<hr />
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| align="center" style="background:#c4dbea" border="0" cellpading="0" cellspacing="0"|<br />
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<br><br />
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<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Organism=<br />
Most of our work this summer is founded upon the diverse metabolism of the bacterium ''Shewanella oneidensis MR-1''. Throughout the summer, we came to better understand how to work with this organism, and we hope our findings will help establish ''S. oneidensis'' as a interesting chasis for synthetic biology.<br />
==''Shewanella oneidensis MR-1'': an Introduction==<br />
<br />
This summer we worked with ''Shewanella oneidensis MR-1'', a gram-negative facultative anaerobe (Myers and Myers 1997). Under anaerobic conditions, it reduces a number of electron acceptors such as MN(IV). This ability can be harnessed by microbial fuel cells (MFC) to produce an electric current (Bretschger et al. 2007). When the bacteria are grown anaerobically in the anode chamber of an MFC, they release electrons onto the electrode, creating an electrical current. These diverse respiratory capabilities require a complex electron transport systems, including 39 c-type cytochromes (Heidelberg et al. 2002). These interesting characteristics of ''S. oneidensis MR-1'' make it an important model organism for both studies of bioremediation as well as biotechnology applications.<br />
<br />
==Molecular Biology with <i>Shewanella oneidensis</i>==<br />
<br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; text-align:justify" width="100%"<br />
|<br />
The ''Shewanella oneidensis MR-1'' genome was sequenced in 2002, greatly increasing its usefulness as a model organism. It was found that it had a 4,969,803 base pair circular chromosome and a 161,613 base pair plasmid (Heidelberg et al. 2002). When cloning in ''S. oneidensis MR-1'', it has also been shown that plasmids with p15A origins replicate freely, whereas plasmids with a pMB1 origin of replication do not (Myers and Myers 1997). We further found that the pSC101* origin from Lutz and Bujard (2007) and the CloDF3 origin on the pCDF-Duet vector from Novagen work in ''Shewanella''. ''S. oneidensis MR-1'' grows at 30 ºC, can be electroporated (see protocol in our [[Team:Harvard/GenProtocols| Notebook]]) and forms round orange pink colonies on plates. It is resistant to ampicillin, but other resistance markers work (Daad). Together, these characteristics make ''S. oneidensis MR-1'' a genetically tractable organism good for exploring the possibilities of regulated bacterial electrical output.</p><br />
|[[Image:Shewanella colonies growing on plate.JPG|thumb|300px|''S. oneidensis MR-1'' colonies from a transformation]]<br />
|-<br />
|}<br />
<br />
==Online resources for working with ''S. oneidensis MR-1''==<br />
*[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=211586&aa=11&style=N| Codon usage table]<br />
*[http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org=gsp| Annotated genome, with information on RBS, terminators, protein domains, gene ontology, etc]<br />
*[http://archaea.ucsc.edu/cgi-bin/hgPcr?org=Shewanella+oneidensis&db=shewOnei&hgsid=159191| ''In silico'' PCR of ''S. oneidensis'' genome]<br />
<br />
==Chassis and the Registry==<br />
To facilitate easy manipulation in different organisms, it may be advantageous to standardize a specification sheet. Below, we provide a quick summary of ''S. oneidensis MR-1'' following what we think may be a suitable format. Since iGEM teams frequently work with species other than ''E. coli'', if only to clone some interesting gene product, a set of such sheets could be built up to facilitate synthetic biology in a more diverse set of organisms.<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Lauren