http://2008.igem.org/wiki/index.php?title=Special:Contributions/Mamut&feed=atom&limit=50&target=Mamut&year=&month=2008.igem.org - User contributions [en]2024-03-29T10:03:41ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:Harvard/Parts/TempsenseciTeam:Harvard/Parts/Tempsenseci2008-10-30T04:58:37Z<p>Mamut: /* BBa_K098988 */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Thermoinducible cI System=<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<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 />
We had previously tried to use the thermoinducible lac system in the Registry ([http://partsregistry.org/Part:BBa_J06912 BBa_J06912] and [http://partsregistry.org/Part:BBa_J06911 BBa_J06911]). However, induction tests, a [https://2008.igem.org/Team:Harvard/Dailybook/Week6/Chemical_and_Light#Western_Blot Western blot], and sequencing confirmed that these parts are not functional. We thus focused our efforts on creating a thermosensitive cI system. <br />
<br />
===BBa_K098995===<br />
This is a thermosensitive cI inducible system driven by a strong promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:122.png|thumb|650px|center|BBa_K098995]]</div><br />
<br />
[http://partsregistry.org/Part:BBa_K098993 BBa_K098993] is similar, with the strong promoter at the beginning of the part replaced by a weak promoter.<br />
<br />
==Induction Test for Thermosensitive cI Systems with GFP Reporters==<br />
An induction test was designed to test the inducibility of the heat sensitive cI systems. Two constructs were made:<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a strong promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:124.png|thumb|650px|center|BBa_K098988]]</div><br />
<br />
[http://partsregistry.org/Part:BBa_K098987 BBa_K098987] is equivalent, except that a weak promoter replaces the strong promoter at the beginning of the BBa_K099988.<br />
<br />
===Experimental Design===<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo.png|thumb|150px|Thermoinduction Experimental Design]]</div><br />
Starter cultures of E. coli with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098988| BBa_K098988] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098987| BBa_K098987] were grown overnight. They were then diluted and grown to OD 0.2 before separation into induced (40 ºC) and uninduced cultures (30 ºC). OD and GFP readings were taken at time 0, 2, and 4 hours. Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in their respective incubators until the end of the experiment, when OD and GFP readings were taken.<br />
<br />
===Results===<br />
Induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. While levels of GFP expression in the GFP+ control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]) went down in the samples at 40 ºC, levels in the inducible systems increased slightly. Note that the ''absolute levels'' of GFP expression do not increase much relative to 30 ºC (see our raw data at at the bottom of the page), but the increase in GFP expression is significantly different behavior from the decrease observed in the GFP+ control. We hypothesize that elevated temperature affects the GFP expression (e.g. by disrupting protein folding), and that even the small increase in GFP expression with our systems indicates effective induction. However, this does suggest that such a system is not optimal for inducing the expressing of heat-sensitive proteins.<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:CIts.png|780px|thumb|center|A comparison of GFP expression following thermoinduction of cells harboring BBa_K098987, BBa_K098988, a constitutive GFP generator (GFP+ control), and a plasmid not encoding GFP (GFP- control). The GFP readings were normalized by culture OD.]]</div><br />
<br />
<br />
Should it help you, we also have the raw values graphed below. Note that a slight decrease in baseline fluorescence was also observed in negative control GFP- cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]).<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo_all.jpg|720px|thumb|center|Thermoinduction, raw data]]</div><br />
<br />
Results from the 1000x dilutions were inconclusive because the E. coli grows much slower at 30 ºC than at 40 ºC, so by the end of the experiment, there was a vast difference in cell concentration between the two sets of samples.<br />
<br />
|}<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/TempsenseciTeam:Harvard/Parts/Tempsenseci2008-10-30T04:58:16Z<p>Mamut: /* BBa_K098988 */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Thermoinducible cI System=<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<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 />
We had previously tried to use the thermoinducible lac system in the Registry ([http://partsregistry.org/Part:BBa_J06912 BBa_J06912] and [http://partsregistry.org/Part:BBa_J06911 BBa_J06911]). However, induction tests, a [https://2008.igem.org/Team:Harvard/Dailybook/Week6/Chemical_and_Light#Western_Blot Western blot], and sequencing confirmed that these parts are not functional. We thus focused our efforts on creating a thermosensitive cI system. <br />
<br />
===BBa_K098995===<br />
This is a thermosensitive cI inducible system driven by a strong promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:122.png|thumb|650px|center|BBa_K098995]]</div><br />
<br />
[http://partsregistry.org/Part:BBa_K098993 BBa_K098993] is similar, with the strong promoter at the beginning of the part replaced by a weak promoter.<br />
<br />
==Induction Test for Thermosensitive cI Systems with GFP Reporters==<br />
An induction test was designed to test the inducibility of the heat sensitive cI systems. Two constructs were made:<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a strong promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:124.png|thumb|650px|center|BBa_K098988]]</div><br />
<br />
[http://partsregistry.org/Part:BBa_K098987 BBa_K098987] is equivalent, except that a weak promoter replaces the strong promoter at the beginning of the BBa_K099987.<br />
<br />
===Experimental Design===<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo.png|thumb|150px|Thermoinduction Experimental Design]]</div><br />
Starter cultures of E. coli with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098988| BBa_K098988] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098987| BBa_K098987] were grown overnight. They were then diluted and grown to OD 0.2 before separation into induced (40 ºC) and uninduced cultures (30 ºC). OD and GFP readings were taken at time 0, 2, and 4 hours. Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in their respective incubators until the end of the experiment, when OD and GFP readings were taken.<br />
<br />
===Results===<br />
Induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. While levels of GFP expression in the GFP+ control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]) went down in the samples at 40 ºC, levels in the inducible systems increased slightly. Note that the ''absolute levels'' of GFP expression do not increase much relative to 30 ºC (see our raw data at at the bottom of the page), but the increase in GFP expression is significantly different behavior from the decrease observed in the GFP+ control. We hypothesize that elevated temperature affects the GFP expression (e.g. by disrupting protein folding), and that even the small increase in GFP expression with our systems indicates effective induction. However, this does suggest that such a system is not optimal for inducing the expressing of heat-sensitive proteins.<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:CIts.png|780px|thumb|center|A comparison of GFP expression following thermoinduction of cells harboring BBa_K098987, BBa_K098988, a constitutive GFP generator (GFP+ control), and a plasmid not encoding GFP (GFP- control). The GFP readings were normalized by culture OD.]]</div><br />
<br />
<br />
Should it help you, we also have the raw values graphed below. Note that a slight decrease in baseline fluorescence was also observed in negative control GFP- cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]).<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo_all.jpg|720px|thumb|center|Thermoinduction, raw data]]</div><br />
<br />
Results from the 1000x dilutions were inconclusive because the E. coli grows much slower at 30 ºC than at 40 ºC, so by the end of the experiment, there was a vast difference in cell concentration between the two sets of samples.<br />
<br />
|}<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/TempsenseciTeam:Harvard/Parts/Tempsenseci2008-10-30T04:56:28Z<p>Mamut: /* BBa_K098995 */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Thermoinducible cI System=<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<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 />
We had previously tried to use the thermoinducible lac system in the Registry ([http://partsregistry.org/Part:BBa_J06912 BBa_J06912] and [http://partsregistry.org/Part:BBa_J06911 BBa_J06911]). However, induction tests, a [https://2008.igem.org/Team:Harvard/Dailybook/Week6/Chemical_and_Light#Western_Blot Western blot], and sequencing confirmed that these parts are not functional. We thus focused our efforts on creating a thermosensitive cI system. <br />
<br />
===BBa_K098995===<br />
This is a thermosensitive cI inducible system driven by a strong promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:122.png|thumb|650px|center|BBa_K098995]]</div><br />
<br />
[http://partsregistry.org/Part:BBa_K098993 BBa_K098993] is similar, with the strong promoter at the beginning of the part replaced by a weak promoter.<br />
<br />
==Induction Test for Thermosensitive cI Systems with GFP Reporters==<br />
An induction test was designed to test the inducibility of the heat sensitive cI systems. Two constructs were made:<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a strong promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:124.png|thumb|650px|center|BBa_K098988]]</div><br />
<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a weak promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:125.png|thumb|650px|center|BBa_K098987]]</div><br />
<br />
===Experimental Design===<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo.png|thumb|150px|Thermoinduction Experimental Design]]</div><br />
Starter cultures of E. coli with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098988| BBa_K098988] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098987| BBa_K098987] were grown overnight. They were then diluted and grown to OD 0.2 before separation into induced (40 ºC) and uninduced cultures (30 ºC). OD and GFP readings were taken at time 0, 2, and 4 hours. Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in their respective incubators until the end of the experiment, when OD and GFP readings were taken.<br />
<br />
===Results===<br />
Induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. While levels of GFP expression in the GFP+ control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]) went down in the samples at 40 ºC, levels in the inducible systems increased slightly. Note that the ''absolute levels'' of GFP expression do not increase much relative to 30 ºC (see our raw data at at the bottom of the page), but the increase in GFP expression is significantly different behavior from the decrease observed in the GFP+ control. We hypothesize that elevated temperature affects the GFP expression (e.g. by disrupting protein folding), and that even the small increase in GFP expression with our systems indicates effective induction. However, this does suggest that such a system is not optimal for inducing the expressing of heat-sensitive proteins.<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:CIts.png|780px|thumb|center|A comparison of GFP expression following thermoinduction of cells harboring BBa_K098987, BBa_K098988, a constitutive GFP generator (GFP+ control), and a plasmid not encoding GFP (GFP- control). The GFP readings were normalized by culture OD.]]</div><br />
<br />
<br />
Should it help you, we also have the raw values graphed below. Note that a slight decrease in baseline fluorescence was also observed in negative control GFP- cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]).<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo_all.jpg|720px|thumb|center|Thermoinduction, raw data]]</div><br />
<br />
Results from the 1000x dilutions were inconclusive because the E. coli grows much slower at 30 ºC than at 40 ºC, so by the end of the experiment, there was a vast difference in cell concentration between the two sets of samples.<br />
<br />
|}<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/TempsenseciTeam:Harvard/Parts/Tempsenseci2008-10-30T04:56:17Z<p>Mamut: /* BBa_K098993 */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Thermoinducible cI System=<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<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 />
We had previously tried to use the thermoinducible lac system in the Registry ([http://partsregistry.org/Part:BBa_J06912 BBa_J06912] and [http://partsregistry.org/Part:BBa_J06911 BBa_J06911]). However, induction tests, a [https://2008.igem.org/Team:Harvard/Dailybook/Week6/Chemical_and_Light#Western_Blot Western blot], and sequencing confirmed that these parts are not functional. We thus focused our efforts on creating a thermosensitive cI system. <br />
<br />
===BBa_K098995===<br />
This is a thermosensitive cI inducible system driven by a strong promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:122.png|thumb|650px|center|BBa_K098995]]</div><br />
<br />
<br />
[http://partsregistry.org/Part:BBa_K098993 BBa_K098993] is similar, with the strong promoter at the beginning of the part replaced by a weak promoter.<br />
<br />
==Induction Test for Thermosensitive cI Systems with GFP Reporters==<br />
An induction test was designed to test the inducibility of the heat sensitive cI systems. Two constructs were made:<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a strong promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:124.png|thumb|650px|center|BBa_K098988]]</div><br />
<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a weak promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:125.png|thumb|650px|center|BBa_K098987]]</div><br />
<br />
===Experimental Design===<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo.png|thumb|150px|Thermoinduction Experimental Design]]</div><br />
Starter cultures of E. coli with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098988| BBa_K098988] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098987| BBa_K098987] were grown overnight. They were then diluted and grown to OD 0.2 before separation into induced (40 ºC) and uninduced cultures (30 ºC). OD and GFP readings were taken at time 0, 2, and 4 hours. Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in their respective incubators until the end of the experiment, when OD and GFP readings were taken.<br />
<br />
===Results===<br />
Induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. While levels of GFP expression in the GFP+ control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]) went down in the samples at 40 ºC, levels in the inducible systems increased slightly. Note that the ''absolute levels'' of GFP expression do not increase much relative to 30 ºC (see our raw data at at the bottom of the page), but the increase in GFP expression is significantly different behavior from the decrease observed in the GFP+ control. We hypothesize that elevated temperature affects the GFP expression (e.g. by disrupting protein folding), and that even the small increase in GFP expression with our systems indicates effective induction. However, this does suggest that such a system is not optimal for inducing the expressing of heat-sensitive proteins.<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:CIts.png|780px|thumb|center|A comparison of GFP expression following thermoinduction of cells harboring BBa_K098987, BBa_K098988, a constitutive GFP generator (GFP+ control), and a plasmid not encoding GFP (GFP- control). The GFP readings were normalized by culture OD.]]</div><br />
<br />
<br />
Should it help you, we also have the raw values graphed below. Note that a slight decrease in baseline fluorescence was also observed in negative control GFP- cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]).<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo_all.jpg|720px|thumb|center|Thermoinduction, raw data]]</div><br />
<br />
Results from the 1000x dilutions were inconclusive because the E. coli grows much slower at 30 ºC than at 40 ºC, so by the end of the experiment, there was a vast difference in cell concentration between the two sets of samples.<br />
<br />
|}<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T04:53:39Z<p>Mamut: /* LacI Inducible System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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|[http://partsregistry.org/Part:BBa_K098984 BBa_K098984] with BioBrick Prefix and Suffix, an example of the circuity we engineered. [http://partsregistry.org/Part:BBa_K098983 BBa_K098983] is similar, with a weaker promoter driving lacI expression.]]</div><br />
<br />
We didn't have enough time to finish thoroughly testing the above system, as the cloning of mtrB (toxic in ''E. coli'') was rather difficult. Our preliminary findings are quite interesting, though, so check out our [[Team:Harvard/Project| Project page]].<br />
<br />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.<br />
<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]<br>This data is also available in [https://static.igem.org/mediawiki/2008/f/fa/Baseline-corrected_of_lac_system.pdf PDF format].]]</div><br />
<br />
<br />
<br />
Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/OtherTeam:Harvard/Parts/Other2008-10-30T04:51:39Z<p>Mamut: /* Light-induced System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Our other parts=<br />
==TetR-based Inducible System==<br />
The TetR system we hoped to develop would have been similar to the lacI system. The pTet promoter which controls mtrB expression is regulated by the repressor TetR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0040| BBa_C0040]). Expression of mtrB would have occurred when the system is induced with anhydrotetracycline which binds TetR. Unfortunately, as mtrB is toxic, we could not clone successful clone this system.<br />
<br />
However, we did submit parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098980 BBa_K098980] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981 BBa_K098981] in case you might find them useful.<br />
==Light-induced System==<br />
We tried to create a light-induced mtrB expression system using the [http://partsregistry.org/Featured_Parts:Light_Sensor light sensor] in the registry. This requires an EnvZ deficient strain of ''S. oneidensis'' which we could not successfully generate. Additionally, the registry versions of some of the parts appear wrong (in sequence) or nonfunctional. We thus provide the following two parts for future teams' use:<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098011 ''E. coli'' ompR for introduction into other species of bacteria]<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098010 A HO-pcyA protein generator]<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/OtherTeam:Harvard/Parts/Other2008-10-30T04:51:05Z<p>Mamut: /* Light-induced System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Our other parts=<br />
==TetR-based Inducible System==<br />
The TetR system we hoped to develop would have been similar to the lacI system. The pTet promoter which controls mtrB expression is regulated by the repressor TetR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0040| BBa_C0040]). Expression of mtrB would have occurred when the system is induced with anhydrotetracycline which binds TetR. Unfortunately, as mtrB is toxic, we could not clone successful clone this system.<br />
<br />
However, we did submit parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098980 BBa_K098980] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981 BBa_K098981] in case you might find them useful.<br />
==Light-induced System==<br />
We tried to create a light-induced mtrB expression system using the [http://partsregistry.org/Featured_Parts:Light_Sensor light sensor] in the registry. This requires an EnvZ deficient strain of ''S. oneidensis'' which we could not successfully generate. Additionally, the registry versions of some of the parts appear wrong (sequence) or nonfunction. We thus provide the following two parts for future teams' use:<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098011 ''E. coli'' ompR for introduction into other species of bacteria]<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098010 A HO-pcyA protein generator]<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/OtherTeam:Harvard/Parts/Other2008-10-30T04:50:14Z<p>Mamut: /* Light-induced System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Our other parts=<br />
==TetR-based Inducible System==<br />
The TetR system we hoped to develop would have been similar to the lacI system. The pTet promoter which controls mtrB expression is regulated by the repressor TetR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0040| BBa_C0040]). Expression of mtrB would have occurred when the system is induced with anhydrotetracycline which binds TetR. Unfortunately, as mtrB is toxic, we could not clone successful clone this system.<br />
<br />
However, we did submit parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098980 BBa_K098980] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981 BBa_K098981] in case you might find them useful.<br />
==Light-induced System==<br />
We tried to create a light-induced mtrB expression system using the [http://partsregistry.org/Featured_Parts:Light_Sensor light sensor] in the registry. This requires an EnvZ deficient strain of ''S. oneidensis'' which we could not successfully generate. Additionally, the registry versions of some of the parts appear wrong (sequence) or nonfunction. We thus provide the following two parts for future teams' use:<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098011 ''E. coli'' ompR for introduction into other species of bacteria<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098010 A HO-pcyA protein generator]<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T04:48:56Z<p>Mamut: /* LacI Inducible System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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, an example of the circuity we engineered. BBa_K098983 is similar, with a weaker promoter driving lacI expression.]]</div><br />
<br />
We didn't have enough time to finish thoroughly testing the above system, as the cloning of mtrB (toxic in ''E. coli'') was rather difficult. Our preliminary findings are quite interesting, though, so check out our [[Team:Harvard/Project| Project page]].<br />
<br />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.<br />
<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]<br>This data is also available in [https://static.igem.org/mediawiki/2008/f/fa/Baseline-corrected_of_lac_system.pdf PDF format].]]</div><br />
<br />
<br />
<br />
Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T04:48:43Z<p>Mamut: /* LacI Inducible System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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, an example of the circuity we engineered. BBa_K098983 is similar, with a weaker promoter driving lacI expression.]]</div><br />
<br />
We didn't have enough time to finish thoroughly testing the above system, as the cloning of mtrB (toxic in ''E. coli'') was rather difficult. Our preliminary findings are quite interesting, though, so check out our [Team:Harvard/Project| Project page].<br />
<br />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.<br />
<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]<br>This data is also available in [https://static.igem.org/mediawiki/2008/f/fa/Baseline-corrected_of_lac_system.pdf PDF format].]]</div><br />
<br />
<br />
<br />
Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T04:48:24Z<p>Mamut: /* LacI Inducible System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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, an example of the circuity we engineered. BBa_K098983 is similar, with a weaker promoter driving lacI expression.]]</div><br />
<br />
We didn't have enough time to finish thoroughly testing the above system, as the cloning of mtrB (toxic in ''E. coli'') was rather difficult. Our preliminary findings are quite interesting, though, so check out our [Team:Harvard/Project Project page].<br />
<br />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.<br />
<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]<br>This data is also available in [https://static.igem.org/mediawiki/2008/f/fa/Baseline-corrected_of_lac_system.pdf PDF format].]]</div><br />
<br />
<br />
<br />
Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T04:44:19Z<p>Mamut: /* Co-Culture Experiment */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<br />
The metabolically versatile bacterium ''Shewanella oneidensis'' adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, ''S. oneidensis'' transfers electrons to an electrode of a microbial fuel cell. We sought to engineer ''S. oneidensis'' to report variations in environmental conditions through changes in current production. A previous study has shown that ''S. oneidensis'' mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.<br />
<br />
==Experimental overview==<br />
<br />
We attempted to develop three inducible systems for electrical current production in ''S. oneidensis''. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression in mtrB knock-out (KO) ''S. oneidensis'' can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach uses temperature-senstive cI which would allow for an increase in electrical output in response to heat. The third is a light-inducible system based on the 2005 UT Austin biological camera.<br />
<br />
Using the LacI system, we attempted to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we built, measuring the effects of IPTG on current production in wild type and mtrB KO ''S. oneidensis'' containing plasmids expressing inducible or constitutive mtrB.<br />
<br />
==Results==<br />
<br />
===Wildtype vs. mtrB deficient ''S. oneidensis''===<br />
<br />
''Overview: By genetically manipulating S. oneidensis, it has been found that noticeable differences of current production can be detected. We wish to control current production by introducing inducible systems in these mutant strains of S. oneidensis. These differences of current production can be detected by a computer, allowing biological systems and electrical devices to be integrated.''<br />
<br />
The basis of our project lies in the discovery that by knocking out the mtrB gene, a current production gene of S. oneidensis, noticeable differences of current production can be detected. Therefore, this allows the current production of S. oneidensis to act more than just an on/off switch.<br />
<br />
The first step of our project was then to test this hypothesis and also to understand the current production behavior of wildtype S. oneidensis and the mutant S. oneidensis (annotated as mtrB). Therefore, we ran a test with microbial fuel cells, one strain in each cell, and allowed the current production to be measured for approximately one day. In literature, mtrB produces approximately 20 to 25 percent of the current or wildtype strains.<br />
<br />
<br />
[[Image: wt_mtrB.jpg|700px]]<br />
<br />
<br />
As shown in the graph above, the results of our experiment matched those in literature. We also observed that wildtype and mtrB strains have very different current production behavior, with wildtype producing current immediately after lactate injection, and mtrB producing current much more gradually. This difference in behavior can allow for more accurate differentiation of current production between wildtype and mtrB strains when integrated with electrical devices. We wish then build on these results and build inducible systems that will allow us to control the current production.<br />
<br />
===Lac-inducible Strains===<br />
<br />
''Overview: We genetically engineered mtrB knock-out S. oneidensis MR-1 by introducing the mtrB gene on a lactose-inducible system. Specifically, we tested engineered mtrB knock-out S. oneidensis MR-1 with high lacQPI at the p15A origin. Our results show the possibility that we successfully complemented the mtrB knock-out as high levels of current were detected in one such strain.''<br />
<br />
Our end goal was to develop inducible systems for electrical current production in S. oneidensis MR-1. In our microbial fuel cells, we were able to test a lac-inducible system where LacI repression of the current-production gene expression in mtrB knock-out S. oneidensis MR-1 would be alleviated by the addition of IPTG. That is, the addition if IPTG to such a system would induce current production as the bacteria would begin breaking down lactate.<br />
<br />
In this experiment, we tested the following combinations:<br />
<br />
1. wt S. oneidensis MR-1 + lactate <br />
<br />
2. mtrB knock-out S. oneidensis MR-1 with lactate <br />
<br />
3. mtrB (with high Lac promoter) with lactate and IPTG<br />
<br />
4. mtrB (with high Lac promoter) with lactate<br />
<br />
We expected combination 1 to be highest, peaking between 100 to 200 microAmps. Combination 2 would set the baseline for the mtrB knock-out strain, usually peaking at 20 to 25 percent of wt S. oneidensis MR-1’s level. If the introduction of mtrB on a lactose-inducible system was successful, then combination 3 would also be high, roughly around the level of wt S. oneidensis MR-1. Combination 4, however, would be expected to peak around the mtrB knock-out strain’s current level as it did not receive IPTG and would not be induced.<br />
<br />
[[ Image: Lacinducible.jpg | 700px]]<br />
<br />
As expected, wt S. oneidensis MR-1 and the mtrB knock-out strain reached their expected levels. Interestingly, combination 4, but not combination 3, reached current levels around 200 microAmps, even greater than wt S. oneidensis MR-1’s level. That is, the engineered mtrB knock-out S. oneidensis MR-1 that did not receive IPTG resulted in elevated current production. The temporal dynamics of the curve was what we would expect from IPTG induction. That is, a delay would be observed as IPTG induction took place turning on the promoter relative to the non-inducible wt S. oneidensis MR-1. <br />
<br />
A possible, and exciting, explanation for this observance may stem from the fact that the repressor used in our engineered strain had an LVA tag which marks it for degradation. As the experiment progressed, the repressor proteins in combination 4 may have degraded, which allowed for lactate breakdown and thus current production. The implication is that if the current production observed is due to the degradation of the repressor protein, then we successfully complemented the mtrB knock-out. We must note that if this were the case, then we should have seen an increase in all strains of the engineered mtrB knock-out if the repressor protein was being degraded. It may be that we would have observed this increase if we had run the experiment for a longer period. Future experiments may shed more light on this observance.<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 | 750px ]]<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>Mamuthttp://2008.igem.org/File:Picture_5.pngFile:Picture 5.png2008-10-30T04:43:41Z<p>Mamut: uploaded a new version of "Image:Picture 5.png"</p>
<hr />
<div></div>Mamuthttp://2008.igem.org/File:Hspacer.pngFile:Hspacer.png2008-10-30T04:38:50Z<p>Mamut: uploaded a new version of "Image:Hspacer.png"</p>
<hr />
<div></div>Mamuthttp://2008.igem.org/Team:HarvardTeam:Harvard2008-10-30T04:38:20Z<p>Mamut: </p>
<hr />
<div><html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #cccccc;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
<br />
<div style="position: absolute; left: 60px; top: 320px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/cc/Bactricity.jpg"><br />
</div><br />
<br />
<div style="position: absolute; left: 100px; top: 880px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/0/06/Thanks_border.jpg"><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 85px; top: 950px; height: 400px; width: 400px; padding: 1em;"><br />
<center><br />
<font style="line-height:170%"><br />
Alain Viel,<br><br />
Orianna Bretschger,<br />
<br>Daad Saffarini,<br />
<br>Helen White,<br />
<br>Remy Chait,<br />
<br>Natalie Farny,<br />
<br>Christina Agapakis,<br />
<br>Jason Lohmueller,<br />
<br>Kim de Mora,<br />
<br>Colleen Hansel,<br />
<br>Peter Girguis,<br />
<br>Christopher Marx,<br />
<br>George Church,<br />
<br>Jagesh V. Shah,<br />
<br>Pam Silver,<br />
<br>Tamara Brenner,<br />
<br>Harvard BioLabs<br />
</font><br />
</center><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 580px; top: 300px; width:300px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Project"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d3/Bactricitynutshell.jpg"></a><br />
</div><br />
<div style="position: absolute; left: 640px; top: 487px; width:180px; padding: .5em;"><br />
<font size=1>Our project sought to combine the detecting capabilities of bacteria with the speed and ubiquity of electricity by creating an inducible system in Shewanella oneidensis MR-1 with an electrical output, allowing for the direct integration of this biosensor with electrical circuits via microbial fuel cells.</font><br />
</div><br />
<br />
<br />
<div style="position: absolute; left: 480px; top: 700px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Shewie"><br />
<img src="https://static.igem.org/mediawiki/2008/6/65/Mainshewie.gif"></a><br />
</div><br />
<div style="position: absolute; left: 510px; top: 800px; width=200px; padding: 1em;"><br />
<font size=1><br />
Shewanella oneidensis MR-1 <br><br />
(fondly referred to as Shewie)<br><br />
is a metabolically versatile, <br><br />
and genetically tractable, gram-<br><br />
negative facultative anaerobe which under <br><br />
anaerobic conditions reduces a number of electron <br><br />
acceptors. This ability can be harnessed by <br><br />
microbial fuel cells to produce an electric current.<br />
</font><br />
</div><br />
<div style="position: absolute; left: 480px; top: 1000px; padding: 1em;"><br />
<a href ="https://2008.igem.org/Team:Harvard/Hardware"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d8/Fuelcellfun.gif"><br />
</a><br />
</div><br />
<div style="position: absolute; left: 550px; top: 1190px; width:200px; padding: 1em;"><br />
<font size=1><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. The answer? Microbial fuel cells.<br />
</div><br />
</div><br />
<div style="position: absolute; left: 100px; top: 800px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/d/da/Igemthanks.jpg"><br />
</div><br />
<br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; font-color:#cccccc; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<font color=#ffffff>ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss </font><br />
<br><br />
[[Image:hspacer.png]]<br />
<br><br />
<br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/File:Hspacer.pngFile:Hspacer.png2008-10-30T04:37:47Z<p>Mamut: uploaded a new version of "Image:Hspacer.png"</p>
<hr />
<div></div>Mamuthttp://2008.igem.org/Team:HarvardTeam:Harvard2008-10-30T04:36:53Z<p>Mamut: </p>
<hr />
<div><html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #cccccc;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
<br />
<div style="position: absolute; left: 60px; top: 320px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/cc/Bactricity.jpg"><br />
</div><br />
<br />
<div style="position: absolute; left: 100px; top: 880px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/0/06/Thanks_border.jpg"><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 85px; top: 950px; height: 400px; width: 400px; padding: 1em;"><br />
<center><br />
<font style="line-height:170%"><br />
Alain Viel,<br><br />
Orianna Bretschger,<br />
<br>Daad Saffarini,<br />
<br>Helen White,<br />
<br>Remy Chait,<br />
<br>Natalie Farny,<br />
<br>Christina Agapakis,<br />
<br>Jason Lohmueller,<br />
<br>Kim de Mora,<br />
<br>Colleen Hansel,<br />
<br>Peter Girguis,<br />
<br>Christopher Marx,<br />
<br>George Church,<br />
<br>Jagesh V. Shah,<br />
<br>Pam Silver,<br />
<br>Tamara Brenner,<br />
<br>Harvard BioLabs<br />
</font><br />
</center><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 580px; top: 300px; width:300px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Project"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d3/Bactricitynutshell.jpg"></a><br />
</div><br />
<div style="position: absolute; left: 640px; top: 495px; width:180px; padding: .5em;"><br />
<font size=1>Our project sought to combine the detecting capabilities of bacteria with the speed and ubiquity of electricity by creating an inducible system in Shewanella oneidensis MR-1 with an electrical output, allowing for the direct integration of this biosensor with electrical circuits via microbial fuel cells.</font><br />
</div><br />
<br />
<br />
<div style="position: absolute; left: 480px; top: 700px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Shewie"><br />
<img src="https://static.igem.org/mediawiki/2008/6/65/Mainshewie.gif"></a><br />
</div><br />
<div style="position: absolute; left: 510px; top: 800px; width=200px; padding: 1em;"><br />
<font size=1><br />
Shewanella oneidensis MR-1 <br><br />
(fondly referred to as Shewie)<br><br />
is a metabolically versatile, <br><br />
and genetically tractable, gram-<br><br />
negative facultative anaerobe which under <br><br />
anaerobic conditions reduces a number of electron <br><br />
acceptors. This ability can be harnessed by <br><br />
microbial fuel cells to produce an electric current.<br />
</font><br />
</div><br />
<div style="position: absolute; left: 480px; top: 1000px; padding: 1em;"><br />
<a href ="https://2008.igem.org/Team:Harvard/Hardware"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d8/Fuelcellfun.gif"><br />
</a><br />
</div><br />
<div style="position: absolute; left: 550px; top: 1190px; width:200px; padding: 1em;"><br />
<font size=1><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. The answer? Microbial fuel cells.<br />
</div><br />
</div><br />
<div style="position: absolute; left: 100px; top: 800px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/d/da/Igemthanks.jpg"><br />
</div><br />
<br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; font-color:#cccccc; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<font color=#ffffff>ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss </font><br />
<br><br />
[[Image:hspacer.png]]<br />
<br><br />
<br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:HarvardTeam:Harvard2008-10-30T04:35:47Z<p>Mamut: Undo revision 104825 by Mamut (Talk)</p>
<hr />
<div><html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #cccccc;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
<br />
<div style="position: absolute; left: 60px; top: 320px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/cc/Bactricity.jpg"><br />
</div><br />
<br />
<div style="position: absolute; left: 100px; top: 880px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/0/06/Thanks_border.jpg"><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 85px; top: 950px; height: 400px; width: 400px; padding: 1em;"><br />
<center><br />
<font style="line-height:170%"><br />
Alain Viel,<br><br />
Orianna Bretschger,<br />
<br>Daad Saffarini,<br />
<br>Helen White,<br />
<br>Remy Chait,<br />
<br>Natalie Farny,<br />
<br>Christina Agapakis,<br />
<br>Jason Lohmueller,<br />
<br>Kim de Mora,<br />
<br>Colleen Hansel,<br />
<br>Peter Girguis,<br />
<br>Christopher Marx,<br />
<br>George Church,<br />
<br>Jagesh V. Shah,<br />
<br>Pam Silver,<br />
<br>Tamara Brenner,<br />
<br>Harvard BioLabs<br />
</font><br />
</center><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 580px; top: 300px; width:300px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Project"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d3/Bactricitynutshell.jpg"></a><br />
</div><br />
<div style="position: absolute; left: 640px; top: 495px; width:180px; padding: .5em;"><br />
<font size=1>Our project sought to combine the detecting capabilities of bacteria with the speed and ubiquity of electricity by creating an inducible system in Shewanella oneidensis MR-1 with an electrical output, allowing for the direct integration of this biosensor with electrical circuits via microbial fuel cells.</font><br />
</div><br />
<br />
<br />
<div style="position: absolute; left: 480px; top: 700px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Shewie"><br />
<img src="https://static.igem.org/mediawiki/2008/6/65/Mainshewie.gif"></a><br />
</div><br />
<div style="position: absolute; left: 510px; top: 800px; width=200px; padding: 1em;"><br />
<font size=1><br />
Shewanella oneidensis MR-1 <br><br />
(fondly referred to as Shewie)<br><br />
is a metabolically versatile, <br><br />
and genetically tractable, gram-<br><br />
negative facultative anaerobe which under <br><br />
anaerobic conditions reduces a number of electron <br><br />
acceptors. This ability can be harnessed by <br><br />
microbial fuel cells to produce an electric current.<br />
</font><br />
</div><br />
<div style="position: absolute; left: 480px; top: 1000px; padding: 1em;"><br />
<a href ="https://2008.igem.org/Team:Harvard/Hardware"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d8/Fuelcellfun.gif"><br />
</a><br />
</div><br />
<div style="position: absolute; left: 550px; top: 1190px; width:200px; padding: 1em;"><br />
<font size=1><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. The answer? Microbial fuel cells.<br />
</div><br />
</div><br />
<div style="position: absolute; left: 100px; top: 800px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/d/da/Igemthanks.jpg"><br />
</div><br />
<br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; font-color:#cccccc; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<font color=#ffffff>ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss </font><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:HarvardTeam:Harvard2008-10-30T04:34:41Z<p>Mamut: Undo revision 104713 by Mamut (Talk)</p>
<hr />
<div><html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #cccccc;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
<br />
<div style="position: absolute; left: 60px; top: 320px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/cc/Bactricity.jpg"><br />
</div><br />
<br />
<div style="position: absolute; left: 100px; top: 880px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/0/06/Thanks_border.jpg"><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 85px; top: 950px; height: 400px; width: 400px; padding: 1em;"><br />
<center><br />
<font style="line-height:170%"><br />
Alain Viel,<br><br />
Orianna Bretschger,<br />
<br>Daad Saffarini,<br />
<br>Helen White,<br />
<br>Remy Chait,<br />
<br>Natalie Farny,<br />
<br>Christina Agapakis,<br />
<br>Jason Lohmueller,<br />
<br>Kim de Mora,<br />
<br>Colleen Hansel,<br />
<br>Peter Girguis,<br />
<br>Christopher Marx,<br />
<br>George Church,<br />
<br>Jagesh V. Shah,<br />
<br>Pam Silver,<br />
<br>Tamara Brenner,<br />
<br>Harvard BioLabs<br />
</font><br />
</center><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 580px; top: 300px; width:300px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Project"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d3/Bactricitynutshell.jpg"></a><br />
</div><br />
<div style="position: absolute; left: 640px; top: 495px; width:180px; padding: .5em;"><br />
<font size=1>Our project sought to combine the detecting capabilities of bacteria with the speed and ubiquity of electricity by creating an inducible system in Shewanella oneidensis MR-1 with an electrical output, allowing for the direct integration of this biosensor with electrical circuits via microbial fuel cells.</font><br />
</div><br />
<br />
<br />
<div style="position: absolute; left: 480px; top: 700px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Shewie"><br />
<img src="https://static.igem.org/mediawiki/2008/6/65/Mainshewie.gif"></a><br />
</div><br />
<div style="position: absolute; left: 510px; top: 800px; width=200px; padding: 1em;"><br />
<font size=1><br />
Shewanella oneidensis MR-1 <br><br />
(fondly referred to as Shewie)<br><br />
is a metabolically versatile, <br><br />
and genetically tractable, gram-<br><br />
negative facultative anaerobe which under <br><br />
anaerobic conditions reduces a number of electron <br><br />
acceptors. This ability can be harnessed by <br><br />
microbial fuel cells to produce an electric current.<br />
</font><br />
</div><br />
<div style="position: absolute; left: 480px; top: 1000px; padding: 1em;"><br />
<a href ="https://2008.igem.org/Team:Harvard/Hardware"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d8/Fuelcellfun.gif"><br />
</a><br />
</div><br />
<div style="position: absolute; left: 550px; top: 1190px; width:200px; padding: 1em;"><br />
<font size=1><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. The answer? Microbial fuel cells.<br />
</div><br />
</div><br />
<div style="position: absolute; left: 100px; top: 800px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/d/da/Igemthanks.jpg"><br />
</div><br />
<br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; font-color:#cccccc; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
[[Image:Hspacer.png]]<br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:HarvardTeam:Harvard2008-10-30T04:34:33Z<p>Mamut: </p>
<hr />
<div><html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #cccccc;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
<br />
<div style="position: absolute; left: 60px; top: 320px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/cc/Bactricity.jpg"><br />
</div><br />
<br />
<div style="position: absolute; left: 100px; top: 880px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/0/06/Thanks_border.jpg"><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 85px; top: 950px; height: 400px; width: 400px; padding: 1em;"><br />
<center><br />
<font style="line-height:170%"><br />
Alain Viel,<br><br />
Orianna Bretschger,<br />
<br>Daad Saffarini,<br />
<br>Helen White,<br />
<br>Remy Chait,<br />
<br>Natalie Farny,<br />
<br>Christina Agapakis,<br />
<br>Jason Lohmueller,<br />
<br>Kim de Mora,<br />
<br>Colleen Hansel,<br />
<br>Peter Girguis,<br />
<br>Christopher Marx,<br />
<br>George Church,<br />
<br>Jagesh V. Shah,<br />
<br>Pam Silver,<br />
<br>Tamara Brenner,<br />
<br>Harvard BioLabs<br />
</font><br />
</center><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 580px; top: 300px; width:300px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Project"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d3/Bactricitynutshell.jpg"></a><br />
</div><br />
<div style="position: absolute; left: 640px; top: 487px; width:180px; padding: .5em;"><br />
<font size=1>Our project sought to combine the detecting capabilities of bacteria with the speed and ubiquity of electricity by creating an inducible system in Shewanella oneidensis MR-1 with an electrical output, allowing for the direct integration of this biosensor with electrical circuits via microbial fuel cells.</font><br />
</div><br />
<br />
<br />
<div style="position: absolute; left: 480px; top: 700px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Shewie"><br />
<img src="https://static.igem.org/mediawiki/2008/6/65/Mainshewie.gif"></a><br />
</div><br />
<div style="position: absolute; left: 510px; top: 800px; width=200px; padding: 1em;"><br />
<font size=1><br />
Shewanella oneidensis MR-1 <br><br />
(fondly referred to as Shewie)<br><br />
is a metabolically versatile, <br><br />
and genetically tractable, gram-<br><br />
negative facultative anaerobe which under <br><br />
anaerobic conditions reduces a number of electron <br><br />
acceptors. This ability can be harnessed by <br><br />
microbial fuel cells to produce an electric current.<br />
</font><br />
</div><br />
<div style="position: absolute; left: 480px; top: 1000px; padding: 1em;"><br />
<a href ="https://2008.igem.org/Team:Harvard/Hardware"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d8/Fuelcellfun.gif"><br />
</a><br />
</div><br />
<div style="position: absolute; left: 550px; top: 1190px; width:200px; padding: 1em;"><br />
<font size=1><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. The answer? Microbial fuel cells.<br />
</div><br />
</div><br />
<div style="position: absolute; left: 100px; top: 800px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/d/da/Igemthanks.jpg"><br />
</div><br />
<br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; font-color:#cccccc; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
[[Image:Hspacer.png]]<br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/File:Hspacer.pngFile:Hspacer.png2008-10-30T04:33:42Z<p>Mamut: </p>
<hr />
<div></div>Mamuthttp://2008.igem.org/Team:HarvardTeam:Harvard2008-10-30T04:25:29Z<p>Mamut: </p>
<hr />
<div><html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #cccccc;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
<br />
<div style="position: absolute; left: 60px; top: 320px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/cc/Bactricity.jpg"><br />
</div><br />
<br />
<div style="position: absolute; left: 100px; top: 880px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/0/06/Thanks_border.jpg"><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 85px; top: 950px; height: 400px; width: 400px; padding: 1em;"><br />
<center><br />
<font style="line-height:170%"><br />
Alain Viel,<br><br />
Orianna Bretschger,<br />
<br>Daad Saffarini,<br />
<br>Helen White,<br />
<br>Remy Chait,<br />
<br>Natalie Farny,<br />
<br>Christina Agapakis,<br />
<br>Jason Lohmueller,<br />
<br>Kim de Mora,<br />
<br>Colleen Hansel,<br />
<br>Peter Girguis,<br />
<br>Christopher Marx,<br />
<br>George Church,<br />
<br>Jagesh V. Shah,<br />
<br>Pam Silver,<br />
<br>Tamara Brenner,<br />
<br>Harvard BioLabs<br />
</font><br />
</center><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 580px; top: 300px; width:300px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Project"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d3/Bactricitynutshell.jpg"></a><br />
</div><br />
<div style="position: absolute; left: 640px; top: 487px; width:180px; padding: .5em;"><br />
<font size=1>Our project sought to combine the detecting capabilities of bacteria with the speed and ubiquity of electricity by creating an inducible system in Shewanella oneidensis MR-1 with an electrical output, allowing for the direct integration of this biosensor with electrical circuits via microbial fuel cells.</font><br />
</div><br />
<br />
<br />
<div style="position: absolute; left: 480px; top: 700px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Shewie"><br />
<img src="https://static.igem.org/mediawiki/2008/6/65/Mainshewie.gif"></a><br />
</div><br />
<div style="position: absolute; left: 510px; top: 800px; width=200px; padding: 1em;"><br />
<font size=1><br />
Shewanella oneidensis MR-1 <br><br />
(fondly referred to as Shewie)<br><br />
is a metabolically versatile, <br><br />
and genetically tractable, gram-<br><br />
negative facultative anaerobe which under <br><br />
anaerobic conditions reduces a number of electron <br><br />
acceptors. This ability can be harnessed by <br><br />
microbial fuel cells to produce an electric current.<br />
</font><br />
</div><br />
<div style="position: absolute; left: 480px; top: 1000px; padding: 1em;"><br />
<a href ="https://2008.igem.org/Team:Harvard/Hardware"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d8/Fuelcellfun.gif"><br />
</a><br />
</div><br />
<div style="position: absolute; left: 550px; top: 1190px; width:200px; padding: 1em;"><br />
<font size=1><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. The answer? Microbial fuel cells.<br />
</div><br />
</div><br />
<div style="position: absolute; left: 100px; top: 800px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/d/da/Igemthanks.jpg"><br />
</div><br />
<br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; font-color:#cccccc; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<font color=#ffffff>ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss </font><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:HarvardTeam:Harvard2008-10-30T04:24:42Z<p>Mamut: </p>
<hr />
<div><html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #cccccc;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
<br />
<div style="position: absolute; left: 60px; top: 320px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/cc/Bactricity.jpg"><br />
</div><br />
<br />
<div style="position: absolute; left: 100px; top: 880px; height: 400px; width: 100px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/0/06/Thanks_border.jpg"><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 85px; top: 950px; height: 400px; width: 400px; padding: 1em;"><br />
<center><br />
<font style="line-height:170%"><br />
Alain Viel,<br><br />
Orianna Bretschger,<br />
<br>Daad Saffarini,<br />
<br>Helen White,<br />
<br>Remy Chait,<br />
<br>Natalie Farny,<br />
<br>Christina Agapakis,<br />
<br>Jason Lohmueller,<br />
<br>Kim de Mora,<br />
<br>Colleen Hansel,<br />
<br>Peter Girguis,<br />
<br>Christopher Marx,<br />
<br>George Church,<br />
<br>Jagesh V. Shah,<br />
<br>Pam Silver,<br />
<br>Tamara Brenner,<br />
<br>Harvard BioLabs<br />
</font><br />
</center><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<div style="position: absolute; left: 580px; top: 300px; width:300px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Project"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d3/Bactricitynutshell.jpg"></a><br />
</div><br />
<div style="position: absolute; left: 640px; top: 495px; width:180px; padding: .5em;"><br />
<font size=1>Our project sought to combine the detecting capabilities of bacteria with the speed and ubiquity of electricity by creating an inducible system in Shewanella oneidensis MR-1 with an electrical output, allowing for the direct integration of this biosensor with electrical circuits via microbial fuel cells.</font><br />
</div><br />
<br />
<br />
<div style="position: absolute; left: 480px; top: 700px; padding: 1em;"><br />
<a href="https://2008.igem.org/Team:Harvard/Shewie"><br />
<img src="https://static.igem.org/mediawiki/2008/6/65/Mainshewie.gif"></a><br />
</div><br />
<div style="position: absolute; left: 510px; top: 800px; width=200px; padding: 1em;"><br />
<font size=1><br />
Shewanella oneidensis MR-1 <br><br />
(fondly referred to as Shewie)<br><br />
is a metabolically versatile, <br><br />
and genetically tractable, gram-<br><br />
negative facultative anaerobe which under <br><br />
anaerobic conditions reduces a number of electron <br><br />
acceptors. This ability can be harnessed by <br><br />
microbial fuel cells to produce an electric current.<br />
</font><br />
</div><br />
<div style="position: absolute; left: 480px; top: 1000px; padding: 1em;"><br />
<a href ="https://2008.igem.org/Team:Harvard/Hardware"><br />
<img src="https://static.igem.org/mediawiki/2008/d/d8/Fuelcellfun.gif"><br />
</a><br />
</div><br />
<div style="position: absolute; left: 550px; top: 1190px; width:200px; padding: 1em;"><br />
<font size=1><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. The answer? Microbial fuel cells.<br />
</div><br />
</div><br />
<div style="position: absolute; left: 100px; top: 800px; padding: 1em;"><br />
<img src="https://static.igem.org/mediawiki/2008/d/da/Igemthanks.jpg"><br />
</div><br />
<br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt; font-color:#cccccc; text-align:justify" cellpadding="50" width="90%"<br />
|<br />
<font color=#ffffff>ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss ssssss </font><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/File:Bactricitynutshell.jpgFile:Bactricitynutshell.jpg2008-10-30T04:23:15Z<p>Mamut: uploaded a new version of "Image:Bactricitynutshell.jpg"</p>
<hr />
<div></div>Mamuthttp://2008.igem.org/Team:Harvard/TeamTeam:Harvard/Team2008-10-30T04:10:34Z<p>Mamut: Undo revision 104484 by Mamut (Talk)</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Team=<br />
<br />
<br />
[[Image:Harvardigem2008teampic.JPG|800px]]<br />
<br />
Left to right, from the back row: Jason, Dan, Sam, Joy, Natalie, Thilini, Meng Xiao, Erica, and Remy. Front row: Amy, Lauren, and Anna Marie.<br />
<br />
==Students==<br />
* Thilini Ariyawansa<br />
* Joy Ding<br />
* Dan Gong<br />
* Meng Xiao He<br />
* Amy Li<br />
* Erica Lin<br />
* Lauren Schumacher<br />
* Anna Marie Wagner<br />
* Sam Workman <br />
<br />
==Teaching Fellows==<br />
<br />
* Remy Chait<br />
* Natalie Farny<br />
* Christina Agapakis<br />
* Jason Lohmueller<br />
* Kim de Mora<br />
<br />
==Advisors==<br />
<br />
* Colleen Hansel<br />
* Peter Girguis<br />
* Christopher Marx<br />
* George Church<br />
* Jagesh V. Shah <br />
* Pamela Silver <br />
* Alain Viel <br />
<br />
==Education Advisor==<br />
<br />
* Tamara Brenner<br />
|}<br />
<br><br><br />
<br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/TeamTeam:Harvard/Team2008-10-30T04:09:44Z<p>Mamut: /* Team */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Team=<br />
<br />
<br />
[[Image:Harvardigem2008teampic.JPG|800px]]<br />
<br />
Left to right, from the back row: Jason, Dan, Sam, Joy, Natalie, Thilini, Meng Xiao, Erica, and Remy. <br />
<br />
Front row: Amy, Lauren, and Anna Marie.<br />
<br />
==Students==<br />
* Thilini Ariyawansa<br />
* Joy Ding<br />
* Dan Gong<br />
* Meng Xiao He<br />
* Amy Li<br />
* Erica Lin<br />
* Lauren Schumacher<br />
* Anna Marie Wagner<br />
* Sam Workman <br />
<br />
==Teaching Fellows==<br />
<br />
* Remy Chait<br />
* Natalie Farny<br />
* Christina Agapakis<br />
* Jason Lohmueller<br />
* Kim de Mora<br />
<br />
==Advisors==<br />
<br />
* Colleen Hansel<br />
* Peter Girguis<br />
* Christopher Marx<br />
* George Church<br />
* Jagesh V. Shah <br />
* Pamela Silver <br />
* Alain Viel <br />
<br />
==Education Advisor==<br />
<br />
* Tamara Brenner<br />
|}<br />
<br><br><br />
<br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T03:58:25Z<p>Mamut: /* mtrB */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- 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 />
==''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on ''mtrB'', which encodes a 679-amino-acid-long outer membrane protein 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 />
Since we found construction intermediates from the registry to be especially useful, we provided our intermediates with mtrB with RBS, terminator, or both.<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 should occur. In this case, the inducer binds the repressor protein, preventing it from binding to the operator within the promoter. RNA polymerase is therefore able to bind to the promoter (green), 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 tried to create such systems capable of being induced by [[Team:Harvard/Parts/LacI| IPTG]], <br />
[[Team:Harvard/Parts/Tempsenseci| heat]], [[Team:Harvard/Parts/Other| tetracycline, and light]].</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/TempsenseciTeam:Harvard/Parts/Tempsenseci2008-10-30T03:57:32Z<p>Mamut: /* Experimental Design */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Thermoinducible cI System=<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<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 />
We had previously tried to use the thermoinducible lac system in the Registry ([http://partsregistry.org/Part:BBa_J06912 BBa_J06912] and [http://partsregistry.org/Part:BBa_J06911 BBa_J06911]). However, induction tests, a [https://2008.igem.org/Team:Harvard/Dailybook/Week6/Chemical_and_Light#Western_Blot Western blot], and sequencing confirmed that these parts are not functional. We thus focused our efforts on creating a thermosensitive cI system. <br />
<br />
===BBa_K098995===<br />
This is a thermosensitive cI inducible system driven by a strong promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:122.png|thumb|650px|center|BBa_K098995]]</div><br />
<br />
===BBa_K098993===<br />
This is a thermosensitive cI inducible system driven by a weak promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:123.png|thumb|650px|center|BBa_K098993]]</div><br />
<br />
==Induction Test for Thermosensitive cI Systems with GFP Reporters==<br />
An induction test was designed to test the inducibility of the heat sensitive cI systems. Two constructs were made:<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a strong promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:124.png|thumb|650px|center|BBa_K098988]]</div><br />
<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a weak promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:125.png|thumb|650px|center|BBa_K098987]]</div><br />
<br />
===Experimental Design===<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo.png|thumb|150px|Thermoinduction Experimental Design]]</div><br />
Starter cultures of E. coli with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098988| BBa_K098988] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098987| BBa_K098987] were grown overnight. They were then diluted and grown to OD 0.2 before separation into induced (40 ºC) and uninduced cultures (30 ºC). OD and GFP readings were taken at time 0, 2, and 4 hours. Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in their respective incubators until the end of the experiment, when OD and GFP readings were taken.<br />
<br />
===Results===<br />
Induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. While levels of GFP expression in the GFP+ control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]) went down in the samples at 40 ºC, levels in the inducible systems increased slightly. Note that the ''absolute levels'' of GFP expression do not increase much relative to 30 ºC (see our raw data at at the bottom of the page), but the increase in GFP expression is significantly different behavior from the decrease observed in the GFP+ control. We hypothesize that elevated temperature affects the GFP expression (e.g. by disrupting protein folding), and that even the small increase in GFP expression with our systems indicates effective induction. However, this does suggest that such a system is not optimal for inducing the expressing of heat-sensitive proteins.<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:CIts.png|780px|thumb|center|A comparison of GFP expression following thermoinduction of cells harboring BBa_K098987, BBa_K098988, a constitutive GFP generator (GFP+ control), and a plasmid not encoding GFP (GFP- control). The GFP readings were normalized by culture OD.]]</div><br />
<br />
<br />
Should it help you, we also have the raw values graphed below. Note that a slight decrease in baseline fluorescence was also observed in negative control GFP- cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]).<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo_all.jpg|720px|thumb|center|Thermoinduction, raw data]]</div><br />
<br />
Results from the 1000x dilutions were inconclusive because the E. coli grows much slower at 30 ºC than at 40 ºC, so by the end of the experiment, there was a vast difference in cell concentration between the two sets of samples.<br />
<br />
|}<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T03:56:39Z<p>Mamut: /* LacI Inducible System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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, an example of the circuity we engineered. BBa_K098983 is similar, with a weaker promoter driving lacI expression.]]</div><br />
<br />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.<br />
<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]<br>This data is also available in [https://static.igem.org/mediawiki/2008/f/fa/Baseline-corrected_of_lac_system.pdf PDF format].]]</div><br />
<br />
<br />
<br />
Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T03:56:27Z<p>Mamut: /* LacI Inducible System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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]], an example of the circuity we engineered. BBa_K098983 is similar, with a weaker promoter driving lacI expression.</div><br />
<br />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.<br />
<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]<br>This data is also available in [https://static.igem.org/mediawiki/2008/f/fa/Baseline-corrected_of_lac_system.pdf PDF format].]]</div><br />
<br />
<br />
<br />
Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/OtherTeam:Harvard/Parts/Other2008-10-30T03:54:38Z<p>Mamut: /* TetR-based Inducible System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Our other parts=<br />
==TetR-based Inducible System==<br />
The TetR system we hoped to develop would have been similar to the lacI system. The pTet promoter which controls mtrB expression is regulated by the repressor TetR ([http://partsregistry.org/wiki/index.php?title=Part:BBa_C0040| BBa_C0040]). Expression of mtrB would have occurred when the system is induced with anhydrotetracycline which binds TetR. Unfortunately, as mtrB is toxic, we could not clone successful clone this system.<br />
<br />
However, we did submit parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098980 BBa_K098980] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981 BBa_K098981] in case you might find them useful.<br />
==Light-induced System==<br />
We tried to create a light-induced mtrB expression system using the [http://partsregistry.org/Featured_Parts:Light_Sensor| light sensor] in the registry. This requires an EnvZ deficient strain of ''S. oneidensis'' which we could not successfully generate. Additionally, the registry versions of some of the parts appear wrong (sequence) or nonfunction. We thus provide the following two parts for future teams' use:<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098011 ''E. coli'' ompR for introduction into other species of bacteria<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098010 A HO-pcyA protein generator]<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/ProjectTeam:Harvard/Project2008-10-30T03:49:12Z<p>Mamut: /* Wildtype vs. mtrB */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
==BACTRICITY: Bacterial Biosensors with Electrical Output==<br />
<br />
<br />
The metabolically versatile bacterium ''Shewanella oneidensis'' adapts to anaerobic environments by transporting electrons to its exterior, reducing a variety of environmental substrates. When grown anaerobically and provided with lactate as a carbon source, ''S. oneidensis'' transfers electrons to an electrode of a microbial fuel cell. We sought to engineer ''S. oneidensis'' to report variations in environmental conditions through changes in current production. A previous study has shown that ''S. oneidensis'' mutants deficient in the mtrB gene produce less current than the wildtype strain, and that current production in these mutants can be restored by the addition of exogenous mtrB. We attempted to control current production in mtrB knockouts by introducing mtrB on lactose, tetracycline, and heat inducible systems. These novel biosensors integrate directly with electrical circuits, paving the way for the development of automated, biological measurement and reporter systems.<br />
<br />
==Experimental overview==<br />
<br />
We attempted to develop three inducible systems for electrical current production in ''S. oneidensis''. The first is a chemically inducible system, where LacI or TetR repression of the current-production gene expression in mtrB knock-out (KO) ''S. oneidensis'' can be alleviated by the addition of IPTG or anhydrotetracycline, respectively. Our second approach uses temperature-senstive cI which would allow for an increase in electrical output in response to heat. The third is a light-inducible system based on the 2005 UT Austin biological camera.<br />
<br />
Using the LacI system, we attempted to interact with engineered ''S. oneidensis'' in multiple anaerobic microbial fuel cells which we built, measuring the effects of IPTG on current production in wild type and mtrB KO ''S. oneidensis'' containing plasmids expressing inducible or constitutive mtrB.<br />
<br />
==Results==<br />
<br />
===Wildtype vs. mtrB deficient ''S. oneidensis''===<br />
[[Image: wt_mtrB.jpg|700px]]<br />
<br />
===Co-Culture Experiment===<br />
<br />
''Overview: One possible system for achieving inducible current could be to couple a inducible-lacZ system in E. coli to current production by wildtype S. oneidensis MR1 through the conversion of lactose to lactate. This leaves open the possibility of using a pre-existing E. coli lacZ reporter or creating our own.<br />
''<br />
<br />
In addition to genetically engineering S. oneidensis MR-1 to respond to chemicals and heat, we also sought to take advantage of its natural metabolic pathway of breaking down lactate to produce current. From our wildtype versus mtrB knockout experiments, we found that feeding S. oneidensis MR-1 lactate led to significant current production almost instantly. In wildtype S. oneidensis MR-1, the current production would increase and stay elevated for a period of twelve hours. This result occurred consistently, thus we sought to use controlled lactate release as a way to control current output. <br />
<br />
We also knew that wildtype E. coli takes lactose and breaks down lactate. Thus, we hypothesized that it might be possible to couple E. coli’s lactate production with S. oneidensis MR-1’s lactate breakdown to produce current. In addition, a great deal of genetic engineering has already been done on E. coli and the up-regulation or down-regulation of its Lac operon. This means that instead of genetically modifying S. oneidensis MR-1 to respond to chemicals or heat, we can instead take advantage of the vast library of E. coli genetics and couple genetically engineered E. coli with wildtype S. oneidensis MR-1. <br />
<br />
In this experiment, we tested different combinations of wildtype E. coli MG1655, S. oneidensis MR-1, and Lac-operon knockout E. coli MC4100 as follows:<br />
<br />
1. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactose <br />
<br />
2. wt E. coli (MG1655) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
3. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactose<br />
<br />
4. Lac-operon knockout E. coli (MC4100) + wt S. oneidensis (MR1) + lactate pos. control<br />
<br />
5. wt S. oneidensis MR-1 + lactose neg. control<br />
<br />
6. wt E. coli (MG1655) + lactose neg. control<br />
<br />
7. Lac-operon knockout E. coli (MC4100) + lactose neg. control<br />
<br />
Based on previous experiments, we would expect current production in combinations 2 and 4 as they both have wildtype S. oneidensis MR-1 and receive lactate. In addition, however, we would expect combination 1 to also produce current. As described above, wt E. coli would break down lactose into lactate, and S. oneidensis MR-1 would break down lactate to produce current.<br />
<br />
[[ Image: picture 5.png | 800px ]]<br />
<br />
From the data above, we found that combination 1 did indeed produce current with a delay relative to the positive control. The delay can be attributed to the time it takes for E. coli to break down lactose into lactate, thus adding an extra step in the carbon source to current production pathway compared to our positive controls. These results are exciting in that they show a possibility of taking advantage of this cooperative effort to achieve inducible current.<br />
<br />
==Future directions==<br />
<br />
Our work with creating a system of inducible electrical output in ''S. oneidensis'' has laid the foundations for many different exciting avenues of further inquiry which look to take advantage of a bacteria-computer interface that combines the amazing sensitivity and adaptability of bacteria with the speed and analytical abilities of electricity and computers.<br />
<br />
Using the same principles underlying the lac system, the [http://parts.mit.edu/wiki/index.php/University_of_Edinburgh_2006 arsenic biosensor] developed by the University of Edinburgh iGEM 2006 team could be introduced into ''S. oneidensis'', allowing for the coupling of arsenic sensing to an electrical output, a form of a data which is easier to automate and transmit. This could be further extended to other chemical sensing systems, resulting ultimately in an array of different strains ''S. oneidensis'' which all respond to the presence of different chemicals with an electrical output that can be monitored by a computer. This could theoretically allow for the remote sensing and analysis of the chemical composition of an environment over time.<br />
<br />
Another interesting direction would be the linking of the light-sensing system developed by the UT Austin iGEM team with electrical output in ''S. oneidensis''. In response to variations in light, the amount of electricity produced by ''S. oneidensis'' would change. This would allow for the intriguing possibility of not only ''S. oneidensis'' conveying information to the computer, but also the computer responding to the ''S. oneidensis''. A simple example would be that in response to a chemical input, ''S. oneidensis'' may increase its electrical output. Sensing this increase, the computer could turn on or off a light directed at the ''S. oneidensis'', modifying ''S. oneidensis'''s output, creating interesting feedback loops. This could ultimately be developed into more complex communications systems between bacteria and computers. We tried constructing this system over summer, but as the process requires making an EnvZ knockout strain of ''S. oneidensis'', we could not finish it. We did, however, make a few parts to facilitate future attempts.<br />
<br />
The possibilities are further broadened by our observations of co-cultures of ''E. coli'' and ''S. oneidensis''. Either of the systems described above could be pursued through an alternative alternative strategy of co-cultures. For instance, an array of ''E. coli'' which respond to different chemicals by breaking down lactose into lactate could be cultured with ''S. oneidensis''. In response to an increase in lactate, ''S. oneidensis'' would begin to produce higher levels of electricity. Co-cultures could also allow for more complex bacteria-computer interactions. This strategy could enable the coupling of almost any ''E. coli'' ability to electrical output.<br />
<br />
These future directions in which our research can be taken demonstrate some of the exciting possibilities of BACTRICITY!<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T03:46:48Z<p>Mamut: /* The Genetic Circuitry */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- 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 />
==''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on ''mtrB'', which encodes a 679-amino-acid-long outer membrane protein 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 should occur. In this case, the inducer binds the repressor protein, preventing it from binding to the operator within the promoter. RNA polymerase is therefore able to bind to the promoter (green), 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 tried to create such systems capable of being induced by [[Team:Harvard/Parts/LacI| IPTG]], <br />
[[Team:Harvard/Parts/Tempsenseci| heat]], [[Team:Harvard/Parts/Other| tetracycline, and light]].</div>Mamuthttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T03:46:08Z<p>Mamut: /* The Genetic Circuitry */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- 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 />
==''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on ''mtrB'', which encodes a 679-amino-acid-long outer membrane protein 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 should 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 tried to create such systems capable of being induced by [[Team:Harvard/Parts/LacI| IPTG]], <br />
[[Team:Harvard/Parts/Tempsenseci| heat]], [[Team:Harvard/Parts/Other| tetracycline, and light]].</div>Mamuthttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T03:33:16Z<p>Mamut: /* mtrB */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- 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 />
==''mtrB''==<br />
Many genes are involved in ''S. oneidensis''’s complex respiratory system (Heidelberg et al. 2002). We focused on ''mtrB'', which encodes a 679-amino-acid-long outer membrane protein 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 tried to create such systems capable of being induced by [[Team:Harvard/Parts/LacI| IPTG]], <br />
[[Team:Harvard/Parts/Tempsenseci| heat]], [[Team:Harvard/Parts/Other| tetracycline, and light]].</div>Mamuthttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T03:32:42Z<p>Mamut: /* The Critical Gene: mtrB */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- 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 />
==''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 tried to create such systems capable of being induced by [[Team:Harvard/Parts/LacI| IPTG]], <br />
[[Team:Harvard/Parts/Tempsenseci| heat]], [[Team:Harvard/Parts/Other| tetracycline, and light]].</div>Mamuthttp://2008.igem.org/Team:Harvard/ShewieTeam:Harvard/Shewie2008-10-30T03:10:08Z<p>Mamut: /* So, who is this "Shewie"? */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
<br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:white;<br />
}<br />
<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<br />
| align="center" style="background:#c4dbea" border="0" cellpading="0" cellspacing="0"|<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 />
<br />
{|style="color:#1b2c8a;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- 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 />
==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 />
{|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 ''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 />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/File:Harvardigem2008teampic.JPGFile:Harvardigem2008teampic.JPG2008-10-30T03:03:12Z<p>Mamut: uploaded a new version of "Image:Harvardigem2008teampic.JPG": red reduced, cropped, feel free to revert</p>
<hr />
<div></div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/TempsenseciTeam:Harvard/Parts/Tempsenseci2008-10-30T03:01:56Z<p>Mamut: /* Thermoinducible cI System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Thermoinducible cI System=<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<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 />
We had previously tried to use the thermoinducible lac system in the Registry ([http://partsregistry.org/Part:BBa_J06912 BBa_J06912] and [http://partsregistry.org/Part:BBa_J06911 BBa_J06911]). However, induction tests, a [https://2008.igem.org/Team:Harvard/Dailybook/Week6/Chemical_and_Light#Western_Blot Western blot], and sequencing confirmed that these parts are not functional. We thus focused our efforts on creating a thermosensitive cI system. <br />
<br />
===BBa_K098995===<br />
This is a thermosensitive cI inducible system driven by a strong promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:122.png|thumb|650px|center|BBa_K098995]]</div><br />
<br />
===BBa_K098993===<br />
This is a thermosensitive cI inducible system driven by a weak promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:123.png|thumb|650px|center|BBa_K098993]]</div><br />
<br />
==Induction Test for Thermosensitive cI Systems with GFP Reporters==<br />
An induction test was designed to test the inducibility of the heat sensitive cI systems. Two constructs were made:<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a strong promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:124.png|thumb|650px|center|BBa_K098988]]</div><br />
<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a weak promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:125.png|thumb|650px|center|BBa_K098987]]</div><br />
<br />
===Experimental Design===<br />
[[Image:Thermo.png|thumb|150px|Thermo Induction Experimental Design]]<br><br />
Starter cultures of E. coli with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098988| BBa_K098988] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098987| BBa_K098987] were grown overnight. They were then diluted and grown to OD 0.2 before separation into induced (40 ºC) and uninduced cultures (30 ºC). OD and GFP readings were taken at time 0, 2, and 4 hours. Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in their respective incubators until the end of the experiment, when OD and GFP readings were taken.<br />
<br />
===Results===<br />
Induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. While levels of GFP expression in the GFP+ control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]) went down in the samples at 40 ºC, levels in the inducible systems increased slightly. Note that the ''absolute levels'' of GFP expression do not increase much relative to 30 ºC (see our raw data at at the bottom of the page), but the increase in GFP expression is significantly different behavior from the decrease observed in the GFP+ control. We hypothesize that elevated temperature affects the GFP expression (e.g. by disrupting protein folding), and that even the small increase in GFP expression with our systems indicates effective induction. However, this does suggest that such a system is not optimal for inducing the expressing of heat-sensitive proteins.<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:CIts.png|780px|thumb|center|A comparison of GFP expression following thermoinduction of cells harboring BBa_K098987, BBa_K098988, a constitutive GFP generator (GFP+ control), and a plasmid not encoding GFP (GFP- control). The GFP readings were normalized by culture OD.]]</div><br />
<br />
<br />
Should it help you, we also have the raw values graphed below. Note that a slight decrease in baseline fluorescence was also observed in negative control GFP- cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]).<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo_all.jpg|720px|thumb|center|Thermoinduction, raw data]]</div><br />
<br />
Results from the 1000x dilutions were inconclusive because the E. coli grows much slower at 30 ºC than at 40 ºC, so by the end of the experiment, there was a vast difference in cell concentration between the two sets of samples.<br />
<br />
|}<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/TempsenseciTeam:Harvard/Parts/Tempsenseci2008-10-30T03:01:32Z<p>Mamut: /* Thermoinducible cI System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=Thermoinducible cI System=<br />
<br />
This system uses a a temperature sensitive variant of cI lambda to regulate the lambda promoter.<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 />
We had previously tried to use the thermoinducible lac system in the Registry ([http://partsregistry.org/Part:BBa_J06912 BBa_J06912] and [http://partsregistry.org/Part:BBa_J06911 BBa_J06911]). However, induction tests, a [https://2008.igem.org/Team:Harvard/Dailybook/Week6/Chemical_and_Light#Western_Blot| Western blot], and sequencing confirmed that these parts are not functional. We thus focused our efforts on creating a thermosensitive cI system. <br />
<br />
===BBa_K098995===<br />
This is a thermosensitive cI inducible system driven by a strong promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:122.png|thumb|650px|center|BBa_K098995]]</div><br />
<br />
===BBa_K098993===<br />
This is a thermosensitive cI inducible system driven by a weak promoter.<br />
<div style="text-indent:0pt;color:black">[[Image:123.png|thumb|650px|center|BBa_K098993]]</div><br />
<br />
==Induction Test for Thermosensitive cI Systems with GFP Reporters==<br />
An induction test was designed to test the inducibility of the heat sensitive cI systems. Two constructs were made:<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a strong promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:124.png|thumb|650px|center|BBa_K098988]]</div><br />
<br />
===BBa_K098988===<br />
This is a thermosensitive cI inducible system driven by a weak promoter and with a GFP indicator.<br />
<div style="text-indent:0pt;color:black">[[Image:125.png|thumb|650px|center|BBa_K098987]]</div><br />
<br />
===Experimental Design===<br />
[[Image:Thermo.png|thumb|150px|Thermo Induction Experimental Design]]<br><br />
Starter cultures of E. coli with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098988| BBa_K098988] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098987| BBa_K098987] were grown overnight. They were then diluted and grown to OD 0.2 before separation into induced (40 ºC) and uninduced cultures (30 ºC). OD and GFP readings were taken at time 0, 2, and 4 hours. Additionally, after diluting T=2hrs samples to OD 0.2 for accurate GFP measurements, samples were further diluted 1000x, induced (or not induced) again, and placed back in their respective incubators until the end of the experiment, when OD and GFP readings were taken.<br />
<br />
===Results===<br />
Induction of GFP expression was observed at both 2 and 4 hours after moving samples to 40 ºC. While levels of GFP expression in the GFP+ control ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098991| BBa_K098991]) went down in the samples at 40 ºC, levels in the inducible systems increased slightly. Note that the ''absolute levels'' of GFP expression do not increase much relative to 30 ºC (see our raw data at at the bottom of the page), but the increase in GFP expression is significantly different behavior from the decrease observed in the GFP+ control. We hypothesize that elevated temperature affects the GFP expression (e.g. by disrupting protein folding), and that even the small increase in GFP expression with our systems indicates effective induction. However, this does suggest that such a system is not optimal for inducing the expressing of heat-sensitive proteins.<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:CIts.png|780px|thumb|center|A comparison of GFP expression following thermoinduction of cells harboring BBa_K098987, BBa_K098988, a constitutive GFP generator (GFP+ control), and a plasmid not encoding GFP (GFP- control). The GFP readings were normalized by culture OD.]]</div><br />
<br />
<br />
Should it help you, we also have the raw values graphed below. Note that a slight decrease in baseline fluorescence was also observed in negative control GFP- cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K098981| BBa_K098981]).<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Thermo_all.jpg|720px|thumb|center|Thermoinduction, raw data]]</div><br />
<br />
Results from the 1000x dilutions were inconclusive because the E. coli grows much slower at 30 ºC than at 40 ºC, so by the end of the experiment, there was a vast difference in cell concentration between the two sets of samples.<br />
<br />
|}<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/ReferencesTeam:Harvard/References2008-10-30T02:59:42Z<p>Mamut: /* Acknowledgments */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=References=<br />
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 />
Cho YK, Donohue TJ, Tejedor I, Anderson MA, McMahon KD, and Noguera DR. Development of a solar-powered microbial fuel cell. J Appl Microbiol 2008 Mar; 104(3) 640-50.<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 />
Lovley DR. Bug juice: harvesting electricity with microorganisms. Nat Rev Microbiol 2006 Jul; 4(7) 497-508.<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.<br />
<br />
<br />
<br />
<br />
|}<br />
<br><br><br />
<br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:59:11Z<p>Mamut: /* The Genetic Circuitry */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- 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 tried to create such systems capable of being induced by [[Team:Harvard/Parts/LacI| IPTG]], <br />
[[Team:Harvard/Parts/Tempsenseci| heat]], [[Team:Harvard/Parts/Other| tetracycline, and light]].</div>Mamuthttp://2008.igem.org/Team:Harvard/PartsTeam:Harvard/Parts2008-10-30T02:51:08Z<p>Mamut: /* The Genetic Circuitry */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: #333333;<br />
color:#333333;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: #333333;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:#333333;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#333333;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- 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 tried 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>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T02:49:57Z<p>Mamut: /* Results */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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 />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.<br />
<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]<br>This data is also available in [https://static.igem.org/mediawiki/2008/f/fa/Baseline-corrected_of_lac_system.pdf PDF format].]]</div><br />
<br />
<br />
<br />
Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T02:48:47Z<p>Mamut: /* Results */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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 />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.<br />
<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]]]</div><br />
<br />
<br />
<br />
Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T02:47:35Z<p>Mamut: /* Induction Test for LacI System with GFP */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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 />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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]). Note that in the graph below, it appears that such an induction method is not reliable at 6 hours.<br />
<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Baseline-corrected_of_lac_system.png|720px|thumb|center|IPTG successfully induces higher levels of GFP expression in cells containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982]]]</div><br />
<br />
<br />
<br />
Additionally, it appears that even the uninduced state of such a system has significant expression of GFP. Tighter control may require higher levels of lacI expression. This leakiness may also explain the difficulty we had in cloning mtrB (a gene toxic to ''E. coli'').<br />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|650px|thumb|center|IPTG induction raw data]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T02:43:52Z<p>Mamut: /* LacI Inducible System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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 />
==Induction Test for LacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor (LacI) is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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 />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|720px|thumb|center|Lac Inducibility Results]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T02:43:21Z<p>Mamut: /* LacI Inducible System */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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. This should allow us to control the expression of mtrB. 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 />
==Induction Test for lacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor (LacI) is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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 />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|720px|thumb|center|Lac Inducibility Results]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/Team:Harvard/Parts/LacITeam:Harvard/Parts/LacI2008-10-30T02:42:37Z<p>Mamut: /* Induction Test for lacI System with GFP */</p>
<hr />
<div>__NOTOC__<br />
<html><br />
<head><br />
<style><br />
table {<br />
background-color: #c4dbea;<br />
font-color: white;<br />
color:white;<br />
}<br />
a.menu {<br />
background-color: #c4dbea;<br />
color: white;<br />
width: 12em;<br />
}<br />
<br />
.firstHeading {<br />
color:white;<br />
}<br />
<br />
#bodyContent {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#content {<br />
background-color: #c4dbea;<br />
}<br />
<br />
#footer-box {<br />
background-color: #c4dbea;<br />
}<br />
p {<br />
color:#333333;<br />
}<br />
body {<br />
background-color:#c4dbea;<br />
}<br />
.firstHeading {<br />
display:none;<br />
}<br />
</style><br />
</head><br />
</html><br />
<br />
<br />
{|<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 />
<br />
{| style="color:#1b2c8a;background-color:#FFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
<!--- body here---><br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="50" width="90%"<br />
|<br />
=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 />
==Induction Test for lacI System with GFP==<br />
In preparing to make the lacI system for mtrB, we tested the IPTG inducibility of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K098982 BBa_K098982] in ''E. coli''. In this system, the repressor (LacI) is driven by a weak promoter.<br />
===Method===<br />
<br />
Our test method is diagrammed below.<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.png|thumb|800px|center|]]</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 />
<br />
<div style="text-indent:0pt;color:black">[[Image:Lac.jpg|720px|thumb|center|Lac Inducibility Results]]</div><br />
<br />
<br />
|}<br />
<br><br><br />
<!--- end body ---><br />
|}</div>Mamuthttp://2008.igem.org/File:Baseline-corrected_of_lac_system.pngFile:Baseline-corrected of lac system.png2008-10-30T02:41:25Z<p>Mamut: </p>
<hr />
<div></div>Mamuthttp://2008.igem.org/File:Baseline-corrected_of_lac_system.pdfFile:Baseline-corrected of lac system.pdf2008-10-30T02:41:20Z<p>Mamut: </p>
<hr />
<div></div>Mamut