http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=PM&year=&month=2010.igem.org - User contributions [en]2024-03-28T18:40:51ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_CharacterisationTeam:Cambridge/Bioluminescence/Firefly Characterisation2010-10-28T00:32:55Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325219 BBa_K325219], Red luciferase and LRE operon from L. cruciata under pBAD.<br />
It also gives a comparison of the light ouput of the different [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour coloured mutants] we produced. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Luciferin_to_light|Luciferin to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Effect_of_pH|Effect of pH]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Coloured_outputs|Coloured outputs]]<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata) as well as the luciferin regenerating enzyme (LRE). It is under the control of an Arabinose induced promoter. D-Luciferin has to be added to obtain light output.<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Transfer function of <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/2/26/IntegratedactiPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 11 values obtained for light output at 30 min interval from 450 min to 750 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 33 data points centred around the mean value. <br />
<br />
'''Maximum luminescence output of <partinfo>K325219</partinfo> as a function of Arabinose concentration.'''<br />
<br />
[http://partsregistry.org/wiki/images/0/05/MaximumlumPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/0/05/MaximumlumPP%2BLRE.png/569px-MaximumlumPP%2BLRE.png]<br />
<br />
<br />
These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/4/4c/TimePP%2BLRE.png http://partsregistry.org/wiki/images/thumb/4/4c/TimePP%2BLRE.png/569px-TimePP%2BLRE.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/8/82/BBa_K325219ArabinosetoLight.xls Media:BBa_K325219ArabinosetoLight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
=Luciferin to light=<br />
<br />
The light output is also a function of the concentration of D-Luciferin the media. This page describes the relationship between D-Luciferin concentration and light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Effect of D-Luciferin concentration on light output from <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/c/c3/Luciferineffect.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 31 values obtained for light output at 30 min interval from 1500 min to 2400 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 31 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/b/b9/Luctimecourse.png http://partsregistry.org/wiki/images/thumb/b/b9/Luctimecourse.png/569px-Luctimecourse.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/9/9a/BBa_K325219luciferintolight.xls Media:BBa_K325219luciferintolight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
<br />
=Effect of pH=<br />
<br />
Both the intensity and the spectrum emitted by the luciferase-luciferin reaction has been shown to be higly dependent on the pH of the medium. The main characterisation experiments have been performed in LB Broth at pH 7, so in order to assess this effect cultures with LB and a citrate buffer were prepared (pH = 5.3, pH 6.1 and pH = 7) This page describes the results of these experiments. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Maximum light output within 5 hours of D-luciferin injection at different pH values.'''<br />
<br />
[http://partsregistry.org/wiki/images/e/e8/Phhistogram.png http://partsregistry.org/wiki/images/thumb/e/e8/Phhistogram.png/569px-Phhistogram.png]<br />
<br />
These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of light output at different values of pH.'''<br />
<br />
[http://partsregistry.org/wiki/images/d/d8/Phtimecourse.png http://partsregistry.org/wiki/images/thumb/d/d8/Phtimecourse.png/569px-Phtimecourse.png]<br />
<br />
Measurements are taken every 20 min. These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/6/64/BBa_K325219pheffect.xls Media:BBa_K325219pheffect.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
#The protocol can be found as [https://2010.igem.org/Team:Cambridge/Notebook/Week11 Experiment 110].<br />
<br />
=Coloured outputs=<br />
<br />
In this section we compare the intensity of the different coloured mutants we developed during our project.<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:Outputpower.jpg|thumb|569px|center|'''Figure 1 - Maximum light output within 5 hours of D-Luciferin injection of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour coloured mutants] we developed. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
<br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:CambridgeiGEMcolouredoutputs.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
<br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.<br />
<br />
<br />
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_CharacterisationTeam:Cambridge/Bioluminescence/Firefly Characterisation2010-10-28T00:28:19Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325219 BBa_K325219], Red luciferase and LRE operon from L. cruciata under pBAD.<br />
It also gives a comparison of the light ouput of the different [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour coloured mutants] we produced. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Luciferin_to_light|Luciferin to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Effect_of_pH|Effect of pH]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Colour_outputs|Coloured outputs]]<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata) as well as the luciferin regenerating enzyme (LRE). It is under the control of an Arabinose induced promoter. D-Luciferin has to be added to obtain light output.<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Transfer function of <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/2/26/IntegratedactiPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 11 values obtained for light output at 30 min interval from 450 min to 750 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 33 data points centred around the mean value. <br />
<br />
'''Maximum luminescence output of <partinfo>K325219</partinfo> as a function of Arabinose concentration.'''<br />
<br />
[http://partsregistry.org/wiki/images/0/05/MaximumlumPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/0/05/MaximumlumPP%2BLRE.png/569px-MaximumlumPP%2BLRE.png]<br />
<br />
<br />
These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/4/4c/TimePP%2BLRE.png http://partsregistry.org/wiki/images/thumb/4/4c/TimePP%2BLRE.png/569px-TimePP%2BLRE.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/8/82/BBa_K325219ArabinosetoLight.xls Media:BBa_K325219ArabinosetoLight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
=Luciferin to light=<br />
<br />
The light output is also a function of the concentration of D-Luciferin the media. This page describes the relationship between D-Luciferin concentration and light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Effect of D-Luciferin concentration on light output from <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/c/c3/Luciferineffect.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 31 values obtained for light output at 30 min interval from 1500 min to 2400 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 31 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/b/b9/Luctimecourse.png http://partsregistry.org/wiki/images/thumb/b/b9/Luctimecourse.png/569px-Luctimecourse.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/9/9a/BBa_K325219luciferintolight.xls Media:BBa_K325219luciferintolight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
<br />
=Effect of pH=<br />
<br />
Both the intensity and the spectrum emitted by the luciferase-luciferin reaction has been shown to be higly dependent on the pH of the medium. The main characterisation experiments have been performed in LB Broth at pH 7, so in order to assess this effect cultures with LB and a citrate buffer were prepared (pH = 5.3, pH 6.1 and pH = 7) This page describes the results of these experiments. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Maximum light output within 5 hours of D-luciferin injection at different pH values.'''<br />
<br />
[http://partsregistry.org/wiki/images/e/e8/Phhistogram.png http://partsregistry.org/wiki/images/thumb/e/e8/Phhistogram.png/569px-Phhistogram.png]<br />
<br />
These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of light output at different values of pH.'''<br />
<br />
[http://partsregistry.org/wiki/images/d/d8/Phtimecourse.png http://partsregistry.org/wiki/images/thumb/d/d8/Phtimecourse.png/569px-Phtimecourse.png]<br />
<br />
Measurements are taken every 20 min. These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/6/64/BBa_K325219pheffect.xls Media:BBa_K325219pheffect.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
#The protocol can be found as [https://2010.igem.org/Team:Cambridge/Notebook/Week11 Experiment 110].<br />
<br />
=Coloured outputs=<br />
<br />
In this section we compare the intensity of the different coloured mutants we developed during our project.<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:Outputpower.jpg|thumb|569px|center|'''Figure 1 - Maximum light output within 5 hours of D-Luciferin injection of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour coloured mutants] we developed. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
<br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:CambridgeiGEMcolouredoutputs.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
<br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.<br />
<br />
<br />
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_CharacterisationTeam:Cambridge/Bioluminescence/Firefly Characterisation2010-10-28T00:22:55Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325219 BBa_K325219], Red luciferase and LRE operon from L. cruciata under pBAD.<br />
It also gives a comparison of the light ouput of the different [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour coloured mutants] we produced. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Luciferin_to_light|Luciferin to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Effect_of_pH|Effect of pH]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Colour_Mutations|Coloured outputs]]<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata) as well as the luciferin regenerating enzyme (LRE). It is under the control of an Arabinose induced promoter. D-Luciferin has to be added to obtain light output.<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Transfer function of <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/2/26/IntegratedactiPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 11 values obtained for light output at 30 min interval from 450 min to 750 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 33 data points centred around the mean value. <br />
<br />
'''Maximum luminescence output of <partinfo>K325219</partinfo> as a function of Arabinose concentration.'''<br />
<br />
[http://partsregistry.org/wiki/images/0/05/MaximumlumPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/0/05/MaximumlumPP%2BLRE.png/569px-MaximumlumPP%2BLRE.png]<br />
<br />
<br />
These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/4/4c/TimePP%2BLRE.png http://partsregistry.org/wiki/images/thumb/4/4c/TimePP%2BLRE.png/569px-TimePP%2BLRE.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/8/82/BBa_K325219ArabinosetoLight.xls Media:BBa_K325219ArabinosetoLight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
=Luciferin to light=<br />
<br />
The light output is also a function of the concentration of D-Luciferin the media. This page describes the relationship between D-Luciferin concentration and light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Effect of D-Luciferin concentration on light output from <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/c/c3/Luciferineffect.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 31 values obtained for light output at 30 min interval from 1500 min to 2400 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 31 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/b/b9/Luctimecourse.png http://partsregistry.org/wiki/images/thumb/b/b9/Luctimecourse.png/569px-Luctimecourse.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/9/9a/BBa_K325219luciferintolight.xls Media:BBa_K325219luciferintolight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
<br />
=Effect of pH=<br />
<br />
Both the intensity and the spectrum emitted by the luciferase-luciferin reaction has been shown to be higly dependent on the pH of the medium. The main characterisation experiments have been performed in LB Broth at pH 7, so in order to assess this effect cultures with LB and a citrate buffer were prepared (pH = 5.3, pH 6.1 and pH = 7) This page describes the results of these experiments. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Maximum light output within 5 hours of D-luciferin injection at different pH values.'''<br />
<br />
[http://partsregistry.org/wiki/images/e/e8/Phhistogram.png http://partsregistry.org/wiki/images/thumb/e/e8/Phhistogram.png/569px-Phhistogram.png]<br />
<br />
These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of light output at different values of pH.'''<br />
<br />
[http://partsregistry.org/wiki/images/d/d8/Phtimecourse.png http://partsregistry.org/wiki/images/thumb/d/d8/Phtimecourse.png/569px-Phtimecourse.png]<br />
<br />
Measurements are taken every 20 min. These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/6/64/BBa_K325219pheffect.xls Media:BBa_K325219pheffect.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
#The protocol can be found as [https://2010.igem.org/Team:Cambridge/Notebook/Week11 Experiment 110].<br />
<br />
=Coloured outputs=<br />
<br />
In this section we compare the intensity of the different coloured mutants we developed during our project.<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:Outputpower.jpg|thumb|569px|center|'''Figure 1 - Maximum light output within 5 hours of D-Luciferin injection of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour coloured mutants] we developed. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
<br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:CambridgeiGEMcolouredoutputs.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.<br />
<br />
<br />
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/File:Outputpower.jpgFile:Outputpower.jpg2010-10-28T00:20:23Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_CharacterisationTeam:Cambridge/Bioluminescence/Firefly Characterisation2010-10-28T00:19:32Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325219 BBa_K325219], Red luciferase and LRE operon from L. cruciata under pBAD.<br />
It also gives a comparison of the light ouput of the different coloroured mutants we produced. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Luciferin_to_light|Luciferin to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Effect_of_pH|Effect of pH]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Colour_Mutations|Coloured outputs]]<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata) as well as the luciferin regenerating enzyme (LRE). It is under the control of an Arabinose induced promoter. D-Luciferin has to be added to obtain light output.<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Transfer function of <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/2/26/IntegratedactiPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 11 values obtained for light output at 30 min interval from 450 min to 750 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 33 data points centred around the mean value. <br />
<br />
'''Maximum luminescence output of <partinfo>K325219</partinfo> as a function of Arabinose concentration.'''<br />
<br />
[http://partsregistry.org/wiki/images/0/05/MaximumlumPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/0/05/MaximumlumPP%2BLRE.png/569px-MaximumlumPP%2BLRE.png]<br />
<br />
<br />
These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/4/4c/TimePP%2BLRE.png http://partsregistry.org/wiki/images/thumb/4/4c/TimePP%2BLRE.png/569px-TimePP%2BLRE.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/8/82/BBa_K325219ArabinosetoLight.xls Media:BBa_K325219ArabinosetoLight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
=Luciferin to light=<br />
<br />
The light output is also a function of the concentration of D-Luciferin the media. This page describes the relationship between D-Luciferin concentration and light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Effect of D-Luciferin concentration on light output from <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/c/c3/Luciferineffect.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 31 values obtained for light output at 30 min interval from 1500 min to 2400 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 31 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/b/b9/Luctimecourse.png http://partsregistry.org/wiki/images/thumb/b/b9/Luctimecourse.png/569px-Luctimecourse.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/9/9a/BBa_K325219luciferintolight.xls Media:BBa_K325219luciferintolight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
<br />
=Effect of pH=<br />
<br />
Both the intensity and the spectrum emitted by the luciferase-luciferin reaction has been shown to be higly dependent on the pH of the medium. The main characterisation experiments have been performed in LB Broth at pH 7, so in order to assess this effect cultures with LB and a citrate buffer were prepared (pH = 5.3, pH 6.1 and pH = 7) This page describes the results of these experiments. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Maximum light output within 5 hours of D-luciferin injection at different pH values.'''<br />
<br />
[http://partsregistry.org/wiki/images/e/e8/Phhistogram.png http://partsregistry.org/wiki/images/thumb/e/e8/Phhistogram.png/569px-Phhistogram.png]<br />
<br />
These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of light output at different values of pH.'''<br />
<br />
[http://partsregistry.org/wiki/images/d/d8/Phtimecourse.png http://partsregistry.org/wiki/images/thumb/d/d8/Phtimecourse.png/569px-Phtimecourse.png]<br />
<br />
Measurements are taken every 20 min. These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/6/64/BBa_K325219pheffect.xls Media:BBa_K325219pheffect.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
#The protocol can be found as [https://2010.igem.org/Team:Cambridge/Notebook/Week11 Experiment 110].<br />
<br />
=Coloured outputs=<br />
<br />
In this section we compare the intensity of the different coloured mutants we developed during our project.<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:Outputpower.jpg|thumb|569px|center|'''Figure 1 - Maximum light output within 5 hours of D-Luciferin injection of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour coloured mutants] we developed. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
<br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:CambridgeiGEMcolouredoutputs.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.<br />
<br />
<br />
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_CharacterisationTeam:Cambridge/Bioluminescence/Firefly Characterisation2010-10-28T00:16:34Z<p>PM: /* Coloured outputs */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325219 BBa_K325219], Red luciferase and LRE operon from L. cruciata under pBAD.<br />
It also gives a comparison of the light ouput of the different coloroured mutants we produced. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Luciferin_to_light|Luciferin to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Effect_of_pH|Effect of pH<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Colour_Mutations|Coloured outputs]]<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata) as well as the luciferin regenerating enzyme (LRE). It is under the control of an Arabinose induced promoter. D-Luciferin has to be added to obtain light output.<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Transfer function of <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/2/26/IntegratedactiPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 11 values obtained for light output at 30 min interval from 450 min to 750 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 33 data points centred around the mean value. <br />
<br />
'''Maximum luminescence output of <partinfo>K325219</partinfo> as a function of Arabinose concentration.'''<br />
<br />
[http://partsregistry.org/wiki/images/0/05/MaximumlumPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/0/05/MaximumlumPP%2BLRE.png/569px-MaximumlumPP%2BLRE.png]<br />
<br />
<br />
These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/4/4c/TimePP%2BLRE.png http://partsregistry.org/wiki/images/thumb/4/4c/TimePP%2BLRE.png/569px-TimePP%2BLRE.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/8/82/BBa_K325219ArabinosetoLight.xls Media:BBa_K325219ArabinosetoLight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
=Luciferin to light=<br />
<br />
The light output is also a function of the concentration of D-Luciferin the media. This page describes the relationship between D-Luciferin concentration and light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Effect of D-Luciferin concentration on light output from <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/c/c3/Luciferineffect.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 31 values obtained for light output at 30 min interval from 1500 min to 2400 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 31 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/b/b9/Luctimecourse.png http://partsregistry.org/wiki/images/thumb/b/b9/Luctimecourse.png/569px-Luctimecourse.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/9/9a/BBa_K325219luciferintolight.xls Media:BBa_K325219luciferintolight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
<br />
=Effect of pH=<br />
<br />
Both the intensity and the spectrum emitted by the luciferase-luciferin reaction has been shown to be higly dependent on the pH of the medium. The main characterisation experiments have been performed in LB Broth at pH 7, so in order to assess this effect cultures with LB and a citrate buffer were prepared (pH = 5.3, pH 6.1 and pH = 7) This page describes the results of these experiments. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Maximum light output within 5 hours of D-luciferin injection at different pH values.'''<br />
<br />
[http://partsregistry.org/wiki/images/e/e8/Phhistogram.png http://partsregistry.org/wiki/images/thumb/e/e8/Phhistogram.png/569px-Phhistogram.png]<br />
<br />
These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of light output at different values of pH.'''<br />
<br />
[http://partsregistry.org/wiki/images/d/d8/Phtimecourse.png http://partsregistry.org/wiki/images/thumb/d/d8/Phtimecourse.png/569px-Phtimecourse.png]<br />
<br />
Measurements are taken every 20 min. These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/6/64/BBa_K325219pheffect.xls Media:BBa_K325219pheffect.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
#The protocol can be found as [https://2010.igem.org/Team:Cambridge/Notebook/Week11 Experiment 110].<br />
<br />
=Coloured outputs=<br />
<br />
In this section we compare the intensity of the different coloured mutants we developed during our project.<br />
<br />
[[Image:Outputpower.jpg|thumb|569px|center|'''Figure 1 - Maximum light output within 5 hours of D-Luciferin injection of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour coloured mutants] we developed. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
<br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[CambridgeiGEMcolouredoutputs.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.<br />
<br />
<br />
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_CharacterisationTeam:Cambridge/Bioluminescence/Firefly Characterisation2010-10-28T00:13:09Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325219 BBa_K325219], Red luciferase and LRE operon from L. cruciata under pBAD.<br />
It also gives a comparison of the light ouput of the different coloroured mutants we produced. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Luciferin_to_light|Luciferin to light]]<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Effect_of_pH|Effect of pH<br />
*[[Team:Cambridge/Bioluminescence/Firefly_Characterisation#Colour_Mutations|Coloured outputs]]<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata) as well as the luciferin regenerating enzyme (LRE). It is under the control of an Arabinose induced promoter. D-Luciferin has to be added to obtain light output.<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Transfer function of <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/2/26/IntegratedactiPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 11 values obtained for light output at 30 min interval from 450 min to 750 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 33 data points centred around the mean value. <br />
<br />
'''Maximum luminescence output of <partinfo>K325219</partinfo> as a function of Arabinose concentration.'''<br />
<br />
[http://partsregistry.org/wiki/images/0/05/MaximumlumPP%2BLRE.png http://partsregistry.org/wiki/images/thumb/0/05/MaximumlumPP%2BLRE.png/569px-MaximumlumPP%2BLRE.png]<br />
<br />
<br />
These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/4/4c/TimePP%2BLRE.png http://partsregistry.org/wiki/images/thumb/4/4c/TimePP%2BLRE.png/569px-TimePP%2BLRE.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/8/82/BBa_K325219ArabinosetoLight.xls Media:BBa_K325219ArabinosetoLight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
=Luciferin to light=<br />
<br />
The light output is also a function of the concentration of D-Luciferin the media. This page describes the relationship between D-Luciferin concentration and light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Effect of D-Luciferin concentration on light output from <partinfo>K325219</partinfo>.'''<br />
<br />
[http://partsregistry.org/wiki/images/c/c3/Luciferineffect.png http://partsregistry.org/wiki/images/thumb/c/c3/Luciferineffect.png/569px-Luciferineffect.png]<br />
<br />
The data points represent the mean of 31 values obtained for light output at 30 min interval from 1500 min to 2400 min after injection of D-Luciferin. These values are the mean of 3 readings as shown in Figure 3. The corresponding error bars represent an interval of twice the standard deviation across the 31 data points centred around the mean value.<br />
<br />
'''Evolution of luminescence with time at different Arabinose concentrations.'''<br />
<br />
[http://partsregistry.org/wiki/images/b/b9/Luctimecourse.png http://partsregistry.org/wiki/images/thumb/b/b9/Luctimecourse.png/569px-Luctimecourse.png]<br />
<br />
The interval between measurements is 30 min. Mean values and error bars are based on 3 time repeats.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/9/9a/BBa_K325219luciferintolight.xls Media:BBa_K325219luciferintolight.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
<br />
=Effect of pH=<br />
<br />
Both the intensity and the spectrum emitted by the luciferase-luciferin reaction has been shown to be higly dependent on the pH of the medium. The main characterisation experiments have been performed in LB Broth at pH 7, so in order to assess this effect cultures with LB and a citrate buffer were prepared (pH = 5.3, pH 6.1 and pH = 7) This page describes the results of these experiments. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
'''Maximum light output within 5 hours of D-luciferin injection at different pH values.'''<br />
<br />
[http://partsregistry.org/wiki/images/e/e8/Phhistogram.png http://partsregistry.org/wiki/images/thumb/e/e8/Phhistogram.png/569px-Phhistogram.png]<br />
<br />
These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<br />
'''Evolution of light output at different values of pH.'''<br />
<br />
[http://partsregistry.org/wiki/images/d/d8/Phtimecourse.png http://partsregistry.org/wiki/images/thumb/d/d8/Phtimecourse.png/569px-Phtimecourse.png]<br />
<br />
Measurements are taken every 20 min. These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/6/64/BBa_K325219pheffect.xls Media:BBa_K325219pheffect.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
#The protocol can be found as [https://2010.igem.org/Team:Cambridge/Notebook/Week11 Experiment 110].<br />
<br />
=Coloured outputs=<br />
<br />
In this section we compare the intensity of the different coloured mutants we developed during our project.<br />
<br />
[[Image:Outputpower.jpg|thumb|569px|center|'''Figure 1 - Maximum light output within 5 hours of D-Luciferin injection of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Colour coloured mutants] we developed. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
<br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[CambridgeiGEMcolouredoutputs.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.<br />
<br />
<br />
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/File:CambridgeiGEMcolouredoutputs.xlsFile:CambridgeiGEMcolouredoutputs.xls2010-10-28T00:11:51Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/File:Ouputpower.jpgFile:Ouputpower.jpg2010-10-28T00:06:46Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/PublicityTeam:Cambridge/Publicity2010-10-27T21:31:23Z<p>PM: /* French Documentary on ARTE */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#386abc|title=Publicity}}<br />
{{:Team:Cambridge/Templates/rightpic|src=Cambridge-Tv.png}}<br />
=Spreading the Word=<br />
As a team we thought it was really imporant to spread the word about iGEM. Synthetic biology is a new and developing field and the world should know about it. We feel it's especially important that synthetic biology is presented in an accessible way to people so that it is embraced by the general public. We also feel it's especially important to promote the open source nature of iGEM in the hope that as the field develops it can adopt these principles. The following are the main ways in which we have been spreading the word about iGEM over the summer. <br />
<br />
===French Documentary on ARTE===<br />
<br />
Through Daisy Ginsberg, we were approached by a French TV crew from the channel [http://www.arte.tv/fr/70.html ARTE] filming a documentary on synthetic biology. They visited us for a day of filming where they recorded us in the lab in the morning and then in the afternoon filmed us as we discussed the human practices and ethical side of synthetic biology, facilited by Daisy's interesting presentation. <br />
<br />
The film crew will also be filming at the jamboree, so look out for them there!<br />
<br />
===Telling Companies About iGEM===<br />
Through the generous sponsorship of all our [[Team:Cambridge/Partners|partners]] we have had the opportunity to spread the word about iGEM. We have written an article for [http://www.sterilin.co.uk/ Sterilin] which will be featured in the next edition of their brochure all about iGEM and the Cambridge team's project this year. <br />
<br />
[http://www.biolegio.com/ Biolegio]'s generous contribution and provision of t-shirts also enabled us to take some amusing photographs to raise iGEM's profile. <br />
<br />
===Gibson Assembly Video===<br />
What started out as an idea to make a Cambridge iGEM band, and to promote the [[Team:Cambridge/Gibson/Introduction|Gibson Assembly]] technique we had been using all summer, materialised into the [[Team:Cambridge/Videos|Gibson Assembly Song]], which is publicly available and has been viewed by over 3000 people so far (24/10/10). A great way to promote having fun in the lab over the summer! Our [[Team:Cambridge/Videos|Bacterial Bubble Lamp]] is also publicly available on youtube.<br />
<br />
===Artist Interest===<br />
We have also been contacted by several artists, including a student from Delft University of Technology, who are interested in the idea of lighting the world with bacterial luminescence. This idea has obviously captured the imagination of many artists who are also looking into the feasiblity of the idea and appreciate how beneficial it could be to the world around us.</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T21:21:20Z<p>PM: /* Compatibility */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325909 BBa K325909], the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_Luciferases ''lux operon'' from ''Vibrio fischeri'']. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#H-NS_mutants|H-NS mutants]]<br />
<br />
<br />
<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This page described the lux operon from Vibrio fischeri. To relieve LuxR control we placed Lux C, D, A, B, E under the pBad promoter.<br />
<br />
<br />
<br />
[[Image:250px-Cambridge-iGEMpixels.jpg|thumb|569px|center|'''Figure 1 - E.coli cells (Invitrogen TOP 10) transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] in a 96 well plate. ''']]<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909Aracurve.png|thumb|569px|center|'''Figure 1 - Light output of <partinfo>K325909</partinfo> as a function of Arabinose concentration in the media. The values correspond to the peak intensity within 5 hours of adding Arabinose to the media. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
[[Image:BBa_K325909timecourse.png|thumb|569px|center|'''Figure 2 - Evolution of luminescence with time at different Arabinose concentrations for <partinfo>K325909</partinfo>. Measurements were taken every 30 minutes. Data points and error bars correspond to the mean and standard deviation of 3 time repeats. ''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909ArabinosetoLight.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
=H-NS mutants=<br />
<br />
It has been shown that the expression of the Vibrio fischeri lux operon when cloned into E. coli was repressed. This repression was linked to the nucleoid protein H-NS. To investigate this effect we cloned the operon into mutant E.coli cells in which the expression of the H-NS protein had been modified. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909AraK28.png|thumb|569px|center|'''Figure 1 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats. ''']]<br />
[[Image:BBa_K325909timecourseK28.png|thumb|569px|center|'''Figure 2 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10 with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes. ''']]<br />
[[Image:BBa_K325909AraR28.png|thumb|569px|center|'''Figure 3 - Figure 3 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan]. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats.''']]<br />
[[Image:BBa_K325909timecourseR28.png|thumb|569px|center|'''Figure 4 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan] with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes.''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909Mutants.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)'', [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan] and H-NS mutant JM 230 H-NS -205::tn10.<br><br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
<br />
[http://www.jstor.org/stable/4449975 '''[1&#x5d;:'''] J. Slock, (1995) Transformation Experiments Using Bioluminescence Genes of ''Vibrio fischeri'',''The American Biology Teacher'', '''57''', 225-227.<br />
<br />
[http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.42.100188.001055 '''[2&#x5d;:'''] E.A. Meighen (1988) Enzymes and genes from the ''lux'' operons of bioluminescent bacteria, ''Annual Reviews in Microbiology'' '''42''', 151-176.<br />
<br />
[http://www.annualreviews.org/doi/pdf/10.1146/annurev.ge.28.120194.001001 '''[3&#x5d;:'''] E.A. Meighen, (1994) Genetics of bacterial bioluminescence, ''Annual Reviews of Genetics'', '''28''', 117-139.<br />
<br />
[http://onlinelibrary.wiley.com/doi/10.1002/%28SICI%291099-1271%28199807/08%2913:4%3C185::AID-BIO486%3E3.0.CO;2-U/abstract '''[4&#x5d;:'''] S. Ulitzur, (1998) H-NS controls the transcription of three promoters of ''Vibrio fischeri lux'' cloned in ''Escherichia coli'',''Journal of Bioluminescence and Chemiluminescence'', '''13'''(4), 185-188.<br />
<br />
[http://www.nature.com/nature/journal/v444/n7117/full/nature05283.html '''[5&#x5d;:'''] R.T. Dame ''et al.'', (2006) Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation,''Nature'', '''444''', 387-390.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/File:BBa_K325909Mutants.xlsFile:BBa K325909Mutants.xls2010-10-27T21:19:31Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T21:14:30Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325909 BBa K325909], the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_Luciferases ''lux operon'' from ''Vibrio fischeri'']. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#H-NS_mutants|H-NS mutants]]<br />
<br />
<br />
<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This page described the lux operon from Vibrio fischeri. To relieve LuxR control we placed Lux C, D, A, B, E under the pBad promoter.<br />
<br />
<br />
<br />
[[Image:250px-Cambridge-iGEMpixels.jpg|thumb|569px|center|'''Figure 1 - E.coli cells (Invitrogen TOP 10) transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] in a 96 well plate. ''']]<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909Aracurve.png|thumb|569px|center|'''Figure 1 - Light output of <partinfo>K325909</partinfo> as a function of Arabinose concentration in the media. The values correspond to the peak intensity within 5 hours of adding Arabinose to the media. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
[[Image:BBa_K325909timecourse.png|thumb|569px|center|'''Figure 2 - Evolution of luminescence with time at different Arabinose concentrations for <partinfo>K325909</partinfo>. Measurements were taken every 30 minutes. Data points and error bars correspond to the mean and standard deviation of 3 time repeats. ''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909ArabinosetoLight.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
=H-NS mutants=<br />
<br />
It has been shown that the expression of the Vibrio fischeri lux operon when cloned into E. coli was repressed. This repression was linked to the nucleoid protein H-NS. To investigate this effect we cloned the operon into mutant E.coli cells in which the expression of the H-NS protein had been modified. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909AraK28.png|thumb|569px|center|'''Figure 1 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats. ''']]<br />
[[Image:BBa_K325909timecourseK28.png|thumb|569px|center|'''Figure 2 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10 with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes. ''']]<br />
[[Image:BBa_K325909AraR28.png|thumb|569px|center|'''Figure 3 - Figure 3 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan]. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats.''']]<br />
[[Image:BBa_K325909timecourseR28.png|thumb|569px|center|'''Figure 4 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan] with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes.''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909Mutants.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)'', [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan] and H-NS mutant JM 230 H-NS -205::tn10.<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
<br />
[http://www.jstor.org/stable/4449975 '''[1&#x5d;:'''] J. Slock, (1995) Transformation Experiments Using Bioluminescence Genes of ''Vibrio fischeri'',''The American Biology Teacher'', '''57''', 225-227.<br />
<br />
[http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.42.100188.001055 '''[2&#x5d;:'''] E.A. Meighen (1988) Enzymes and genes from the ''lux'' operons of bioluminescent bacteria, ''Annual Reviews in Microbiology'' '''42''', 151-176.<br />
<br />
[http://www.annualreviews.org/doi/pdf/10.1146/annurev.ge.28.120194.001001 '''[3&#x5d;:'''] E.A. Meighen, (1994) Genetics of bacterial bioluminescence, ''Annual Reviews of Genetics'', '''28''', 117-139.<br />
<br />
[http://onlinelibrary.wiley.com/doi/10.1002/%28SICI%291099-1271%28199807/08%2913:4%3C185::AID-BIO486%3E3.0.CO;2-U/abstract '''[4&#x5d;:'''] S. Ulitzur, (1998) H-NS controls the transcription of three promoters of ''Vibrio fischeri lux'' cloned in ''Escherichia coli'',''Journal of Bioluminescence and Chemiluminescence'', '''13'''(4), 185-188.<br />
<br />
[http://www.nature.com/nature/journal/v444/n7117/full/nature05283.html '''[5&#x5d;:'''] R.T. Dame ''et al.'', (2006) Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation,''Nature'', '''444''', 387-390.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T21:11:08Z<p>PM: /* References */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325909 BBa K325909], the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_Luciferases ''lux operon'' from ''Vibrio fischeri'']. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#H-NS_mutants|H-NS mutants]]<br />
<br />
<br />
<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This page described the lux operon from Vibrio fischeri. To relieve LuxR control we placed Lux C, D, A, B, E under the pBad promoter.<br />
<br />
<br />
<br />
[[Image:250px-Cambridge-iGEMpixels.jpg|thumb|569px|center|'''Figure 1 - E.coli cells (Invitrogen TOP 10) transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] in a 96 well plate. ''']]<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909Aracurve.png|thumb|569px|center|'''Figure 1 - Light output of <partinfo>K325909</partinfo> as a function of Arabinose concentration in the media. The values correspond to the peak intensity within 5 hours of adding Arabinose to the media. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
[[Image:BBa_K325909timecourse.png|thumb|569px|center|'''Figure 2 - Evolution of luminescence with time at different Arabinose concentrations for <partinfo>K325909</partinfo>. Measurements were taken every 30 minutes. Data points and error bars correspond to the mean and standard deviation of 3 time repeats. ''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909ArabinosetoLight.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
=H-NS mutants=<br />
<br />
It has been shown that the expression of the Vibrio fischeri lux operon when cloned into E. coli was repressed. This repression was linked to the nucleoid protein H-NS. To investigate this effect we cloned the operon into mutant E.coli cells in which the expression of the H-NS protein had been modified. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909AraK28.png|thumb|569px|center|'''Figure 1 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats. ''']]<br />
[[Image:BBa_K325909timecourseK28.png|thumb|569px|center|'''Figure 2 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10 with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes. ''']]<br />
[[Image:BBa_K325909AraR28.png|thumb|569px|center|'''Figure 3 - Figure 3 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan]. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats.''']]<br />
[[Image:BBa_K325909timecourseR28.png|thumb|569px|center|'''Figure 4 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan] with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes.''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909Mutants.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
<br />
[http://www.jstor.org/stable/4449975 '''[1&#x5d;:'''] J. Slock, (1995) Transformation Experiments Using Bioluminescence Genes of ''Vibrio fischeri'',''The American Biology Teacher'', '''57''', 225-227.<br />
<br />
[http://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.42.100188.001055 '''[2&#x5d;:'''] E.A. Meighen (1988) Enzymes and genes from the ''lux'' operons of bioluminescent bacteria, ''Annual Reviews in Microbiology'' '''42''', 151-176.<br />
<br />
[http://www.annualreviews.org/doi/pdf/10.1146/annurev.ge.28.120194.001001 '''[3&#x5d;:'''] E.A. Meighen, (1994) Genetics of bacterial bioluminescence, ''Annual Reviews of Genetics'', '''28''', 117-139.<br />
<br />
[http://onlinelibrary.wiley.com/doi/10.1002/%28SICI%291099-1271%28199807/08%2913:4%3C185::AID-BIO486%3E3.0.CO;2-U/abstract '''[4&#x5d;:'''] S. Ulitzur, (1998) H-NS controls the transcription of three promoters of ''Vibrio fischeri lux'' cloned in ''Escherichia coli'',''Journal of Bioluminescence and Chemiluminescence'', '''13'''(4), 185-188.<br />
<br />
[http://www.nature.com/nature/journal/v444/n7117/full/nature05283.html '''[5&#x5d;:'''] R.T. Dame ''et al.'', (2006) Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation,''Nature'', '''444''', 387-390.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T21:08:53Z<p>PM: /* References */</p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325909 BBa K325909], the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_Luciferases ''lux operon'' from ''Vibrio fischeri'']. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#H-NS_mutants|H-NS mutants]]<br />
<br />
<br />
<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This page described the lux operon from Vibrio fischeri. To relieve LuxR control we placed Lux C, D, A, B, E under the pBad promoter.<br />
<br />
<br />
<br />
[[Image:250px-Cambridge-iGEMpixels.jpg|thumb|569px|center|'''Figure 1 - E.coli cells (Invitrogen TOP 10) transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] in a 96 well plate. ''']]<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909Aracurve.png|thumb|569px|center|'''Figure 1 - Light output of <partinfo>K325909</partinfo> as a function of Arabinose concentration in the media. The values correspond to the peak intensity within 5 hours of adding Arabinose to the media. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
[[Image:BBa_K325909timecourse.png|thumb|569px|center|'''Figure 2 - Evolution of luminescence with time at different Arabinose concentrations for <partinfo>K325909</partinfo>. Measurements were taken every 30 minutes. Data points and error bars correspond to the mean and standard deviation of 3 time repeats. ''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909ArabinosetoLight.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
=H-NS mutants=<br />
<br />
It has been shown that the expression of the Vibrio fischeri lux operon when cloned into E. coli was repressed. This repression was linked to the nucleoid protein H-NS. To investigate this effect we cloned the operon into mutant E.coli cells in which the expression of the H-NS protein had been modified. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909AraK28.png|thumb|569px|center|'''Figure 1 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats. ''']]<br />
[[Image:BBa_K325909timecourseK28.png|thumb|569px|center|'''Figure 2 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10 with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes. ''']]<br />
[[Image:BBa_K325909AraR28.png|thumb|569px|center|'''Figure 3 - Figure 3 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan]. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats.''']]<br />
[[Image:BBa_K325909timecourseR28.png|thumb|569px|center|'''Figure 4 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan] with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes.''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909Mutants.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.<br />
<br />
<br />
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.<br />
<br />
[http://onlinelibrary.wiley.com/doi/10.1002/%28SICI%291099-1271%28199807/08%2913:4%3C185::AID-BIO486%3E3.0.CO;2-U/abstract '''[4&#x5d;:'''] S. Ulitzur, (1998) H-NS controls the transcription of three promoters of ''Vibrio fischeri lux'' cloned in ''Escherichia coli'',''Journal of Bioluminescence and Chemiluminescence'', '''13'''(4), 185-188.<br />
<br />
[http://www.nature.com/nature/journal/v444/n7117/full/nature05283.html '''[5&#x5d;:'''] R.T. Dame ''et al.'', (2006) Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation,''Nature'', '''444''', 387-390.<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T21:07:16Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
<br />
This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325909 BBa K325909], the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_Luciferases ''lux operon'' from ''Vibrio fischeri'']. <br />
<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#H-NS_mutants|H-NS mutants]]<br />
<br />
<br />
<br />
=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This page described the lux operon from Vibrio fischeri. To relieve LuxR control we placed Lux C, D, A, B, E under the pBad promoter.<br />
<br />
<br />
<br />
[[Image:250px-Cambridge-iGEMpixels.jpg|thumb|569px|center|'''Figure 1 - E.coli cells (Invitrogen TOP 10) transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] in a 96 well plate. ''']]<br />
<br />
=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909Aracurve.png|thumb|569px|center|'''Figure 1 - Light output of <partinfo>K325909</partinfo> as a function of Arabinose concentration in the media. The values correspond to the peak intensity within 5 hours of adding Arabinose to the media. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
[[Image:BBa_K325909timecourse.png|thumb|569px|center|'''Figure 2 - Evolution of luminescence with time at different Arabinose concentrations for <partinfo>K325909</partinfo>. Measurements were taken every 30 minutes. Data points and error bars correspond to the mean and standard deviation of 3 time repeats. ''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909ArabinosetoLight.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
=H-NS mutants=<br />
<br />
It has been shown that the expression of the Vibrio fischeri lux operon when cloned into E. coli was repressed. This repression was linked to the nucleoid protein H-NS. To investigate this effect we cloned the operon into mutant E.coli cells in which the expression of the H-NS protein had been modified. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
<br />
[[Image:BBa_K325909AraK28.png|thumb|569px|center|'''Figure 1 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats. ''']]<br />
[[Image:BBa_K325909timecourseK28.png|thumb|569px|center|'''Figure 2 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10 with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes. ''']]<br />
[[Image:BBa_K325909AraR28.png|thumb|569px|center|'''Figure 3 - Figure 3 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan]. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats.''']]<br />
[[Image:BBa_K325909timecourseR28.png|thumb|569px|center|'''Figure 4 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan] with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes.''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909Mutants.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
<br />
<br />
=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
<br />
=References=<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.<br />
<br />
<br />
[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/File:BBa_K325909timecourseR28.pngFile:BBa K325909timecourseR28.png2010-10-27T21:04:25Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/File:BBa_K325909AraR28.pngFile:BBa K325909AraR28.png2010-10-27T21:03:27Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/File:BBa_K325909timecourseK28.pngFile:BBa K325909timecourseK28.png2010-10-27T21:02:13Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/File:BBa_K325909AraK28.pngFile:BBa K325909AraK28.png2010-10-27T21:01:33Z<p>PM: </p>
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<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T20:59:39Z<p>PM: /* H-NS mutants */</p>
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<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}}<br />
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This page describes characterisation for part [http://partsregistry.org/Part:BBa_K325909 BBa K325909], the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Bacterial_Luciferases ''lux operon'' from ''Vibrio fischeri'']. <br />
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*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Description|Description]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#Arabinose_to_light|Arabinose to light]]<br />
*[[Team:Cambridge/Bioluminescence/Vibrio_Characterisation#H-NS_mutants|H-NS mutants]]<br />
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=Description=<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-low.jpg|caption=E.Coli (Invitrogen TOP 10) cells transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] (blue light bulb) and [http://partsregistry.org/Part:BBa_K325219 BBa 325219] (red light bulb)}}<br />
This page described the lux operon from Vibrio fischeri. To relieve LuxR control we placed Lux C, D, A, B, E under the pBad promoter.<br />
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[[Image:250px-Cambridge-iGEMpixels.jpg|thumb|569px|center|'''Figure 1 - E.coli cells (Invitrogen TOP 10) transformed with [http://partsregistry.org/Part:BBa_K325909 BBa K325909] in a 96 well plate. ''']]<br />
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=Arabinose to light=<br />
This page describes the relationship between Arabinose concentration in the medium with light output. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocol and plate reader settings used are given below. <br />
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{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
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[[Image:BBa_K325909Aracurve.png|thumb|569px|center|'''Figure 1 - Light output of <partinfo>K325909</partinfo> as a function of Arabinose concentration in the media. The values correspond to the peak intensity within 5 hours of adding Arabinose to the media. Data points and error bars correspond to the mean and the standard deviation of 3 time repeats. ''']]<br />
[[Image:BBa_K325909timecourse.png|thumb|569px|center|'''Figure 2 - Evolution of luminescence with time at different Arabinose concentrations for <partinfo>K325909</partinfo>. Measurements were taken every 30 minutes. Data points and error bars correspond to the mean and standard deviation of 3 time repeats. ''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909ArabinosetoLight.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
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=H-NS mutants=<br />
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It has been shown that the expression of the Vibrio fischeri lux operon when cloned into E. coli was repressed. This repression was linked to the nucleoid protein H-NS. To investigate this effect we cloned the operon into mutant E.coli cells in which the expression of the H-NS protein had been modified. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
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{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
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[[Image:BBa_K325909AraK28.png|thumb|569px|center|'''Figure 1 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats. ''']]<br />
[[Image:BBa_K325909timecourseK28.png|thumb|569px|center|'''Figure 2 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant JM 230 H-NS -205::tn10 with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes. ''']]<br />
[[Image:BBa_K325909AraR28.png|thumb|569px|center|'''Figure 3 - Figure 3 - Peak light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan]. The data points and the error bar are the mean and standard deviation obtained by 3 tim repeats.''']]<br />
[[Image:BBa_K325909timecourseR28.png|thumb|569px|center|'''Figure 4 - Evolution of light output from <partinfo>K325909</partinfo> cloned into H-NS mutant [http://www.ecoliwiki.net/colipedia/index.php/BW25113 BW25113 DELTA H-NS::kan] with time at different Arabinose concentrations. The data points and the error bar are the mean and standard deviation obtained by 3 time repeats. Measurements were taken every 30 minutes.''']]<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[[Media:BBa_K325909Mutants.xls]]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
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=Effect of pH=<br />
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Both the intensity and the spectrum emitted by the luciferase-luciferin reaction has been shown to be higly dependent on the pH of the medium. The main characterisation experiments have been performed in LB Broth at pH 7, so in order to assess this effect cultures with LB and a citrate buffer were prepared (pH = 5.3, pH 6.1 and pH = 7) This page describes the results of these experiments. We used a [http://www.bmglabtech.com/products/microplate-reader/instruments.cfm?product_id=2 FLUOstar OPTIMA] microplate reader to quantify the light output. Protocols and plate reader settings used are given below. <br />
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{{:Team:Cambridge/Templates/Nolineheader|header=Data}}<br />
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'''Maximum light output within 5 hours of D-luciferin injection at different pH values.'''<br />
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[http://partsregistry.org/wiki/images/e/e8/Phhistogram.png http://partsregistry.org/wiki/images/thumb/e/e8/Phhistogram.png/569px-Phhistogram.png]<br />
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These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
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'''Evolution of light output at different values of pH.'''<br />
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[http://partsregistry.org/wiki/images/d/d8/Phtimecourse.png http://partsregistry.org/wiki/images/thumb/d/d8/Phtimecourse.png/569px-Phtimecourse.png]<br />
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Measurements are taken every 20 min. These values are the mean of 3 readings. The corresponding error bars represent an interval of twice the standard deviation across the 3 data points centred around the mean value.<br />
<center><br />
{|{{Table}}<br />
!Data<br />
!Notes<br />
!Date Uploaded<br />
|-<br />
|[http://partsregistry.org/wiki/images/6/64/BBa_K325219pheffect.xls Media:BBa_K325219pheffect.xls]<br />
|Raw data from experiment<br />
|21/10/2010<br />
|}<br />
</center><br />
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{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
#The protocol can be found as [https://2010.igem.org/Team:Cambridge/Notebook/Week11 Experiment 110].<br />
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=Compatibility=<br />
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=cell ''Chassis:''] Device has been shown to work in ''Top 10 (Invitrogen)''<br />
[[Plasmid backbones|''Plasmids:'']] Device has been shown to work on ''<partinfo>pSB1C3</partinfo>'' <br><br />
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=References=<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18949818 '''[1&#x5d;:'''] S.M. Marques and J.C.G. Esteves da Silva, (2009) Firefly Bioluminescence: A Mechanistic Approach of Luciferase Catalyzed Reactions,''Life'' '''61''', 6-17.<br />
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[http://www.nature.com/nature/journal/v440/n7082/abs/nature04542.html '''[2&#x5d;:'''] T. Nakatsu ''et al.'' (2006) Structural Basis for the spectral difference in luciferase bioluminescence, ''Nature'' '''440'''(16), 372-376.<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/11457857 '''[3&#x5d;:'''] K. Gomi and N. Kajiyama, (2001) Oxyluciferin, a Luminescence Product of Firefly Luciferase, Is Enzymatically Regenerated into Luciferin, ''The Journal of Biological Chemistry'', '''276'''(39), 36508-36513.<br />
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{{:Team:Cambridge/Templates/footer}}</div>PM