http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=PM2010.igem.org - User contributions [en]2024-03-29T10:09:38ZFrom 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>
<hr />
<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>
<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 />
=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 />
=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:250px-Cambridge-iGEMpixels.jpgFile:250px-Cambridge-iGEMpixels.jpg2010-10-27T20:51:51Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T20:51:13Z<p>PM: /* Description */</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 />
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 />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
#Three 5 ml cultures of [http://openwetware.org/wiki/Endy:M9_media/supplemented supplemented M9 medium] and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing <partinfo>BBa_T9002</partinfo>. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing a <partinfo>BBa_T9002</partinfo> mutant (T9002m) lacking a GFP expression device described in the [http://partsregistry.org/Part:BBa_F2620:Stability stability section].<br />
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.<br />
#Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.<br />
#Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br />
#2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]]), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]. Three wells were each filled with 200 µl of medium to measure the absorbance background. Three further wells were each filled with 200 µl of the <partinfo>BBa_T9002</partinfo> mutant culture to measure fluorescent background.<br />
#The plate was incubated in a [http://openwetware.org/wiki/Endy:Victor3_plate_reader Wallac Victor3 multi-well fluorimeter] (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s. Approximately 6 min elapsed between beginning addition of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] to the wells and the first plate reader measurement. [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] was added in order of increasing concentration to minimize GFP synthesis during plate loading. Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).<br />
#We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate. <br />
#We processed the data to compute the PoPS output from <partinfo>BBa_F2620</partinfo> as described on the [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data analysis page]. The data for each colony tested was averaged across the three replicate wells. The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3. Following an initial transient response, device output reached an approximate steady state. <br />
#The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line). Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples. The cyan shaded region represents the range of the nine independent samples.<br />
#To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data. A Hill equation derived from simple biochemical equations describes the data well (R<sup>2</sup>=0.99). In the equation (shown below), P<sub>out</sub> is the PoPS per cell output of <partinfo>BBa_F2620</partinfo>, P<sub>max</sub> is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.<br />
#To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above. Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data. The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations.<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 />
=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/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T20:49:58Z<p>PM: /* Arabinose to light */</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 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 />
[[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 />
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 />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
#Three 5 ml cultures of [http://openwetware.org/wiki/Endy:M9_media/supplemented supplemented M9 medium] and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing <partinfo>BBa_T9002</partinfo>. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing a <partinfo>BBa_T9002</partinfo> mutant (T9002m) lacking a GFP expression device described in the [http://partsregistry.org/Part:BBa_F2620:Stability stability section].<br />
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.<br />
#Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.<br />
#Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br />
#2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]]), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]. Three wells were each filled with 200 µl of medium to measure the absorbance background. Three further wells were each filled with 200 µl of the <partinfo>BBa_T9002</partinfo> mutant culture to measure fluorescent background.<br />
#The plate was incubated in a [http://openwetware.org/wiki/Endy:Victor3_plate_reader Wallac Victor3 multi-well fluorimeter] (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s. Approximately 6 min elapsed between beginning addition of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] to the wells and the first plate reader measurement. [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] was added in order of increasing concentration to minimize GFP synthesis during plate loading. Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).<br />
#We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate. <br />
#We processed the data to compute the PoPS output from <partinfo>BBa_F2620</partinfo> as described on the [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data analysis page]. The data for each colony tested was averaged across the three replicate wells. The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3. Following an initial transient response, device output reached an approximate steady state. <br />
#The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line). Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples. The cyan shaded region represents the range of the nine independent samples.<br />
#To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data. A Hill equation derived from simple biochemical equations describes the data well (R<sup>2</sup>=0.99). In the equation (shown below), P<sub>out</sub> is the PoPS per cell output of <partinfo>BBa_F2620</partinfo>, P<sub>max</sub> is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.<br />
#To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above. Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data. The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations.<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 />
=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_K325909ArabinosetoLight.xlsFile:BBa K325909ArabinosetoLight.xls2010-10-27T20:49:15Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T20:48:30Z<p>PM: /* Arabinose to light */</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 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 />
[[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 />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
=H-NS mutants=<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 />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
#Three 5 ml cultures of [http://openwetware.org/wiki/Endy:M9_media/supplemented supplemented M9 medium] and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing <partinfo>BBa_T9002</partinfo>. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing a <partinfo>BBa_T9002</partinfo> mutant (T9002m) lacking a GFP expression device described in the [http://partsregistry.org/Part:BBa_F2620:Stability stability section].<br />
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.<br />
#Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.<br />
#Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br />
#2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]]), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]. Three wells were each filled with 200 µl of medium to measure the absorbance background. Three further wells were each filled with 200 µl of the <partinfo>BBa_T9002</partinfo> mutant culture to measure fluorescent background.<br />
#The plate was incubated in a [http://openwetware.org/wiki/Endy:Victor3_plate_reader Wallac Victor3 multi-well fluorimeter] (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s. Approximately 6 min elapsed between beginning addition of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] to the wells and the first plate reader measurement. [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] was added in order of increasing concentration to minimize GFP synthesis during plate loading. Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).<br />
#We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate. <br />
#We processed the data to compute the PoPS output from <partinfo>BBa_F2620</partinfo> as described on the [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data analysis page]. The data for each colony tested was averaged across the three replicate wells. The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3. Following an initial transient response, device output reached an approximate steady state. <br />
#The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line). Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples. The cyan shaded region represents the range of the nine independent samples.<br />
#To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data. A Hill equation derived from simple biochemical equations describes the data well (R<sup>2</sup>=0.99). In the equation (shown below), P<sub>out</sub> is the PoPS per cell output of <partinfo>BBa_F2620</partinfo>, P<sub>max</sub> is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.<br />
#To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above. Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data. The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations.<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 />
=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_K325909timecourse.pngFile:BBa K325909timecourse.png2010-10-27T20:47:19Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T20:44:49Z<p>PM: /* Arabinose to light */</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 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 />
[[Image:BBa_K325909Aracurve.png|thumb|569px|center|'''Figure 1 - Light output of <partinfo>K325219</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>K325219</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 />
<br />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
=H-NS mutants=<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 />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
#Three 5 ml cultures of [http://openwetware.org/wiki/Endy:M9_media/supplemented supplemented M9 medium] and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing <partinfo>BBa_T9002</partinfo>. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing a <partinfo>BBa_T9002</partinfo> mutant (T9002m) lacking a GFP expression device described in the [http://partsregistry.org/Part:BBa_F2620:Stability stability section].<br />
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.<br />
#Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.<br />
#Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br />
#2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]]), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]. Three wells were each filled with 200 µl of medium to measure the absorbance background. Three further wells were each filled with 200 µl of the <partinfo>BBa_T9002</partinfo> mutant culture to measure fluorescent background.<br />
#The plate was incubated in a [http://openwetware.org/wiki/Endy:Victor3_plate_reader Wallac Victor3 multi-well fluorimeter] (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s. Approximately 6 min elapsed between beginning addition of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] to the wells and the first plate reader measurement. [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] was added in order of increasing concentration to minimize GFP synthesis during plate loading. Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).<br />
#We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate. <br />
#We processed the data to compute the PoPS output from <partinfo>BBa_F2620</partinfo> as described on the [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data analysis page]. The data for each colony tested was averaged across the three replicate wells. The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3. Following an initial transient response, device output reached an approximate steady state. <br />
#The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line). Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples. The cyan shaded region represents the range of the nine independent samples.<br />
#To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data. A Hill equation derived from simple biochemical equations describes the data well (R<sup>2</sup>=0.99). In the equation (shown below), P<sub>out</sub> is the PoPS per cell output of <partinfo>BBa_F2620</partinfo>, P<sub>max</sub> is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.<br />
#To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above. Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data. The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations.<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 />
=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/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T20:44:12Z<p>PM: /* Luciferin to light */</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 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 />
=H-NS mutants=<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 />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
#Three 5 ml cultures of [http://openwetware.org/wiki/Endy:M9_media/supplemented supplemented M9 medium] and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing <partinfo>BBa_T9002</partinfo>. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing a <partinfo>BBa_T9002</partinfo> mutant (T9002m) lacking a GFP expression device described in the [http://partsregistry.org/Part:BBa_F2620:Stability stability section].<br />
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.<br />
#Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.<br />
#Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br />
#2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]]), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]. Three wells were each filled with 200 µl of medium to measure the absorbance background. Three further wells were each filled with 200 µl of the <partinfo>BBa_T9002</partinfo> mutant culture to measure fluorescent background.<br />
#The plate was incubated in a [http://openwetware.org/wiki/Endy:Victor3_plate_reader Wallac Victor3 multi-well fluorimeter] (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s. Approximately 6 min elapsed between beginning addition of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] to the wells and the first plate reader measurement. [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] was added in order of increasing concentration to minimize GFP synthesis during plate loading. Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).<br />
#We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate. <br />
#We processed the data to compute the PoPS output from <partinfo>BBa_F2620</partinfo> as described on the [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data analysis page]. The data for each colony tested was averaged across the three replicate wells. The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3. Following an initial transient response, device output reached an approximate steady state. <br />
#The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line). Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples. The cyan shaded region represents the range of the nine independent samples.<br />
#To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data. A Hill equation derived from simple biochemical equations describes the data well (R<sup>2</sup>=0.99). In the equation (shown below), P<sub>out</sub> is the PoPS per cell output of <partinfo>BBa_F2620</partinfo>, P<sub>max</sub> is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.<br />
#To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above. Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data. The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations.<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 />
=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/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T20:42:52Z<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 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 />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
#Three 5 ml cultures of [http://openwetware.org/wiki/Endy:M9_media/supplemented supplemented M9 medium] and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing <partinfo>BBa_T9002</partinfo>. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing a <partinfo>BBa_T9002</partinfo> mutant (T9002m) lacking a GFP expression device described in the [http://partsregistry.org/Part:BBa_F2620:Stability stability section].<br />
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.<br />
#Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.<br />
#Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br />
#2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]]), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]. Three wells were each filled with 200 µl of medium to measure the absorbance background. Three further wells were each filled with 200 µl of the <partinfo>BBa_T9002</partinfo> mutant culture to measure fluorescent background.<br />
#The plate was incubated in a [http://openwetware.org/wiki/Endy:Victor3_plate_reader Wallac Victor3 multi-well fluorimeter] (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s. Approximately 6 min elapsed between beginning addition of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] to the wells and the first plate reader measurement. [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] was added in order of increasing concentration to minimize GFP synthesis during plate loading. Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).<br />
#We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate. <br />
#We processed the data to compute the PoPS output from <partinfo>BBa_F2620</partinfo> as described on the [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data analysis page]. The data for each colony tested was averaged across the three replicate wells. The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3. Following an initial transient response, device output reached an approximate steady state. <br />
#The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line). Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples. The cyan shaded region represents the range of the nine independent samples.<br />
#To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data. A Hill equation derived from simple biochemical equations describes the data well (R<sup>2</sup>=0.99). In the equation (shown below), P<sub>out</sub> is the PoPS per cell output of <partinfo>BBa_F2620</partinfo>, P<sub>max</sub> is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.<br />
#To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above. Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data. The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations.<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 />
=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_K325909Aracurve.pngFile:BBa K325909Aracurve.png2010-10-27T20:39:35Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Vibrio_CharacterisationTeam:Cambridge/Bioluminescence/Vibrio Characterisation2010-10-27T20:25:48Z<p>PM: New page: {{:Team:Cambridge/Templates/headerMinimalprototype}} {{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Characterisation}} This page describes ...</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 />
<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 />
<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 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 />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
#Three 5 ml cultures of [http://openwetware.org/wiki/Endy:M9_media/supplemented supplemented M9 medium] and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing <partinfo>BBa_T9002</partinfo>. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing a <partinfo>BBa_T9002</partinfo> mutant (T9002m) lacking a GFP expression device described in the [http://partsregistry.org/Part:BBa_F2620:Stability stability section].<br />
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.<br />
#Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.<br />
#Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br />
#2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]]), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]. Three wells were each filled with 200 µl of medium to measure the absorbance background. Three further wells were each filled with 200 µl of the <partinfo>BBa_T9002</partinfo> mutant culture to measure fluorescent background.<br />
#The plate was incubated in a [http://openwetware.org/wiki/Endy:Victor3_plate_reader Wallac Victor3 multi-well fluorimeter] (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s. Approximately 6 min elapsed between beginning addition of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] to the wells and the first plate reader measurement. [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] was added in order of increasing concentration to minimize GFP synthesis during plate loading. Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).<br />
#We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate. <br />
#We processed the data to compute the PoPS output from <partinfo>BBa_F2620</partinfo> as described on the [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data analysis page]. The data for each colony tested was averaged across the three replicate wells. The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3. Following an initial transient response, device output reached an approximate steady state. <br />
#The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line). Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples. The cyan shaded region represents the range of the nine independent samples.<br />
#To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data. A Hill equation derived from simple biochemical equations describes the data well (R<sup>2</sup>=0.99). In the equation (shown below), P<sub>out</sub> is the PoPS per cell output of <partinfo>BBa_F2620</partinfo>, P<sub>max</sub> is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.<br />
#To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above. Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data. The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations.<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 />
=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 Characterisation2010-10-27T20:20:15Z<p>PM: /* Arabinose to light */</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 />
<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 />
<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 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 />
{{:Team:Cambridge/Templates/Nolineheader|header=Protocol}}<br />
<br />
#Three 5 ml cultures of [http://openwetware.org/wiki/Endy:M9_media/supplemented supplemented M9 medium] and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing <partinfo>BBa_T9002</partinfo>. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of [http://partsregistry.org/Part:BBa_V1000 MG1655] containing a <partinfo>BBa_T9002</partinfo> mutant (T9002m) lacking a GFP expression device described in the [http://partsregistry.org/Part:BBa_F2620:Stability stability section].<br />
#Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.<br />
#Cultures were diluted 1:1000 into 5.5 ml of fresh medium and grown to an OD600 of 0.15 under the same conditions as before. This growth took on average 4.5 hrs.<br />
#Twenty-four 200 µl aliquots of each of the cultures were transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br />
#2 µl of the stock concentrations of the cognate AHL, 3-oxohexanoyl-homoserine lactone ([http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]]), was added to each well to yield 8 different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). Three replicate wells were measured for each concentration of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL]. Three wells were each filled with 200 µl of medium to measure the absorbance background. Three further wells were each filled with 200 µl of the <partinfo>BBa_T9002</partinfo> mutant culture to measure fluorescent background.<br />
#The plate was incubated in a [http://openwetware.org/wiki/Endy:Victor3_plate_reader Wallac Victor3 multi-well fluorimeter] (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12901 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements was 2 min and 21 s. Approximately 6 min elapsed between beginning addition of [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] to the wells and the first plate reader measurement. [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] was added in order of increasing concentration to minimize GFP synthesis during plate loading. Cells appear to grow exponentially for the duration of the plate reader measurement protocol (see Figure 2 for representative growth curves).<br />
#We repeated steps 1 through 6 on three separate days to obtain data for nine colonies from a single plate. <br />
#We processed the data to compute the PoPS output from <partinfo>BBa_F2620</partinfo> as described on the [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data analysis page]. The data for each colony tested was averaged across the three replicate wells. The mean for each colony was then averaged to obtain a population mean. The time and dose dependent input-output surface is shown above in Figure 3. Following an initial transient response, device output reached an approximate steady state. <br />
#The snapshot transfer function in Figure 1 is the 60 min time-slice from the surface shown in Figure 3 (highlighted as a heavy black line). Error bars in Figure 1 representing the 95% confidence interval in the population for the nine independent samples. The cyan shaded region represents the range of the nine independent samples.<br />
#To estimate parameters that characterize the measured transfer function, we used least squares estimation to fit a simple model to the data. A Hill equation derived from simple biochemical equations describes the data well (R<sup>2</sup>=0.99). In the equation (shown below), P<sub>out</sub> is the PoPS per cell output of <partinfo>BBa_F2620</partinfo>, P<sub>max</sub> is the maximum output level, K is the switch point, and n is the hill coefficient describing the steepness of the transition from low output to high output.<br />
#To gain further information about the transition region of the transfer function, measurements were subsequently taken at two intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations (3.3E-09 M and 3.3E-08 M) using the same protocol defined above. Measurements were simultaneously taken at a subset of the original concentrations to ensure the new data was consistent with the earlier data. The new data was processed simultaneously with the original data, with the exception that only six independent colonies were measured for the intermediate [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] concentrations.<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 />
=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/Luciferin_RegenerationTeam:Cambridge/Bioluminescence/Luciferin Regeneration2010-10-27T00:45:45Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|linkcolour=#6bbe00|colour=#96d446|title=Bioluminescence: Luciferin Regeneration}}<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-MixedPhenotype.jpg|caption=Colonies expressing luciferase and LRE}}<br />
<br />
===Introduction===<br />
Fireflies are easily visible in the evening, whereas colonies of bacteria expressing luciferase are not. We hope to remove this divide by expressing further enzymes exploited by fireflies. In <i>E. coli</i> and in eukaryotes, each molecule of luciferin that emits light is turned into oxyluciferin and cannot be regenerated. A further problem is that this oxyluciferin inhibits the further reactions of luciferase.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 Gomi and Kajiyama (2001)] describe a <strong>luciferin regenerating enzyme (LRE)</strong>, which we believed may solve these problems and allow brighter longer lasting light output without further addition of substrate.<br />
<br />
[[Image:Cam-luci-cycle.jpg|center|500px]]<br />
LRE transforms the inhibitory oxyluciferin to CHBT, which is converted back into the luciferin substrate required for light output. This was believed to occur in one of two ways: non-enzymatically requiring D-cysteine, or via L-luciferin requiring the L-cysteine. We aimed to verify these claims in our lab experiments and to quantify the difference in light output that LRE makes when expressed in ''E. coli''. To formally test the effect of the LRE we would have needed to add Oxyluciferin to the medium. Unfortunately, Oxyluciferin is not available commercially so we were not able to perform this experiment. We however tested other parts of the cycle, by adding CHBT and D-Cysteine to the medium. <br />
<br />
===Our Experiments===<br />
We tested both the ''L. cruciata'' and ''P. pyralis'' luciferases with and without LRE on our plate reader, kindly on loan from [http://www.bmglabtech.com/ BMG Labtech]. The results showed that when D-cysteine was added to the reaction, the light output was brighter and lasted for longer. This proved that both the working of LRE to produce CHBT and the conversion of CHBT into luciferin worked as expected. <br />
<br />
<br />
[[Image:Dcysteineffect.png|thumb|center|700px|'''Figure 1 - Light output as a function of time for [http://partsregistry.org/Part:BBa_K325219 L.Cruciata luciferase and LRE] with and without D-Cysteine added to teh meidum.''']]<br />
<br />
However, the conversion from CHBT to luciferin via L-luciferin by adding L-cysteine was found not to work. Our research suggested that overexpression of thioesterase might help to make this possible, unfortunately our attempts to PCR out the E. coli thioesterase were not successful.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/File:Dcysteineffect.pngFile:Dcysteineffect.png2010-10-27T00:39:43Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Luciferin_RegenerationTeam:Cambridge/Bioluminescence/Luciferin Regeneration2010-10-27T00:38:46Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|linkcolour=#6bbe00|colour=#96d446|title=Bioluminescence: Luciferin Regeneration}}<br />
<br />
{{:Team:Cambridge/Templates/RightImage|image=Cambridge-MixedPhenotype.jpg|caption=Colonies expressing luciferase and LRE}}<br />
<br />
===Introduction===<br />
Fireflies are easily visible in the evening, whereas colonies of bacteria expressing luciferase are not. We hope to remove this divide by expressing further enzymes exploited by fireflies. In <i>E. coli</i> and in eukaryotes, each molecule of luciferin that emits light is turned into oxyluciferin and cannot be regenerated. A further problem is that this oxyluciferin inhibits the further reactions of luciferase.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11457857 Gomi and Kajiyama (2001)] describe a <strong>luciferin regenerating enzyme (LRE)</strong>, which we believed may solve these problems and allow brighter longer lasting light output without further addition of substrate.<br />
<br />
[[Image:Cam-luci-cycle.jpg|center|500px]]<br />
LRE transforms the inhibitory oxyluciferin to CHBT, which is converted back into the luciferin substrate required for light output. This was believed to occur in one of two ways: non-enzymatically requiring D-cysteine, or via L-luciferin requiring the L-cysteine. We aimed to verify these claims in our lab experiments and to quantify the difference in light output that LRE makes when expressed in ''E. coli''. To formally test the effect of the LRE we would have needed to add Oxyluciferin to the medium. Unfortunately, Oxyluciferin is not available commercially so we were not able to perform this experiment. We however tested other parts of the cycle, by adding CHBT and D-Cysteine to the medium. <br />
<br />
===Our Experiments===<br />
We tested both the ''L. cruciata'' and ''P. pyralis'' luciferases with and without LRE on our plate reader, kindly on loan from [http://www.bmglabtech.com/ BMG Labtech]. The results showed that when D-cysteine was added to the reaction, the light output was brighter and lasted for longer. This proved that both the working of LRE to produce CHBT and the conversion of CHBT into luciferin worked as expected. <br />
<br />
However, the conversion from CHBT to luciferin via L-luciferin by adding L-cysteine was found not to work. Our research suggested that overexpression of thioesterase might help to make this possible, unfortunately our attempts to PCR out the E. coli thioesterase were not successful.<br />
<br />
<br />
{{:Team:Cambridge/Templates/footer}}</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_ModellingTeam:Cambridge/Bioluminescence/Firefly Modelling2010-10-27T00:28:25Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Modelling}}<br />
<br />
We modelled the Luciferin cycle as described in the [http://www.ncbi.nlm.nih.gov/pubmed/18949818 literature] We used data from [http://www.ncbi.nlm.nih.gov/pubmed/19859663 S. Inouye]'s review and the paper on [http://www.ncbi.nlm.nih.gov/pubmed/20655239 firefly luciferase inhibition] from J.M Leitão ''et al.''<br />
<br />
We used the [http://www.mathworks.com/products/simbiology/ Matlab Simbiology toolbox] to model the cycle. A diagram of the model we developed can be found below: <br />
<br />
[[Image:Modelluccycle.jpg|center|700px]]<br />
<br />
Some of the parameters could be found in the litterature, but some of the parameters (namely the diffusion of Luciferin into the cell and the regeneration rate were inferred from experimental data.) <br />
<br />
We modelled the system both with and without the effect of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Luciferin_Regeneration Luciferase Regenerating Enzyme] (LRE.) The 4 Figures below show a comparison the model and our data for both cases. <br />
<br />
<br />
[[Image:Modellight.png|thumb|center|700px|'''Figure 1 - Model of the light output as a function of time without the effect of the LRE. ''']]<br />
<br />
<br />
[[Image:Datawithoutreg.png|thumb|center|700px|'''Figure 2 - Light output as a function of time for [http://partsregistry.org/Part:BBa_K325108 P.Pyralis luciferase] under pBad promoter. Arabinose concentration is 10mM and D-Luciferin concentration is 100µM''']]<br />
<br />
<br />
[[Image:Modellight_with_regen.png|thumb|center|700px|'''Figure 3 - Model of the light output as a function of time with the effect of the LRE. ''']]<br />
<br />
<br />
[[Image:Datawithreg.png|thumb|center|700px|'''Figure 4 - Light output as a function of time for [http://partsregistry.org/Part:BBa_K325219 L. Cruciata luciferase with LRE] under pBad promoter. Arabinose concentration is 100 µM and D-Luciferin concentration is 1µM''']]</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_ModellingTeam:Cambridge/Bioluminescence/Firefly Modelling2010-10-27T00:26:55Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Modelling}}<br />
<br />
We modelled the Luciferin cycle as described in the [http://www.ncbi.nlm.nih.gov/pubmed/18949818 literature] We used data from [http://www.ncbi.nlm.nih.gov/pubmed/19859663 S. Inouye]'s review and the paper on [http://www.ncbi.nlm.nih.gov/pubmed/20655239 firefly luciferase inhibition] from J.M Leitão ''et al.''<br />
<br />
We used the [http://www.mathworks.com/products/simbiology/ Matlab Simbiology toolbox] to model the cycle. A diagram of the model we developed can be found below: <br />
<br />
[[Image:Modelluccycle.jpg|center|700px]]<br />
<br />
Some of the parameters could be found in the litterature, but some of the parameters (namely the diffusion of Luciferin into the cell and the regeneration rate were inferred from experimental data.) <br />
<br />
We modelled the system both with and without the effect of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Luciferin_Regeneration Luciferase Regenerating Enzyme] (LRE.) The 4 Figures below show a comparison the model and our data for both cases. <br />
<br />
<br />
[[Image:Modellight.png|center|700px|'''Figure 1 - Model of the light output as a function of time without the effect of the LRE. ''']]<br />
<br />
<br />
[[Image:Datawithoutreg.png|center|700px|'''Figure 2 - Light output as a function of time for [http://partsregistry.org/Part:BBa_K325108 P.Pyralis luciferase] under pBad promoter. Arabinose concentration is 10mM and D-Luciferin concentration is 100µM''']]<br />
<br />
<br />
[[Image:Modellight_with_regen.png|center|700px|'''Figure 3 - Model of the light output as a function of time with the effect of the LRE. ''']]<br />
<br />
<br />
[[Image:Datawithreg.png|center|700px|'''Figure 4 - Light output as a function of time for [http://partsregistry.org/Part:BBa_K325219 L. Cruciata luciferase with LRE] under pBad promoter. Arabinose concentration is 100 µM and D-Luciferin concentration is 1µM''']]</div>PMhttp://2010.igem.org/File:Datawithreg.pngFile:Datawithreg.png2010-10-27T00:24:36Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_ModellingTeam:Cambridge/Bioluminescence/Firefly Modelling2010-10-27T00:21:11Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Modelling}}<br />
<br />
We modelled the Luciferin cycle as described in the [http://www.ncbi.nlm.nih.gov/pubmed/18949818 literature] We used data from [http://www.ncbi.nlm.nih.gov/pubmed/19859663 S. Inouye]'s review and the paper on [http://www.ncbi.nlm.nih.gov/pubmed/20655239 firefly luciferase inhibition] from J.M Leitão ''et al.''<br />
<br />
We used the [http://www.mathworks.com/products/simbiology/ Matlab Simbiology toolbox] to model the cycle. A diagram of the model we developed can be found below: <br />
<br />
[[Image:Modelluccycle.jpg|center|700px]]<br />
<br />
Some of the parameters could be found in the litterature, but some of the parameters (namely the diffusion of Luciferin into the cell and the regeneration rate were inferred from experimental data.) <br />
<br />
We modelled the system both with and without the effect of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Luciferin_Regeneration Luciferase Regenerating Enzyme] (LRE.) The 4 Figures below show a comparison the model and our data for both cases. <br />
<br />
<br />
[[Image:Modellight.png|center|700px|'''Figure 1 - Model of the light output as a function of time without the effect of the LRE. ''']]<br />
<br />
<br />
[[Image:Datawithoutreg.png|center|700px|'''Figure 2 - Light output as a function of time for [http://partsregistry.org/Part:BBa_K325108 P.Pyralis luciferase] under pBad promoter. Arabinose concentration is 10mM and D-Luciferin concentration is 100µM''']]<br />
<br />
<br />
[[Image:Modellight_with_regen.png|center|700px|'''Figure 3 - Model of the light output as a function of time with the effect of the LRE. ''']]</div>PMhttp://2010.igem.org/File:Modellight_with_regen.pngFile:Modellight with regen.png2010-10-27T00:10:18Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_ModellingTeam:Cambridge/Bioluminescence/Firefly Modelling2010-10-27T00:09:02Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Modelling}}<br />
<br />
We modelled the Luciferin cycle as described in the [http://www.ncbi.nlm.nih.gov/pubmed/18949818 literature] We used data from [http://www.ncbi.nlm.nih.gov/pubmed/19859663 S. Inouye]'s review and the paper on [http://www.ncbi.nlm.nih.gov/pubmed/20655239 firefly luciferase inhibition] from J.M Leitão ''et al.''<br />
<br />
We used the [http://www.mathworks.com/products/simbiology/ Matlab Simbiology toolbox] to model the cycle. A diagram of the model we developed can be found below: <br />
<br />
[[Image:Modelluccycle.jpg|center|700px]]<br />
<br />
Some of the parameters could be found in the litterature, but some of the parameters (namely the diffusion of Luciferin into the cell and the regeneration rate were inferred from experimental data.) <br />
<br />
We modelled the system both with and without the effect of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Luciferin_Regeneration Luciferase Regenerating Enzyme] (LRE.) The 4 Figures below show a comparison the model and our data for both cases. <br />
<br />
<br />
[[Image:Modellight.png|center|700px|'''Figure 1 - Model of the light output as a function of time without the effect of the LRE. ''']]<br />
<br />
<br />
[[Image:Datawithoutreg.png|center|700px|'''Figure 1 - Light output as a function of time for [http://partsregistry.org/Part:BBa_K325108 P.Pyralis luciferase] under pBad promoter. Arabinose concentration is 10mM and D-Luciferin concentration is 100µM''']]</div>PMhttp://2010.igem.org/File:Datawithoutreg.pngFile:Datawithoutreg.png2010-10-27T00:06:25Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_ModellingTeam:Cambridge/Bioluminescence/Firefly Modelling2010-10-27T00:00:15Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Modelling}}<br />
<br />
We modelled the Luciferin cycle as described in the [http://www.ncbi.nlm.nih.gov/pubmed/18949818 literature] We used data from [http://www.ncbi.nlm.nih.gov/pubmed/19859663 S. Inouye]'s review and the paper on [http://www.ncbi.nlm.nih.gov/pubmed/20655239 firefly luciferase inhibition] from J.M Leitão ''et al.''<br />
<br />
We used the [http://www.mathworks.com/products/simbiology/ Matlab Simbiology toolbox] to model the cycle. A diagram of the model we developed can be found below: <br />
<br />
[[Image:Modelluccycle.jpg|center|700px]]<br />
<br />
Some of the parameters could be found in the litterature, but some of the parameters (namely the diffusion of Luciferin into the cell and the regeneration rate were inferred from experimental data.) <br />
<br />
We modelled the system both with and without the effect of the [https://2010.igem.org/Team:Cambridge/Bioluminescence/Luciferin_Regeneration Luciferase Regenerating Enzyme] (LRE.) The 4 Figures below show a comparison the model and our data for both cases. <br />
<br />
<br />
[[Image:Modellight.png|center|700px|'''Figure 1 - Model of the light output as a function of time without the effect of the LRE. ''']]</div>PMhttp://2010.igem.org/File:Modellight.pngFile:Modellight.png2010-10-26T23:33:08Z<p>PM: </p>
<hr />
<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_ModellingTeam:Cambridge/Bioluminescence/Firefly Modelling2010-10-26T23:24:31Z<p>PM: </p>
<hr />
<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Modelling}}<br />
<br />
We modelled the Luciferin cycle as described in the [http://www.ncbi.nlm.nih.gov/pubmed/18949818 literature] We used data from [http://www.ncbi.nlm.nih.gov/pubmed/19859663 S. Inouye]'s review and the paper on [http://www.ncbi.nlm.nih.gov/pubmed/20655239 firefly luciferase inhibition] from J.M Leitão ''et al.''<br />
<br />
A Diagram of the model can be found below: <br />
<br />
[[Image:Modelluccycle.jpg|center|700px]]</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_ModellingTeam:Cambridge/Bioluminescence/Firefly Modelling2010-10-26T23:24:10Z<p>PM: </p>
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<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Modelling}}<br />
<br />
We modelled the Luciferin cycle as described in the [http://www.ncbi.nlm.nih.gov/pubmed/18949818 literature] We used data from [http://www.ncbi.nlm.nih.gov/pubmed/19859663 S. Inouye]'s review and the paper on [http://www.ncbi.nlm.nih.gov/pubmed/20655239 firefly luciferase inhibition] from J.M Leitão ''et al.''<br />
<br />
A Diagram of the model can be found below: <br />
<br />
[[Image:Modelluccycle.jpg|center|500px]]</div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_ModellingTeam:Cambridge/Bioluminescence/Firefly Modelling2010-10-26T23:22:54Z<p>PM: </p>
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<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Modelling}}<br />
<br />
We modelled the Luciferin cycle as described in the [http://www.ncbi.nlm.nih.gov/pubmed/18949818 literature] We used data from [http://www.ncbi.nlm.nih.gov/pubmed/19859663 S. Inouye]'s review and the paper on [http://www.ncbi.nlm.nih.gov/pubmed/20655239 firefly luciferase inhibition] from J.M Leitão ''et al.''<br />
<br />
A Diagram of the model can be found below: <br />
<br />
[[Image:Modelluccycle.jpg]]</div>PMhttp://2010.igem.org/File:Modelluccycle.jpgFile:Modelluccycle.jpg2010-10-26T23:22:15Z<p>PM: </p>
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<div></div>PMhttp://2010.igem.org/Team:Cambridge/Bioluminescence/Firefly_ModellingTeam:Cambridge/Bioluminescence/Firefly Modelling2010-10-26T23:19:00Z<p>PM: </p>
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<div>{{:Team:Cambridge/Templates/headerMinimalprototype}}<br />
{{:Team:Cambridge/Templates/headerbar|colour=#96d446|linkcolour=#6bbe00|title=Project Firefly: Modelling}}<br />
<br />
We modelled the Luciferin cycle as described in the [http://www.ncbi.nlm.nih.gov/pubmed/18949818 literature] We used data from [http://www.ncbi.nlm.nih.gov/pubmed/19859663 S. Inouye]'s review and the paper on [http://www.ncbi.nlm.nih.gov/pubmed/20655239 firefly luciferase inhibition] from J.M Leitão ''et al.''</div>PMhttp://2010.igem.org/Team:Cambridge/Protocols/ColonyPCRTeam:Cambridge/Protocols/ColonyPCR2010-09-14T15:28:37Z<p>PM: /* Method */</p>
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<div>{{:Team:Cambridge/Templates/header}}<br />
{{:Team:Cambridge/Protocol Menu}}<br />
=Colony PCR=<br />
<br />
==Materials==<br />
*2x Phusion Mastermic<br />
*Primers<br />
*Nuclease free H2O<br />
*Cell culture!<br />
<br />
==Method==<br />
*Pick a single colony and place in 20µL of H20. <br />
*Cell lysis:<br />
**Incubate 10 min @ 98°C<br />
**Freeze 10 min @ -80°C<br />
**Vortex for 2-5 min<br />
*In a PCR tube mix:<br />
**10µL of 2X Phusion Mastermix<br />
**1µL of DNA template (lysate <br />
**XµL of Primer 1<br />
**XµL of Primer 2<br />
**7µL of Nuclease free H2O<br />
*Run PCR:<br />
**Initial Denaturation: 30s @ 98°C<br />
**Denaturation: 10s @ 98°C<br />
**Annealing: 30s @ Y°C<br />
**Elongation: 30s per kb @ 72°C<br />
**Reapeat steps 2-4, 25 to 35 times<br />
**Final Elongation: 10 min @ 72°C<br />
**Final Hold: ∞ @ 4°C<br />
<br />
==Notes==<br />
*For Biobrick primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 VR]) from James Brown we used the following values: <br />
**X = 1µL<br />
**Y = 65°C<br />
*[http://www.finnzymes.com/pdf/f531_f532_phusion_highfidelity_pcr_mastermix_datasheet_1_9_low.pdf Datasheet for the 2X Phusion Mastermix]<br />
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