http://2009.igem.org/wiki/index.php?title=Special:Contributions/Enememily&feed=atom&limit=50&target=Enememily&year=&month=2009.igem.org - User contributions [en]2024-03-28T20:47:18ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-19T15:04:26Z<p>Enememily: /* Overall project */</p>
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!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
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{|body style="background-color:#7CFC00;"<br />
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== '''Overall project''' ==<br />
<br />
'''The Initial System'''<br />
<br />
<br />
[[Image:Sheff_PCB1.jpg|900px|center|boarder]]<br />
<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. We use the initial system that was mentioned inthe Levskaya et al. which involves phycocyanobilin produced through ho1 and pcyA biosynthesis gene converting haem. Phycocyanobilin then forms part of the photoreceptor which controls phosphorylation of the whole system. <br />
<br />
<br />
However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a photo-spectrometer.<br />
<br />
== Results ==<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
<br />
<br />
'''3D Characterisation plot'''<br />
<br />
[[Image:result2.png|center|900px|border]]<br />
<br />
<br />
<br />
This 3D plot is generated through Matlab, it models the system's LacZ activity over various light intensity over a course of 12 hours.<br />
<br />
==Discussion==<br />
<br />
<br />
This [[link]] shows the table of measurement taken in order to obtain results shown above.<br />
<br />
All the results we have got show trends and strong characterization, which gives an idea how to go about the design.<br />
<br />
The error bias of our result cannot be calculated or shown but; to obtain a more accurate result more readings have to be taken.<br />
<br />
<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-19T15:04:07Z<p>Enememily: /* Overall project */</p>
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{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
'''The Initial System'''<br />
<br />
<br />
[[Image:Sheff_PCB1.jpg|600px|center|boarder]]<br />
<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. We use the initial system that was mentioned inthe Levskaya et al. which involves phycocyanobilin produced through ho1 and pcyA biosynthesis gene converting haem. Phycocyanobilin then forms part of the photoreceptor which controls phosphorylation of the whole system. <br />
<br />
<br />
However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a photo-spectrometer.<br />
<br />
== Results ==<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
<br />
<br />
'''3D Characterisation plot'''<br />
<br />
[[Image:result2.png|center|900px|border]]<br />
<br />
<br />
<br />
This 3D plot is generated through Matlab, it models the system's LacZ activity over various light intensity over a course of 12 hours.<br />
<br />
==Discussion==<br />
<br />
<br />
This [[link]] shows the table of measurement taken in order to obtain results shown above.<br />
<br />
All the results we have got show trends and strong characterization, which gives an idea how to go about the design.<br />
<br />
The error bias of our result cannot be calculated or shown but; to obtain a more accurate result more readings have to be taken.<br />
<br />
<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/File:Sheff_PCB1.jpgFile:Sheff PCB1.jpg2009-10-19T15:03:39Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/File:Sheff_PCB.jpgFile:Sheff PCB.jpg2009-10-19T15:02:32Z<p>Enememily: uploaded a new version of "Image:Sheff PCB.jpg"</p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/File:Sheff_PCB.jpgFile:Sheff PCB.jpg2009-10-19T15:02:04Z<p>Enememily: uploaded a new version of "Image:Sheff PCB.jpg"</p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/File:Sheff_PCB.jpgFile:Sheff PCB.jpg2009-10-19T15:00:34Z<p>Enememily: uploaded a new version of "Image:Sheff PCB.jpg"</p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-19T14:54:54Z<p>Enememily: /* Overall project */</p>
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|[[Image:SHEF LOGO.png|953px|right|border]]<br />
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{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
'''The Initial System'''<br />
<br />
<br />
[[Image:Sheff_PCB.jpg|600px|center|boarder]]<br />
<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. We use the initial system that was mentioned inthe Levskaya et al. which involves phycocyanobilin produced through ho1 and pcyA biosynthesis gene converting haem. Phycocyanobilin then forms part of the photoreceptor which controls phosphorylation of the whole system. <br />
<br />
<br />
However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a photo-spectrometer.<br />
<br />
== Results ==<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
<br />
<br />
'''3D Characterisation plot'''<br />
<br />
[[Image:result2.png|center|900px|border]]<br />
<br />
<br />
<br />
This 3D plot is generated through Matlab, it models the system's LacZ activity over various light intensity over a course of 12 hours.<br />
<br />
==Discussion==<br />
<br />
<br />
This [[link]] shows the table of measurement taken in order to obtain results shown above.<br />
<br />
All the results we have got show trends and strong characterization, which gives an idea how to go about the design.<br />
<br />
The error bias of our result cannot be calculated or shown but; to obtain a more accurate result more readings have to be taken.<br />
<br />
<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-19T14:48:48Z<p>Enememily: /* Overall project */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
'''The Initial System'''<br />
<br />
[Image:Sheff_PCB|600px|center|boarder]<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a photo-spectrometer.<br />
<br />
== Results ==<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
<br />
<br />
'''3D Characterisation plot'''<br />
<br />
[[Image:result2.png|center|900px|border]]<br />
<br />
<br />
<br />
This 3D plot is generated through Matlab, it models the system's LacZ activity over various light intensity over a course of 12 hours.<br />
<br />
==Discussion==<br />
<br />
<br />
This [[link]] shows the table of measurement taken in order to obtain results shown above.<br />
<br />
All the results we have got show trends and strong characterization, which gives an idea how to go about the design.<br />
<br />
The error bias of our result cannot be calculated or shown but; to obtain a more accurate result more readings have to be taken.<br />
<br />
<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/File:Sheff_PCB.jpgFile:Sheff PCB.jpg2009-10-19T14:47:26Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-19T14:45:26Z<p>Enememily: /* Overall project */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
'''The Initial System'''<br />
<br />
<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a photo-spectrometer.<br />
<br />
== Results ==<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
<br />
<br />
'''3D Characterisation plot'''<br />
<br />
[[Image:result2.png|center|900px|border]]<br />
<br />
<br />
<br />
This 3D plot is generated through Matlab, it models the system's LacZ activity over various light intensity over a course of 12 hours.<br />
<br />
==Discussion==<br />
<br />
<br />
This [[link]] shows the table of measurement taken in order to obtain results shown above.<br />
<br />
All the results we have got show trends and strong characterization, which gives an idea how to go about the design.<br />
<br />
The error bias of our result cannot be calculated or shown but; to obtain a more accurate result more readings have to be taken.<br />
<br />
<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/Team:SheffieldTeam:Sheffield2009-10-18T18:13:48Z<p>Enememily: </p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield/Home|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
[[Image:title.png|left|200px|border]]<br />
===SWITCHED ON !!!===<br />
For the second time running [http://www.sheffield.ac.uk/ University of Sheffield] is proud to undertake another remarkable iGEM journey. The team constitute of three members specializing in different fields of engineering such as biomedical and system control. <br />
<br />
<br />
<br />
<br />
===Project Description===<br />
[[Image:Sheffield_logo.jpg|right|150px|border]]<br />
<br />
By modifying E.coli so that it can use a phytochrome- with a light receptor- from cyanobacteria as a trigger of protein generation. This pathway is controlled by a certain wavelength of red light, acting as a system switch for lacZ production. LacZ can react with substrate X-gal and form a blue precipitate as a reporter. However, other reporter genes can be attached to the lacZ gene, so different reporters can be expressed. From the fact that this mechanism is sensitive to a certain wavelength of light, we hope to create a system that can be sensitive to various wavelengths and hence triggering different protein generation. Through this the E.coli can become a wavelength sensor; a different wavelength can trigger a different production of protein, for example various types of fluorescent protein, giving a different a colour-scaled indication of the wavelength of the environment around the E.coli.<br />
<br />
Our aim is to design an E.coli system that is sensitive to multiple wavelengths of light and therefore produce a colour indication of the specific wavelength it is exposed to. A pictorial description is shown below:<br />
<br />
<br />
[[Image:intropic.png|center|600px|border]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
==Sponsors==<br />
<br />
[[Image:sponsor.png|left|500px|border]]<br />
[[Image:bbsrc.png|center|200px|border]]<br />
<br />
<br />
[[Image:chelsi.png|left|500px|border]]<br />
<br />
[[Image:epsrc.png|center|120px|border]]<br />
<br />
<br />
<br />
<br />
Special thanks to:<br />
<br />
Prof Philip Wright, Prof Visakan Kadirkamanathan, Prof Alan Matthews, Dr Jagroop Pandhal, Dr Josselin Noirel, Tara <br />
Baldacchino and everyone in the bioincubator.<br />
<br />
<br />
<br />
<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/File:Sheff_team_pic.jpgFile:Sheff team pic.jpg2009-10-18T18:12:48Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/TeamTeam:Sheffield/Team2009-10-18T18:10:36Z<p>Enememily: /* ALL ABOUT US */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
===ALL ABOUT US===<br />
[[Image:Sheffield_logo.jpg|right|150px|border]]<br />
<br />
Hello there, be my guest as I give you a quick peep at my team. It all started in early July when all interested candidates were asked to gather up for a meeting regarding iGEM and eventually 7 students showed up. As time passed by the number decreased exponentially and now we are left with 3 team members. And may I not officially but rationally declare us as the smallest iGEM team for 2009. That does not mean we are any weaker as each member’s workmanship, enthusiasm and self believe cannot be doubted but driven towards a successful iGEM team.<br />
<br />
We are supported by various personalities from different department in the university. Their combined knowledge and advice is always’ well appreciated and as well; makes us a much stronger integrity.<br />
<br />
<br />
[[Image:Sheff_team_pic.jpg|center|550px|border]]<br />
<br />
===HOW WE OPERATE===<br />
<br />
The two main heads of the project are Professor Philip Wright and Professor Visakan Kadirkamanathan. Any main issues regarding the project are dealt by them. Then comes the supportive postgrads; Jagrob Pandhal and Josselin Noirel who help us out in the biology based aspects, while Tara Baldacchino covers the modelling bit. <br />
<br />
<br />
<br />
<br />
==TEAM GALLERY==<br />
<br />
====PROFESSORS:====<br />
<br />
<br />
[[Image:philip.png|left|100px|border]]<br />
<br />
[http://wrightlab.group.shef.ac.uk/ '''Professor Philip Wright;''']<br />
<br />
Head of Department, Director of Research, Biological and Environmental Systems Group.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:visakan.png|left|100px|border]]<br />
<br />
[http://www.sheffield.ac.uk/acse/staff/vk '''Professor Visakan Kadirkamanathan;''']<br />
<br />
Head of Department of Automatic Control and Systems Engineering<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Sheff_Matthews.jpg|left|100px|border]]<br />
[http://www.shef.ac.uk/materials/staff/amatthews.html '''Professor Allan Matthews;''']<br />
<br />
Head of Department of Engineering Materials<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
====POSTGRADS:====<br />
<br />
[http://www.sheffield.ac.uk/cpe/people/staffprofiles/p_docresear/pandhal/index.html '''Jagrob Pandhal''']<br />
<br />
[http://www.sheffield.ac.uk/cpe/people/staffprofiles/p_docresear/noirel '''Josselin Noirel''']<br />
<br />
'''Tara Baldacchino'''<br />
<br />
<br />
<br />
====GRADUATE====<br />
'''Oluwaseun Awotunde'''<br />
<br />
<br />
<br />
====UNDERGRADS:====<br />
<br />
[[Image:Sheffield_logo.jpg|left|100px|border]]<br />
'''Dmitry;'''<br />
<br />
Biomedical Engineeer with past IGEM experience <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Team_member_1.png|left|100px|border]]<br />
'''Emily;'''<br />
<br />
The Biomedical Engineer<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:krish.png|left|100px|border]]<br />
'''Krish;'''<br />
<br />
The System Control Engineer</div>Enememilyhttp://2009.igem.org/File:Sheff_Team_pic.pngFile:Sheff Team pic.png2009-10-18T18:02:51Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:56:03Z<p>Enememily: /* Barriers still to overcome */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system. Through the characterization of the system, we have a model which shows us the LacZ activity level decrease as the wavelength increases:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
[[Image:Sheff_FP.jpg|400px|center|boarder]]<br />
<br />
<br />
'''Say if we fused EGFP (488nm) onto the LacZ gene.'''<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
<br />
[[Image:Sheff_400nm.jpg|400px|center|boarder]]<br />
<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
[[Image:Sheff_500nm.jpg|400px|center|boarder]]<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is '''''switched on!!!!'''''<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|400px|center|border]]<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to different wavelengths.<br />
<br />
== Barriers still to overcome ==<br />
<br />
There are still many technical details yet still to be investigated, a few main issues are:<br />
<br />
1. Method of fusing a fluorescing protein onto the LacZ gene<br />
<br />
2. What kind or type of fluorescing protein should be used, as there are many types. The choice of the fluorescent protein<br />
<br />
3. How can we fuse more than one fluorescent protein on the LacZ?<br />
<br />
<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:53:59Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
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|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system. Through the characterization of the system, we have a model which shows us the LacZ activity level decrease as the wavelength increases:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
[[Image:Sheff_FP.jpg|400px|center|boarder]]<br />
<br />
<br />
'''Say if we fused EGFP (488nm) onto the LacZ gene.'''<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
<br />
[[Image:Sheff_400nm.jpg|400px|center|boarder]]<br />
<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
[[Image:Sheff_500nm.jpg|400px|center|boarder]]<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is '''''switched on!!!!'''''<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|400px|center|border]]<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to different wavelengths.<br />
<br />
== Barriers still to overcome ==<br />
<br />
There are still many technical details yet still to be investigated, a few main issues are:<br />
<br />
1. Method of fusing a fluorescing protein onto the LacZ gene<br />
<br />
2. What kind or type of fluorescing protein should be used, as there are many types. The choice of the fluorescent protein<br />
<br />
<br />
<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:51:39Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system. Through the characterization of the system, we have a model which shows us the LacZ activity level decrease as the wavelength increases:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
[[Image:Sheff_FP.jpg|400px|center|boarder]]<br />
<br />
<br />
'''Say if we fused EGFP (488nm) onto the LacZ gene.'''<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
<br />
[[Image:Sheff_400nm.jpg|400px|center|boarder]]<br />
<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
[[Image:Sheff_500nm.jpg|400px|center|boarder]]<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is '''switched on!!!!'''<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|400px|center|border]]<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to different wavelengths.<br />
<br />
== Barriers still to overcome ==<br />
<br />
There are still many technical details yet still to be investigated, a few main issues are:<br />
<br />
1. Method of fusing a fluorescing protein onto the LacZ gene<br />
<br />
2. What kind or type of fluorescing protein should be used, as there are many types. The choice of the fluorescent protein<br />
<br />
<br />
<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:51:01Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system. Through the characterization of the system, we have a model which shows us the LacZ activity level decrease as the wavelength increases:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
[[Image:Sheff_FP.jpg|400px|center|boarder]]<br />
<br />
<br />
Say if we fused EGFP (488nm) onto the LacZ gene.<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
<br />
[[Image:Sheff_400nm.jpg|400px|center|boarder]]<br />
<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
[[Image:Sheff_500nm.jpg|400px|center|boarder]]<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is '''switched on!!!!'''<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|400px|center|border]]<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to different wavelengths.<br />
<br />
<br />
<br />
== Barriers still to overcome ==<br />
<br />
There are still many technical details yet still to be investigated, a few main issues are:<br />
<br />
1. Method of fusing a fluorescing protein onto the LacZ gene<br />
<br />
2. What kind or type of fluorescing protein should be used, as there are many types. The choice of the fluorescent protein<br />
<br />
<br />
<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:38:34Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system. Through the characterization of the system, we have a model which shows us the LacZ activity level decrease as the wavelength increases:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
[[Image:Sheff_FP.jpg|400px|center|boarder]]<br />
<br />
<br />
Say if we fused EGFP (488nm) onto the LacZ gene.<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
<br />
[[Image:Sheff_400nm.jpg|400px|center|boarder]]<br />
<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
[[Image:Sheff_500nm.jpg|400px|center|boarder]]<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is switched on<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|400px|center|border]]<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to <br />
different wavelengths.<br />
<br />
<br />
<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/File:Sheff_500nm.jpgFile:Sheff 500nm.jpg2009-10-18T17:35:27Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:29:56Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system. Through the characterization of the system, we have a model which shows us the LacZ activity level decrease as the wavelength increases:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
[[Image:Sheff_FP.jpg|400px|Left|boarder]]<br />
<br />
<br />
Say if we fused EGFP (488nm) onto the LacZ gene.<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
<br />
[[Image:Sheff_400nm.jpg|400px|Left|boarder]]<br />
<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is switched on<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to <br />
different wavelengths.<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|200px|right|border]]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/File:Sheff_400nm.jpgFile:Sheff 400nm.jpg2009-10-18T17:27:14Z<p>Enememily: uploaded a new version of "Image:Sheff 400nm.jpg"</p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/File:Sheff_400nm.jpgFile:Sheff 400nm.jpg2009-10-18T17:27:07Z<p>Enememily: uploaded a new version of "Image:Sheff 400nm.jpg"</p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:26:38Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
[[Image:Sheff_trend.jpg|400px|Left|boarder]]<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system. Through the characterization of the system, we have a model which shows us the LacZ activity level decrease as the wavelength increases:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
[[Image:Sheff_FP.jpg|400px|Left|boarder]]<br />
<br />
<br />
Say if we fused EGFP (488nm) onto the LacZ gene.<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
<br />
[[Image:Sheff_400nm.jpg|400px|Left|boarder]]<br />
<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is switched on<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to <br />
different wavelengths.<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|200px|right|border]]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/File:Sheff_400nm.jpgFile:Sheff 400nm.jpg2009-10-18T17:17:06Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:13:44Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
[[Image:Sheff_trend.jpg|400px|Left|boarder]]<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system. Through the characterization of the system, we have a model which shows us the LacZ activity level decrease as the wavelength increases:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
[[Image:Sheff_FP.jpg|400px|Left|boarder]]<br />
<br />
<br />
Say if we fused EGFP (488nm) onto the LacZ gene.<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is switched on<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to <br />
different wavelengths.<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|200px|right|border]]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/File:Sheff_FP.jpgFile:Sheff FP.jpg2009-10-18T17:11:35Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:11:14Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
[[Image:Sheff_trend.jpg|400px|Left|boarder]]<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system.Through the characterization of the system, we have a model which shows us:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
[[Image:Sheff_FP.jpg|400px|Left|boarder]]<br />
<br />
<br />
Say if we fused EGFP (488nm) onto the LacZ gene.<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is switched on<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to <br />
different wavelengths.<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|200px|right|border]]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:10:03Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
[[Image:Sheff_trend.jpg|400px|Left|boarder]]<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system.Through the characterization of the system, we have a model which shows us:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
Say if we fused EGFP (488nm) onto the LacZ gene.<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is switched on<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to <br />
different wavelengths.<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|200px|right|border]]<br />
<br />
|}</div>Enememilyhttp://2009.igem.org/File:Sheff_trend.jpgFile:Sheff trend.jpg2009-10-18T17:04:38Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:04:06Z<p>Enememily: /* How can we use the results we obtained from the experiments and the model to create a wavelength biosensor? */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
|}<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system.<br />
<br />
Through the characterization of the system, we have a model which shows us:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
Say if we fused EGFP (488nm) onto the LacZ gene.<br />
<br />
<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is switched on<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to <br />
different wavelengths.<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|200px|right|border]]</div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T17:01:04Z<p>Enememily: </p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
|}<br />
<br />
<br />
<br />
=='''How can we use the results we obtained from the experiments and the model to create a wavelength biosensor?'''==<br />
<br />
<br />
<br />
'''This is how we propose that the system would work:'''<br />
<br />
From experiments we have characterised this trend of our initial system:<br />
<br />
<br />
<br />
We can achieve this through fusing different fluorescent proteins onto the system.<br />
<br />
Through the characterization of the system, we have a model which shows us:<br />
<br />
<br />
We also know that different fluorescent proteins have different excitation wavelengths.<br />
<br />
<br />
Say if we fused EGFP (488nm) onto the LacZ gene.<br />
1. When we shine a blue light of 400nm onto our system, the LacZ activity in the system is high, but the wavelength of the light is not high enough to excite the fluorescent protein.<br />
<br />
2. The system will have an initial system that is switched on, but an EGFP that is switched off. Therefore the overall system is switched off<br />
<br />
<br />
3. However if we shine a blue light of 500nm onto our system, the activity of the LacZ is high and the EGFP is also activated and so we have an overall system that is switched on<br />
<br />
Through this method, we can fuse several fluorescent protein with different excitation and emission wavelengths and therefore get a system that can respond to <br />
different wavelengths.<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|200px|right|border]]</div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T16:52:45Z<p>Enememily: </p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
|}<br />
<br />
<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|200px|right|border]]</div>Enememilyhttp://2009.igem.org/File:Sheff_Tally_chart.pngFile:Sheff Tally chart.png2009-10-18T16:51:53Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T16:50:46Z<p>Enememily: </p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
|}<br />
<br />
<br />
<br />
<br />
[[Image:Sheff_Tally_chart.png|953px|right|border]]</div>Enememilyhttp://2009.igem.org/File:Sheff_Tally_chart.jpgFile:Sheff Tally chart.jpg2009-10-18T16:46:42Z<p>Enememily: uploaded a new version of "Image:Sheff Tally chart.jpg"</p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/TeamTeam:Sheffield/Team2009-10-18T16:42:08Z<p>Enememily: /* TEAM GALLERY */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
===ALL ABOUT US===<br />
[[Image:Sheffield_logo.jpg|right|150px|border]]<br />
<br />
Hello there, be my guest as I give you a quick peep at my team. It all started in early July when all interested candidates were asked to gather up for a meeting regarding iGEM and eventually 7 students showed up. As time passed by the number decreased exponentially and now we are left with 3 team members. And may I not officially but rationally declare us as the smallest iGEM team for 2009. That does not mean we are any weaker as each member’s workmanship, enthusiasm and self believe cannot be doubted but driven towards a successful iGEM team.<br />
<br />
We are supported by various personalities from different department in the university. Their combined knowledge and advice is always’ well appreciated and as well; makes us a much stronger integrity. <br />
<br />
<br />
<br />
===HOW WE OPERATE===<br />
<br />
The two main heads of the project are Professor Philip Wright and Professor Visakan Kadirkamanathan. Any main issues regarding the project are dealt by them. Then comes the supportive postgrads; Jagrob Pandhal and Josselin Noirel who help us out in the biology based aspects, while Tara Baldacchino covers the modelling bit. <br />
<br />
<br />
<br />
<br />
==TEAM GALLERY==<br />
<br />
====PROFESSORS:====<br />
<br />
<br />
[[Image:philip.png|left|100px|border]]<br />
<br />
[http://wrightlab.group.shef.ac.uk/ '''Professor Philip Wright;''']<br />
<br />
Head of Department, Director of Research, Biological and Environmental Systems Group.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:visakan.png|left|100px|border]]<br />
<br />
[http://www.sheffield.ac.uk/acse/staff/vk '''Professor Visakan Kadirkamanathan;''']<br />
<br />
Head of Department of Automatic Control and Systems Engineering<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Sheff_Matthews.jpg|left|100px|border]]<br />
[http://www.shef.ac.uk/materials/staff/amatthews.html '''Professor Allan Matthews;''']<br />
Head of Department of Engineering Materials<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
====POSTGRADS:====<br />
<br />
[http://www.sheffield.ac.uk/cpe/people/staffprofiles/p_docresear/pandhal/index.html '''Jagrob Pandhal''']<br />
<br />
[http://www.sheffield.ac.uk/cpe/people/staffprofiles/p_docresear/noirel '''Josselin Noirel''']<br />
<br />
'''Tara Baldacchino'''<br />
<br />
<br />
<br />
====GRADUATE====<br />
'''Oluwaseun Awotunde'''<br />
<br />
<br />
<br />
====UNDERGRADS:====<br />
<br />
[[Image:Sheffield_logo.jpg|left|100px|border]]<br />
'''Dmitry;'''<br />
<br />
Biomedical Engineeer with past IGEM experience <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Team_member_1.png|left|100px|border]]<br />
'''Emily;'''<br />
<br />
The Biomedical Engineer<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:krish.png|left|100px|border]]<br />
'''Krish;'''<br />
<br />
The System Control Engineer</div>Enememilyhttp://2009.igem.org/Team:Sheffield/TeamTeam:Sheffield/Team2009-10-18T16:41:02Z<p>Enememily: /* PROFESSORS: */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
===ALL ABOUT US===<br />
[[Image:Sheffield_logo.jpg|right|150px|border]]<br />
<br />
Hello there, be my guest as I give you a quick peep at my team. It all started in early July when all interested candidates were asked to gather up for a meeting regarding iGEM and eventually 7 students showed up. As time passed by the number decreased exponentially and now we are left with 3 team members. And may I not officially but rationally declare us as the smallest iGEM team for 2009. That does not mean we are any weaker as each member’s workmanship, enthusiasm and self believe cannot be doubted but driven towards a successful iGEM team.<br />
<br />
We are supported by various personalities from different department in the university. Their combined knowledge and advice is always’ well appreciated and as well; makes us a much stronger integrity. <br />
<br />
<br />
<br />
===HOW WE OPERATE===<br />
<br />
The two main heads of the project are Professor Philip Wright and Professor Visakan Kadirkamanathan. Any main issues regarding the project are dealt by them. Then comes the supportive postgrads; Jagrob Pandhal and Josselin Noirel who help us out in the biology based aspects, while Tara Baldacchino covers the modelling bit. <br />
<br />
<br />
<br />
<br />
==TEAM GALLERY==<br />
<br />
====PROFESSORS:====<br />
<br />
<br />
[[Image:philip.png|left|100px|border]]<br />
<br />
[http://wrightlab.group.shef.ac.uk/ '''Professor Philip Wright;''']<br />
<br />
Head of Department, Director of Research, Biological and Environmental Systems Group.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:visakan.png|left|100px|border]]<br />
<br />
[http://www.sheffield.ac.uk/acse/staff/vk '''Professor Visakan Kadirkamanathan;''']<br />
<br />
Head of Department of Automatic Control and Systems Engineering<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Sheff_Matthews.jpg|left|100px|border]]<br />
[http://www.shef.ac.uk/materials/staff/amatthews.html '''Professor Allan Matthews;''']<br />
Head of Department of Engineering Materials<br />
<br />
====POSTGRADS:====<br />
<br />
[http://www.sheffield.ac.uk/cpe/people/staffprofiles/p_docresear/pandhal/index.html '''Jagrob Pandhal''']<br />
<br />
[http://www.sheffield.ac.uk/cpe/people/staffprofiles/p_docresear/noirel '''Josselin Noirel''']<br />
<br />
'''Tara Baldacchino'''<br />
<br />
<br />
<br />
====GRADUATE====<br />
'''Oluwaseun Awotunde'''<br />
<br />
<br />
<br />
====UNDERGRADS:====<br />
<br />
[[Image:Sheffield_logo.jpg|left|100px|border]]<br />
'''Dmitry;'''<br />
<br />
Biomedical Engineeer with past IGEM experience <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Team_member_1.png|left|100px|border]]<br />
'''Emily;'''<br />
<br />
The Biomedical Engineer<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:krish.png|left|100px|border]]<br />
'''Krish;'''<br />
<br />
The System Control Engineer</div>Enememilyhttp://2009.igem.org/Team:Sheffield/TeamTeam:Sheffield/Team2009-10-18T16:40:36Z<p>Enememily: /* PROFESSORS: */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Further Work|Further Work]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
===ALL ABOUT US===<br />
[[Image:Sheffield_logo.jpg|right|150px|border]]<br />
<br />
Hello there, be my guest as I give you a quick peep at my team. It all started in early July when all interested candidates were asked to gather up for a meeting regarding iGEM and eventually 7 students showed up. As time passed by the number decreased exponentially and now we are left with 3 team members. And may I not officially but rationally declare us as the smallest iGEM team for 2009. That does not mean we are any weaker as each member’s workmanship, enthusiasm and self believe cannot be doubted but driven towards a successful iGEM team.<br />
<br />
We are supported by various personalities from different department in the university. Their combined knowledge and advice is always’ well appreciated and as well; makes us a much stronger integrity. <br />
<br />
<br />
<br />
===HOW WE OPERATE===<br />
<br />
The two main heads of the project are Professor Philip Wright and Professor Visakan Kadirkamanathan. Any main issues regarding the project are dealt by them. Then comes the supportive postgrads; Jagrob Pandhal and Josselin Noirel who help us out in the biology based aspects, while Tara Baldacchino covers the modelling bit. <br />
<br />
<br />
<br />
<br />
==TEAM GALLERY==<br />
<br />
====PROFESSORS:====<br />
<br />
<br />
[[Image:philip.png|left|100px|border]]<br />
<br />
[http://wrightlab.group.shef.ac.uk/ '''Professor Philip Wright;''']<br />
<br />
Head of Department, Director of Research, Biological and Environmental Systems Group.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:visakan.png|left|100px|border]]<br />
<br />
[http://www.sheffield.ac.uk/acse/staff/vk '''Professor Visakan Kadirkamanathan;''']<br />
<br />
Head of Department of Automatic Control and Systems Engineering<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Sheff_Matthews.jpg|left|100px|border]]<br />
[http://www.shef.ac.uk/materials/staff/amatthews.html '''Professor Allan Matthews;''']<br />
Head of Department of Engineering Materials<br />
<br />
====POSTGRADS:====<br />
<br />
[http://www.sheffield.ac.uk/cpe/people/staffprofiles/p_docresear/pandhal/index.html '''Jagrob Pandhal''']<br />
<br />
[http://www.sheffield.ac.uk/cpe/people/staffprofiles/p_docresear/noirel '''Josselin Noirel''']<br />
<br />
'''Tara Baldacchino'''<br />
<br />
<br />
<br />
====GRADUATE====<br />
'''Oluwaseun Awotunde'''<br />
<br />
<br />
<br />
====UNDERGRADS:====<br />
<br />
[[Image:Sheffield_logo.jpg|left|100px|border]]<br />
'''Dmitry;'''<br />
<br />
Biomedical Engineeer with past IGEM experience <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Team_member_1.png|left|100px|border]]<br />
'''Emily;'''<br />
<br />
The Biomedical Engineer<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Image:krish.png|left|100px|border]]<br />
'''Krish;'''<br />
<br />
The System Control Engineer</div>Enememilyhttp://2009.igem.org/File:Sheff_Matthews.jpgFile:Sheff Matthews.jpg2009-10-18T16:36:30Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T16:22:25Z<p>Enememily: </p>
<hr />
<div><br />
<br />
[[Sheff_Tally_chart.jpg|center|500px|border]]</div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T16:21:52Z<p>Enememily: </p>
<hr />
<div><br />
<br />
<br />
[[Sheff_Tally_chart.jpg|center|500px|border]]</div>Enememilyhttp://2009.igem.org/Team:Sheffield/Further_WorkTeam:Sheffield/Further Work2009-10-18T16:20:08Z<p>Enememily: New page: border</p>
<hr />
<div><br />
<br />
<br />
<br />
[[Sheff_Tally_chart.jpg|center|500px|border]]</div>Enememilyhttp://2009.igem.org/File:Sheff_Tally_chart.jpgFile:Sheff Tally chart.jpg2009-10-18T16:15:51Z<p>Enememily: uploaded a new version of "Image:Sheff Tally chart.jpg"</p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/File:Sheff_Tally_chart.jpgFile:Sheff Tally chart.jpg2009-10-18T16:11:58Z<p>Enememily: </p>
<hr />
<div></div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-18T14:17:05Z<p>Enememily: /* Further Work */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Parts|Parts]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a ooptical spectrometer.<br />
<br />
== Results ==<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
<br />
<br />
'''3D Characterisation plot'''<br />
<br />
[[Image:result2.png|center|900px|border]]<br />
<br />
<br />
<br />
This 3D plot is generated through Matlab, it models the system's LacZ activity over various light intensity over a course of 12 hours.<br />
<br />
==Discussion==<br />
<br />
<br />
==Further Work==<br />
<br />
After the characterisation of the system, we have a<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-18T14:12:37Z<p>Enememily: /* Results */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Parts|Parts]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a ooptical spectrometer.<br />
<br />
== Results ==<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
<br />
<br />
'''3D Characterisation plot'''<br />
<br />
[[Image:result2.png|center|900px|border]]<br />
<br />
<br />
<br />
This 3D plot is generated through Matlab, it models the system's LacZ activity over various light intensity over a course of 12 hours.<br />
<br />
==Discussion==<br />
<br />
<br />
==Further Work==<br />
<br />
Due to time restrictions, and the few people we had available for this project. We would to continue characterising the parts of this system, as well as repeat our the Miller assay for more values of light intensities and wavelengths.<br />
<br />
We also have the thought of new directions to develop the project after contacting Christopher A. Voigt.<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-14T16:41:34Z<p>Enememily: /* Results */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Parts|Parts]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a ooptical spectrometer.<br />
<br />
== Results ==<br />
<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
<br />
<br />
'''3D Characterisation plot'''<br />
<br />
[[Image:result2.png|center|900px|border]]<br />
<br />
<br />
<br />
This 3D plot is generated through Matlab, it models the system's LacZ activity over various light intensity over a course of 12 hours.<br />
<br />
==Discussion==<br />
<br />
<br />
==Further Work==<br />
<br />
Due to time restrictions, and the few people we had available for this project. We would to continue characterising the parts of this system, as well as repeat our the Miller assay for more values of light intensities and wavelengths.<br />
<br />
We also have the thought of new directions to develop the project after contacting Christopher A. Voigt.<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-14T16:39:09Z<p>Enememily: /* Results */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Parts|Parts]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a ooptical spectrometer.<br />
<br />
== Results ==<br />
<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
<br />
<br />
'''3D Characterisation plot'''<br />
<br />
[[Image:result2.png|center|900px|border]]<br />
<br />
==Discussion==<br />
<br />
<br />
==Further Work==<br />
<br />
Due to time restrictions, and the few people we had available for this project. We would to continue characterising the parts of this system, as well as repeat our the Miller assay for more values of light intensities and wavelengths.<br />
<br />
We also have the thought of new directions to develop the project after contacting Christopher A. Voigt.<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-13T21:10:01Z<p>Enememily: /* Results */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
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!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Parts|Parts]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a ooptical spectrometer.<br />
<br />
== Results ==<br />
<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
The control strain has produced a similar trend to the experimental values.<br />
<br />
==Discussion==<br />
<br />
<br />
==Further Work==<br />
<br />
Due to time restrictions, and the few people we had available for this project. We would to continue characterising the parts of this system, as well as repeat our the Miller assay for more values of light intensities and wavelengths.<br />
<br />
We also have the thought of new directions to develop the project after contacting Christopher A. Voigt.<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]|}</div>Enememilyhttp://2009.igem.org/Team:Sheffield/ProjectTeam:Sheffield/Project2009-10-13T21:07:49Z<p>Enememily: /* Results */</p>
<hr />
<div>{|body style="background-color:#7CFC00;"<br />
|[[Image:SHEF LOGO.png|953px|right|border]]<br />
|-<br />
|<br />
|}<br />
{| style="color:#FFFF00;background-color:#ADFF2F;" cellpadding="1" cellspacing="1" width="100%" align="center"<br />
!align="center"|[[Team:Sheffield|Home]]<br />
!align="center"|[[Team:Sheffield/Team|Team]]<br />
!align="center"|[[Team:Sheffield/Project|Project]]<br />
!align="center"|[[Team:Sheffield/Parts|Parts]]<br />
!align="center"|[[Team:Sheffield/Modeling|Modeling]]<br />
!align="center"|[[Team:Sheffield/Notebook|Notebook]]<br />
|}<br />
{|body style="background-color:#7CFC00;"<br />
|<br />
<br />
<br />
<br />
<br />
== '''Overall project''' ==<br />
<br />
In this project, we originally intended to create an E.coli system that is sensitive to multiple wavelengths of light and produce a colour indication of the specific wavelength it is exposed to. However due to limitations in the time and number of people available, and due to unexpected results the project changed significantly.<br />
<br />
<br />
In the end, our project became: characterizing existing parts, such as BBa_R0082 for beta-galactocidase activity depending on time from the reaction start as well as wavelength and intensity of exposed light.<br />
<br />
== Project Details==<br />
<br />
The first experiment we carried to familiarise ourselves with the strain, was to repeat the light sensing ability of RU1012 as described in the Levskaya et al. paper[1], in its most simple form. Red light switch off the photoreceptor through inhibiting autophosphorylation, and therefore switching off the expression for Lac Z production. We shown borad spectrum light on one sample and kept the other in the dark so that gene expression can not be interefered. The samples were put on seperate agar plates with X-gal on it which acts as a substrate for Lac Z and producing a blue colouration. To our surprise, the result were opposite to what we expected: the illuminated plate produced more pigment than the one in the dark, whereas it should have been the opposite. No control was used in this experiment.<br />
<br />
[[Image:Sheff_Initial_Experiment.jpg|center|500px|border]]<br />
<br />
The same experiment was repeated with a control strain to confirm that the result was systematic. Later on we realise that the wavelength that inhibits the autophosphorylation has to be a specific wavelength, a broadspectrun light that contains that wavelength does not have the same effect. Too solve that problem we have tried various measures:<br />
<br />
<br />
'''Hydrogen lamp'''<br />
<br />
The Balmer lamp provided a narrower band of wavelength, we hoped that it can characterise the E.coli system better than broad spectrum light. Experiments were repeated with Hydrogen lamp, conducted at room temperature - due to set up of the lamp, the experiment can not be carried out in an incubator at 37C. Unfortunately, this has not changed the outcome of the result, the gene expression was still not switched off.<br />
<br />
<br />
To compensate the fact that the experiment was conducted at room temperature, L-arabinose was added to the agar plates as an inducer for the conversion of heam to phycocyanobillin - forms part of the photoreceptor. However, results still remained unchaged. Further investigation was carried out, we contacted authors of the article and asked for their help and extracted the fact that to deactivate the phytochrom of the transformed E.coli RU1012, the wavelength used can only be very narrow band of red light. We also extracted that the intensity of the light is also significant to the deactiation.<br />
<br />
<br />
The response from the authors inspired us to characterise the two gene system further in terms of the optimum wavelength and intensity for gene expression deactivation. The gene expression is also apparently leaky<br />
<br />
[[Image:Wv.PNG|center|400px|border]]<br />
<br />
'''Filter paper'''<br />
<br />
<br />
A range of filter paper which only let through certain band of wavelength was used; blue(450-495nm),red(620-750) and green(495-570). All samples was were wrapped in different filter paper and incubated for 12hrs at 37C, photos of samples were taken as a time series to show the progress of the colouration. Result at 12hrs was left out of the time series because it was identical to 9hrs results.<br />
<br />
<br />
<br />
[[Image:sheff_results_colourfilter.jpg|center|border]]Graduation of colouration can be seen clearly in this results table, the sample under red filter has the least colouration, while the sample in the dark has the most colouration. Blue and green has similar level of colouration judging by eye. This result matches the theory in the paper; that red light inhibits the gene expression the most. However, to complete the charaterisation of the system, quantitative measurements is required. Miller Assay quantifies the amount of beta-galactocidase, this can act as an indicator of how active the gene expression is.<br />
<br />
<br />
'''Varying light intensity'''<br />
<br />
<br />
After verifying that red light inhibits the most gene expression, the characterisation of the intensity can be proceed. According to the research paper, the light intensity can effect the amount of gene expression in this system. To investigate this, 3 sample and a control strain were put into LB broth, was then exposed to red light for 12hrs at the following intensity uEins/m2/s: 2.5, 6, 7, 10. Miller Assay was then performed to measure the amount of beta-galactocidase produced at each intensity by measuring the optical density of each sample in a ooptical spectrometer.<br />
<br />
== Results ==<br />
<br />
<br />
'''Characterisation of the system'''<br />
<br />
<br />
[[Image:result1.png|center|900px|border]]<br />
<br />
<br />
Graphs above shows the model of LacZ activity over different intensity over the course of 12 hours.<br />
<br />
- This model is based on our experimental result and it matches with the model in the paper.<br />
<br />
- An important point is that the amount of black precipitate the system produce is not a representation of the amount of LacZ activity, because the black precipitate does not degrades on the agar plate.<br />
<br />
-Hence why Miller Assay must be carried out to quantify the amount of LacZ activity.<br />
<br />
'''Graph analysis'''<br />
<br />
'''3hrs'''- The system is very unstable, the beginning of translation and transcription of gene, LacZ activity is erratic, does not form a trend proportional to intensity over the first 3 hours.<br />
<br />
'''6hrs'''- The system is still unstable, but relatively calmer. <br />
<br />
'''9hrs'''- The system is more similar to expected trend.<br />
<br />
'''12hrs'''- The system behaves as expected in the paper, LacZ activity decrease as light intensity increase<br />
<br />
==Discussion==<br />
<br />
<br />
==Further Work==<br />
<br />
Due to time restrictions, and the few people we had available for this project. We would to continue characterising the parts of this system, as well as repeat our the Miller assay for more values of light intensities and wavelengths.<br />
<br />
We also have the thought of new directions to develop the project after contacting Christopher A. Voigt.<br />
<br />
==References==<br />
===Papers===<br />
#'''Engineering Escherichia coli to see light'''<br> ''Nature'' 24 November 2005 DOI:10.1038/nature04405<br> A. Levskaya ''et al.''<br> [http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html URL] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16306980 Pubmed] [http://www.hubmed.org/display.cgi?uids=16306980 Hubmed]|}</div>Enememily