Difference between revisions of "Team:Glasgow/Practices"

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        <div class="banner scrollTop">
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        <!--<div class='leftContainer'>-->
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        <p class="links scrollOverview"><a style="color:blue;" href="https://2015.igem.org/Team:Glasgow"> Home</a> > <a style="color:blue;" href="https://2015.igem.org/Team:Glasgow/Project/Overview"> Project</a> > <a style="color:blue;" href="https://2015.igem.org/Team:Glasgow/Project/Overview/UVA">UVA</a></p>
 +
        <div id="sidebar"class="widget widget-categories">
 +
        <table>
 +
            <tr><td class="overview">Summary</td></tr>
 +
            <tr><td class="sensor">Overview</td></tr>
 +
            <tr><td class="survivability">Sensor</td></tr>
 +
            <tr><td class="results">Survivability</td></tr>
 +
            <tr><td class="conclusion">Conclusion</td></tr>     
 +
            <tr><td class="top">Top</td></tr>   
 +
        </table>
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        </div>
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      <!-- </div> -->
 +
       
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        <div class='containerRight'>
 +
           
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            <h2 style="margin-top:13vh;">Summary</h2>
 +
           
 +
            <p class="mainText">
 +
Aims:
 +
<ul>
 +
<li>To characterise the three components of a UV-A sensor system: UirS, UirR, PlsiR.</li>
 +
<li>To investigate E. coli survival rates in UV-A and sunlight.</li>
 +
</ul>
 +
</br>
 +
Results:
 +
</br>
 +
</br>
 +
Parts:
 +
<ul>
 +
<li>Essential:
 +
</br>
 +
• K1725400 (PlsiR)
 +
</br>
 +
• K1725410 (UirS)
 +
</br>
 +
• K172541#(UirS with RBS)
 +
</br>
 +
• K1725420 (UirR)
 +
</br>
 +
• K1725421 (UirR with RBS)
  
 +
</li>
 +
<li>Others:
 +
</br>
 +
• K1725401 (PlsiR.I13500)
 +
</br>
 +
• K1725402 (PlsiR.E5501)
 +
</br>
 +
• K1725422 (J23101.B0032.UirR)
 +
</br>
 +
• K1725423 (J23110.B0032.UirR)
 +
</br>
 +
• K1725424 (J23114.B0032.UirR)
 +
</br>
 +
• K1725425 (J23116.B0032.UirR)
 +
</br>
 +
• K1725426 (J23101.B0032.UirR.B0015)
 +
</br>
 +
• K1725427 (J23110.B0032.UirR.B0015)
 +
</br>
 +
• K1725428 (J23114.B0032.UirR.B0015)
 +
</br>
 +
• K1725429 (J23116.B0032.UirR.B0015)
 +
</br>
 +
• K1725430 (J23101.B0032.UirR.B0015.PlsiR.I13500)
 +
</br>
 +
• K1725431 (J23110.B0032.UirR.B0015.PlsiR.I13500)
 +
</br>
 +
• K1725432 (J23114.B0032.UirR.B0015.PlsiR.I13500)
 +
</br>
 +
• K1725433 (J23116.B0032.UirR.B0015.PlsiR.I13500)
 +
</br>
 +
</li>
 +
</ul>
 +
</br>
 +
 +
 +
 +
 +
 +
<div class="scrollSensor"></div></p>
 +
</br>
 +
</br>
 +
   
 +
    <h2>Overview</h2>
 +
   
 +
            <p class="mainText">
 +
In order to prevent cells permanently shifting their metabolism towards one that favours light production, we decided to repress the bioluminescence genes (our optimised lux operon) in the presence of sunlight. The chosen system recognizes the presence of unidirectional UV-A light and comes from the cyanobacterium Synechocystis sp. PCC6803. The system has not been previously characterized before in iGEM. Three components are required to produce a response to UV-A: UirS (UV intensity response Sensor), UirR (UV intensity response Regulator), and PlsiR (promoter of the light and stress integrating response Regulator). For the system to be fully online K322122 is required. This BioBrick is responsible for the synthesis of phycocyanobillin, a chromophore normally found in cyanobacteria that is necessary for the functioning of nearly all light-sensing proteins.
 +
</br>
 +
</br>
 +
We obtained UirS and UirR from genomic Synechocystis DNA via PCR. Our primers included the BioBrick prefix and suffix as well as a ribosome-binding site (B0032). Due to non-BioBrick compatible restriction sites in the UirS gene PCR mutagenesis was carried out with the use of the TOPO TA plasmid vector. The UirR gene contained no such sites and was therefore inserted directly into the pSB1C3 plasmid. PlsiR also lacked such restriction sites and was therefore inserted into pSB1C3.
 +
</br>
 +
</br>
 +
Due to possible toxicity of the UirS gene, our proposed construct contains UirR and PlsiR with an appropriate coding gene in the high copy number pSB1C3, while UirS, alongside with the phycocyanobillin synthesis operon, was put in the low copy number plasmid pSB3K3. We have decided not to put a terminator between UirS and K322122 because the promoter of K322122 is stronger than that of UirS.
 +
</br>
 +
<img src="https://static.igem.org/mediawiki/2015/c/c5/2015-Glasgow-UVA1.png" height="60%" width="60%"/>
 +
 +
 +
<div style="visibility:hidden; height:0;width:0;" class="scrollSurvivability"></div> </p>
 +
</br>
 +
</br>
 +
        <h2> Sensor</h2>
 +
   
 +
            <p class="mainText">
 +
Originally, the system containing UirS, UirR, and PlsiR accounts for a negative phototactic response to unidirectional UV-A light. The proposed mechanism puts UirS, a transmembrane protein of the CBCR family, as the molecule that perceives UV light. It is suggested that through a physical interaction between UirS and UirR and possibly a phosphotransfer from UirS to UirR, UirR is released from the transmembrane protein. The released UirR can now bind to DNA and UirR, which is similar to other activators of stress responses, was found to be a transcriptional activator of lsiR after binding to its promoter PlsiR .
 +
</br>
 +
</br>
 +
We suggest a system where UV-light triggers the expression of a repressor that acts on the production of bioluminescence genes to alleviate the burden they may cause on the cells’ metabolism if constantly expressed. During the night, the bioluminescence genes are expressed, and produce a green-blue light. As it becomes day, UV-A causes the release and activation of UirR. UirR binds to PlsiR to turn expression of the repressor PhlF. PhlF binds to PPhlF to turn off expression of LuxCDABE, so there is no bioluminescence. As it becomes night again, UirR is no longer bound to PlsiR so expression of PhlF is turned off. As PPhlF is no longer repressed, expression of LuxCDABE is turned and bioluminescence is produced again.
 +
</br>
 +
<img src="https://static.igem.org/mediawiki/2015/0/06/2015-Glasgow-UVA2.png" height="60%" width="60%"/>
 +
</br>
 +
</br>
 +
We have confirmed through the use of a laser scanner that PlsiR is not active when UirS and UirR are absent. PlsiR was ligated to GFP with two ribosome binding sites of different strength and no fluorescence was observed (the parts we used for this experiment were K1725401 and K1725402) (Chart 1). Moreover, cells that possess UirR but lack UirS also did not show levels of fluorescence above the expected for E. coli. Therefore, UirR is not sufficient to drive the activation of PlsiR.
 +
</br>
 +
<img src="https://static.igem.org/mediawiki/2015/d/d7/2015-Glasgow-UVA3.png" height="60%" width="60%"/>
 +
</br>
 +
<figcaption>Chart 1. Relative Fluorescence (Compared to Last Taken Measurement of Constitutively Expressed GFP Control) over Absorbance in DH5α cells. DH5α cells containing the PlsiR promoter with GFP fluoresce no more than the original laboratory strain or cells that have GFP without a promoter.
 +
</figcaption>
 +
 +
 +
<div class="scrollResults"></div></p>
 +
</br>
 +
</br>
 +
            <h2> Survivability </h2>
 +
<div class="box">
 +
<h5>Introduction</h5>
 +
<div class="text">
 +
The fact that UV exposure can be lethal to E.coli is well documented.  After deciding to use a UV sensor to activate our system, it became obvious that we would have to examine the effects of exposing E.coli to the amount of UV needed to activate the sensor over time. To do this we generated a number of survival curves. (Please note, these experiments were not repeated enough to generate statistically significant results.) The graphs have been included as an indication of the thought process that went into guiding the experiments. Given more time we would have repeated these experiments many more times. Genotypes of the strains used can be found <a href = "http://blog.addgene.org/plasmids-101-common-lab-e-coli-strains">here</a> for MG6115 TOP10 and DH5α and <a href = "http://genesdev.cshlp.org/content/14/23/2976/T1.expansion.html">here</a> for DS941.
 +
</div>
 +
</div>
 +
</br>
 +
<div class="box">
 +
<h5>Initial aims</h5>
 +
<div class="text">
 +
Initially we wanted to explore what happened over a relatively small period of exposure, as our assumption was that we would see substantial reduction in a fixed number of E.coli,  even over a relatively small time period. We also wanted to see how the different strains (DH5α ,TOP10) being used would respond compared to each other.
 +
</br>
 +
Since both DH5α and TOP10 are recA negative, and are therefore incapable of certain major kinds of DNA repair, we predicted that these strains would be more acutely affected than DS941 and MG6155.
 +
</div>
 +
</div>
 +
</br>
 +
<div class="box">
 +
<h5>Method</h5>
 +
<div class="text">
 +
1ml 10x serial dilutions were made up to 10-6 from a 5ml overnight of each strain. 10µl spots of each dilution were then spotted onto LB agar plates.
 +
These plates were then exposed to 50µmoles/m2/s of UVA at room temperature in illumination cabinets. Time points were then taken by removing plates from the illumination cabinet.
 +
</br>
 +
After illumination, plates were incubated at 37°C, under the assumption that every single viable cell will form a colony. The length of incubation is irrelevant, provided that every cell is given enough time to form a visible colony. This also forms the basis of our counting system, where a colony is assumed to have come from a single cell. We also make the assumption that cell division at the temperature and time we were running the experiment was negligible/nonexistent.
 +
</br>
 +
After incubation, the colonies on each spot/dilution were counted. The number of colonies from the lowest visible dilution (some dilutions formed a lawn of growth) were then multiplied by the dilution factor to approximate how many cells would be in 10µl of undiluted culture.
 +
</br>
 +
</div>
 +
</div>
 +
</br>
 +
<div class="box">
 +
<h5>Results</h5>
 +
<div class="text">
 +
<img src="https://static.igem.org/mediawiki/2015/7/7d/2015-Glasgow-sur1.png" height="70%" width="70%"/>
 +
<figcaption>Figure 1  mean estimated  cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 50 µmoles/m2/s of  UVA  time points taken at 010 20 30 60 120 .  time points connected  by straight line </figcaption>
 +
</br>
 +
We seemed to be seeing a decrease by 30 mins  and between 60 and 120 at least in the recA positive strains the decrease between 0-30 is much steeper in the reca negative strains  (fig1).
 +
</br>
 +
Since we had no time points between 60 -120 we decided to take time points  at 80 100 120 to better visualise the change (fig 2).
 +
</br>
 +
</br>
 +
<img src="https://static.igem.org/mediawiki/2015/9/98/2015-Glasgow-sur2.png" height="70%" width="70%"/>
 +
<figcaption>Fig 2 mean  estimated cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 50 µmoles/m2/s to UVA time points at 0 80 100 120  time points connected  by straight line </figcaption>
 +
</br>
 +
As noted above figure 2 show this second decline seems to be a feature of  MG6115 and  DS941, may be du e to their recA status
 +
</br>
 +
</br>
 +
<img src="https://static.igem.org/mediawiki/2015/1/19/2015-Glasgow-sur3.png" height="70%" width="70%"/>
 +
<figcaption>Figure 3 estimated  cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 60 µmoles/m2/s to UVA composite figure including mean from  previous 2 graphs </figcaption>
 +
</br>
 +
It’s reported that E.coli suffer lethal effects at around 1000kw  with illumination at  366nminin continuous culture (Berney et al 2006).this corresponds with around 16 hour with the fluence and wavelength we were using.  We decide to illuminate bacteria for 14hours  as we felt that if least some  bacteria could withstand 14 hours  of activating radiation then the idea as using UVA as the input into the toy was at least theoretically feasible 
 +
</br>
 +
</br>
 +
Plates at 14 hours showed growth
 +
 +
 +
</div>
 +
</div>
 +
 +
 +
 +
<div class="scrollConclusion"></div>
 +
      </br>
 +
</br>
 +
 +
            <h2>Conclusion</h2>
 +
     
 +
            <h2 class="readMore">Read More!</h2>
 +
           
 +
        <div class="monsterContainer">
 +
<div class="monster"><a href="https://2015.igem.org/Team:Glasgow/Project/Overview/Protocols"><img class='monsterImg' src="https://static.igem.org/mediawiki/2015/9/9d/Monster2-inverted.png">
 +
            <h3><span class="monsterSpan">Protocols</span></h3></a></div>
 +
           
 +
            <div class="monster"><a href="https://2015.igem.org/Team:Glasgow/Project/Overview/Bioluminesence"><img class='monsterImg' src="https://static.igem.org/mediawiki/2015/d/d4/Monster3-inverted.jpg">
 +
            <h3 style="left:2.5%;"><span class="monsterSpan" style="font-size:20px;">Bioluminescence</span></h3></a></div>
 +
           
 +
            <div class="monster"><a href="https://2015.igem.org/Team:Glasgow/Project/Overview/Terminator"><img class='monsterImg' src="https://static.igem.org/mediawiki/2015/e/e1/Monster4-inverted.jpg">
 +
            <h3><span class="monsterSpan">Terminator</span></h3></a></div>
 +
           
 +
            <div class="monster"><a href="https://2015.igem.org/Team:Glasgow/Project/Overview/Repressors"><img class='monsterImg' src="https://static.igem.org/mediawiki/2015/d/d1/Monster5-inverted.jpg">
 +
            <h3><span class="monsterSpan">Repressors</span></h3></a></div>
 +
       
 +
        </div>
 +
</div>
  
 
<script>
 
<script>

Revision as of 12:16, 18 September 2015

Home > Project > UVA

Summary

Aims:

  • To characterise the three components of a UV-A sensor system: UirS, UirR, PlsiR.
  • To investigate E. coli survival rates in UV-A and sunlight.

Results:

Parts:
  • Essential:
    • K1725400 (PlsiR)
    • K1725410 (UirS)
    • K172541#(UirS with RBS)
    • K1725420 (UirR)
    • K1725421 (UirR with RBS)
  • Others:
    • K1725401 (PlsiR.I13500)
    • K1725402 (PlsiR.E5501)
    • K1725422 (J23101.B0032.UirR)
    • K1725423 (J23110.B0032.UirR)
    • K1725424 (J23114.B0032.UirR)
    • K1725425 (J23116.B0032.UirR)
    • K1725426 (J23101.B0032.UirR.B0015)
    • K1725427 (J23110.B0032.UirR.B0015)
    • K1725428 (J23114.B0032.UirR.B0015)
    • K1725429 (J23116.B0032.UirR.B0015)
    • K1725430 (J23101.B0032.UirR.B0015.PlsiR.I13500)
    • K1725431 (J23110.B0032.UirR.B0015.PlsiR.I13500)
    • K1725432 (J23114.B0032.UirR.B0015.PlsiR.I13500)
    • K1725433 (J23116.B0032.UirR.B0015.PlsiR.I13500)



Overview

In order to prevent cells permanently shifting their metabolism towards one that favours light production, we decided to repress the bioluminescence genes (our optimised lux operon) in the presence of sunlight. The chosen system recognizes the presence of unidirectional UV-A light and comes from the cyanobacterium Synechocystis sp. PCC6803. The system has not been previously characterized before in iGEM. Three components are required to produce a response to UV-A: UirS (UV intensity response Sensor), UirR (UV intensity response Regulator), and PlsiR (promoter of the light and stress integrating response Regulator). For the system to be fully online K322122 is required. This BioBrick is responsible for the synthesis of phycocyanobillin, a chromophore normally found in cyanobacteria that is necessary for the functioning of nearly all light-sensing proteins.

We obtained UirS and UirR from genomic Synechocystis DNA via PCR. Our primers included the BioBrick prefix and suffix as well as a ribosome-binding site (B0032). Due to non-BioBrick compatible restriction sites in the UirS gene PCR mutagenesis was carried out with the use of the TOPO TA plasmid vector. The UirR gene contained no such sites and was therefore inserted directly into the pSB1C3 plasmid. PlsiR also lacked such restriction sites and was therefore inserted into pSB1C3.

Due to possible toxicity of the UirS gene, our proposed construct contains UirR and PlsiR with an appropriate coding gene in the high copy number pSB1C3, while UirS, alongside with the phycocyanobillin synthesis operon, was put in the low copy number plasmid pSB3K3. We have decided not to put a terminator between UirS and K322122 because the promoter of K322122 is stronger than that of UirS.



Sensor

Originally, the system containing UirS, UirR, and PlsiR accounts for a negative phototactic response to unidirectional UV-A light. The proposed mechanism puts UirS, a transmembrane protein of the CBCR family, as the molecule that perceives UV light. It is suggested that through a physical interaction between UirS and UirR and possibly a phosphotransfer from UirS to UirR, UirR is released from the transmembrane protein. The released UirR can now bind to DNA and UirR, which is similar to other activators of stress responses, was found to be a transcriptional activator of lsiR after binding to its promoter PlsiR .

We suggest a system where UV-light triggers the expression of a repressor that acts on the production of bioluminescence genes to alleviate the burden they may cause on the cells’ metabolism if constantly expressed. During the night, the bioluminescence genes are expressed, and produce a green-blue light. As it becomes day, UV-A causes the release and activation of UirR. UirR binds to PlsiR to turn expression of the repressor PhlF. PhlF binds to PPhlF to turn off expression of LuxCDABE, so there is no bioluminescence. As it becomes night again, UirR is no longer bound to PlsiR so expression of PhlF is turned off. As PPhlF is no longer repressed, expression of LuxCDABE is turned and bioluminescence is produced again.


We have confirmed through the use of a laser scanner that PlsiR is not active when UirS and UirR are absent. PlsiR was ligated to GFP with two ribosome binding sites of different strength and no fluorescence was observed (the parts we used for this experiment were K1725401 and K1725402) (Chart 1). Moreover, cells that possess UirR but lack UirS also did not show levels of fluorescence above the expected for E. coli. Therefore, UirR is not sufficient to drive the activation of PlsiR.

Chart 1. Relative Fluorescence (Compared to Last Taken Measurement of Constitutively Expressed GFP Control) over Absorbance in DH5α cells. DH5α cells containing the PlsiR promoter with GFP fluoresce no more than the original laboratory strain or cells that have GFP without a promoter.



Survivability

Introduction
The fact that UV exposure can be lethal to E.coli is well documented. After deciding to use a UV sensor to activate our system, it became obvious that we would have to examine the effects of exposing E.coli to the amount of UV needed to activate the sensor over time. To do this we generated a number of survival curves. (Please note, these experiments were not repeated enough to generate statistically significant results.) The graphs have been included as an indication of the thought process that went into guiding the experiments. Given more time we would have repeated these experiments many more times. Genotypes of the strains used can be found here for MG6115 TOP10 and DH5α and here for DS941.

Initial aims
Initially we wanted to explore what happened over a relatively small period of exposure, as our assumption was that we would see substantial reduction in a fixed number of E.coli, even over a relatively small time period. We also wanted to see how the different strains (DH5α ,TOP10) being used would respond compared to each other.
Since both DH5α and TOP10 are recA negative, and are therefore incapable of certain major kinds of DNA repair, we predicted that these strains would be more acutely affected than DS941 and MG6155.

Method
1ml 10x serial dilutions were made up to 10-6 from a 5ml overnight of each strain. 10µl spots of each dilution were then spotted onto LB agar plates. These plates were then exposed to 50µmoles/m2/s of UVA at room temperature in illumination cabinets. Time points were then taken by removing plates from the illumination cabinet.
After illumination, plates were incubated at 37°C, under the assumption that every single viable cell will form a colony. The length of incubation is irrelevant, provided that every cell is given enough time to form a visible colony. This also forms the basis of our counting system, where a colony is assumed to have come from a single cell. We also make the assumption that cell division at the temperature and time we were running the experiment was negligible/nonexistent.
After incubation, the colonies on each spot/dilution were counted. The number of colonies from the lowest visible dilution (some dilutions formed a lawn of growth) were then multiplied by the dilution factor to approximate how many cells would be in 10µl of undiluted culture.

Results
Figure 1 mean estimated cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 50 µmoles/m2/s of UVA time points taken at 010 20 30 60 120 . time points connected by straight line

We seemed to be seeing a decrease by 30 mins and between 60 and 120 at least in the recA positive strains the decrease between 0-30 is much steeper in the reca negative strains (fig1).
Since we had no time points between 60 -120 we decided to take time points at 80 100 120 to better visualise the change (fig 2).

Fig 2 mean estimated cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 50 µmoles/m2/s to UVA time points at 0 80 100 120 time points connected by straight line

As noted above figure 2 show this second decline seems to be a feature of MG6115 and DS941, may be du e to their recA status

Figure 3 estimated cell count per 10ul of a 5ml overnight in lb broth over time of exposure to 60 µmoles/m2/s to UVA composite figure including mean from previous 2 graphs

It’s reported that E.coli suffer lethal effects at around 1000kw with illumination at 366nminin continuous culture (Berney et al 2006).this corresponds with around 16 hour with the fluence and wavelength we were using. We decide to illuminate bacteria for 14hours as we felt that if least some bacteria could withstand 14 hours of activating radiation then the idea as using UVA as the input into the toy was at least theoretically feasible

Plates at 14 hours showed growth


Conclusion

Read More!

Protocols

Bioluminescence

Terminator

Repressors

Location

Bower Building, Wilkins Teaching Laboratory
University of Glasgow
University Avenue
G12 8QQ

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