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| | <h1>Results</h1> | | <h1>Results</h1> |
| - | Collated here are the results from numerous experiments that have been performed for the DISColi project. Detailed information about the biobricks we made is contained in links in the details of the experiment in which they are used. | + | Collated here are the results from numerous experiments that have been performed for the DISColi project. |
| - | </br>
| + | <table align="center" cellpadding="20"> |
| - | <h2> Properties of <i>P. Aeruginosa Biofilms</i></h2>
| + | |
| - | <b>Aims</b>
| + | |
| - | </br><p>
| + | |
| - | 1. Determine quantitatively the base rate of dissociation from a biofilm. This rate can be compared to the novel biofilm dispersing biobricks rate of dispersal to determine their effectiveness.
| + | |
| - | </br></br>
| + | |
| - | 2. Examine the structure of biofilms after set periods of time. These images can be used to compare the 3D structure of Nissle 1917 biofilm.
| + | |
| - | </br>
| + | |
| - | <table> | + | |
| | <tr> | | <tr> |
| | <td> | | <td> |
| - | <b>Method</b> | + | <h3>Biofilm</h3> |
| - | <p>As we were trying to specifically disperse areas of biofilm it was necessary to establish a base rate of dispersal of the biofilm without using any of the dispersal mechanisms we designed. This would allow us to show quantitativly the increase in rate of dispersal that our different dispersal biobrick could generate when compared to a biofilm of non-transformed bacteria.</p> | + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/Biofilm">Biofilm Overview</a></h4> |
| - | <p>To measure the base rate of dispersal glass slides were put into 50ml tubes containing 20ml of LB broth. The LB covered around a third of the glass slide, this is the area where the biofilm would form. The LB was then inoculated with 20μl of overnight culture of <i>Pseudomonas aeruginosa</i>. These tubes were then left on a bench top shaker at room temperature for a set amount of time (time points ranged from 1hr to 48hrs). After the biofilm had grown its alloted time the glass slide was carefully removed and placed into a fresh 50ml tube with 25ml of LB (which completely covered the biofilm) and left to allow the bacteria to disperse for 1hr. The slide was then transferred to a fresh 50ml tube with 25ml of LB. The biofilm is scraped off the slide using a thin flexible spatula. At this point both the dispersed cell and the biofilm scrapings were sonicated to stop clumping and plated in serial dilutions.</p> | + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/Biofilm/P._aeruginosa"><i>P. aeruginosa</i></a><h5> |
| - | </br></br> | + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/Biofilm/Nissle"><i>E. coli</i> Nissle 1917</a><h5> |
| | + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/BiofilmResults">Biofilm Results</a><h5> |
| | </td> | | </td> |
| | + | |
| | <td> | | <td> |
| - | <div align="center"> | + | <h3>Reporters</h3> |
| - | <img src="https://static.igem.org/mediawiki/2011/c/c7/Biofilmgraph.jpg" /> | + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/LOV2">LOV2 and iLOV Overview</a></h5> |
| - | </br> | + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/Lovcharacterisation">LOV2 Characterisation</a></h5> |
| - | </br> | + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/LOVresults">Reporter Results</a></h5> |
| - | <img src="https://static.igem.org/mediawiki/2011/f/f5/Biofilmtable.jpg" /> | + | </br></br></br></br> |
| - | <div align="left"> | + | |
| - | <p><b>Figure 1: Number of viable cells in biofilm compared to number of cells dispersed from the biofilm in one hour.</b> The base rate of dispersal remains roughly proportional to the number of cells in the biofilm. The data for the time point hour 14 for the biofilm was not available. | + | |
| - | </div> | + | |
| - | </div> | + | |
| | </td> | | </td> |
| - | </tr>
| |
| - | </table>
| |
| - | <p> To make the images of biofilm formation, biofilms were formed on glass slides inserted into 50ml tubes. The tube was filled with 20ml of LB broth and inoculated with 20μl of over night culture of <i>Psuedomonas Aeruginosa</i>. The biofilms were left to form for the time indicated on the images.</p>
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| - | </br>
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| - | <b>Results</b>
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| | + | <td> |
| | + | <h3>Control of Dispersal</h3> |
| | + | <h5>- <a href="https://2011.igem.org/wiki/index.php?title=Team:Glasgow/Control_of_Dispersal/Intro">Control of Dispersal Overview</a></h5> |
| | + | <h5>- <a href="https://2011.igem.org/wiki/index.php?title=Team:Glasgow/Control_of_Dispersal/Results">Control of Dispersal Results</a></h5> |
| | + | </br></br></br></br></br> |
| | + | </td> |
| | | | |
| - | <h2>Base Rate Dispersal of <i>E.coli</i> Nissle 1917</h2>
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| - | <p> The time series was carried out with Nissle (details see lab books) the same way as detailed for <i>P. aeruginosa</i> above. It showed that on average 48% of the cells within a biofilm will disperse once the biofilm is transferred into fresh media. The percentage dispersed was expected to be inversely correlated to the size of the biofilm, because we expected a larger biofilm to have a higher affinity for the cells it holds. However, the data shows that the percentage dispersed is relatively constant, and varies between 30-60% dispersal. </p>
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| - | <div><img src="http://farm7.static.flickr.com/6164/6168708841_f4b9970e7c_z.jpg" alt="Graph of percentage dispersal from biofilm">
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| - | <p><font size="1" font color="grey"> Graph 1: The total percentage of cells dispersed when a biofilm is transferred into new media vs time in hours </font></p> </div>
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| - | <p> The data also showed that over time the amount of cells firmly within a biofilm increases at a similar rate to those that disperse and are not attached to the biofilm.</p>
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| - | <div><img src="http://farm7.static.flickr.com/6171/6169259636_24e606eb08_z.jpg" alt="Cell count of biofilm and planktonic cells vs time">
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| - | <p><font size="1" color="grey"> Graph 2: The estimated number of dispersed cells compared to the number of biofilm cells vs time in hours</font></p></div>
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| - | <p>View the full spreadsheet of <i>E.coli</i> Nissle 1917 dispersal <a href="http://www.scribd.com/doc/65776929/Nissle-Spreadsheet" style="margin: 12px auto 6px auto; font-family: Helvetica,Arial,Sans-serif; font-style: normal; font-variant: normal; font-weight: normal; font-size: 14px; line-height: normal; font-size-adjust: none; font-stretch: normal; -x-system-font: none; display: block; text-decoration: underline;">here</a><iframe class="scribd_iframe_embed" src="http://www.scribd.com/embeds/65776929/content?start_page=1&view_mode=list&access_key=key-11772wo0tyhfolzmirio" data-auto-height="true" data-aspect-ratio="1.04081632653061" scrolling="no" id="doc_20507" width="100%" height="600" frameborder="0"></iframe><script type="text/javascript">(function() { var scribd = document.createElement("script"); scribd.type = "text/javascript"; scribd.async = true; scribd.src = "http://www.scribd.com/javascripts/embed_code/inject.js"; var s = document.getElementsByTagName("script")[0]; s.parentNode.insertBefore(scribd, s); })();</script></p>
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| - | </br>
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| - | <h3><a href="https://2011.igem.org/Team:Glasgow/Results:dispersal">Surfactants</a></h3>
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| - | <p><h2>Modelling</h2>
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| - | The results regarding the biofilm dispersal we acquired using our single-cell models and some additional values are shown in the table below. We also created models for Phosphodiesterase activity and the multi-cell models for Latherin and Colicin E2. For more information, please refer to <a href="https://2011.igem.org/Team:Glasgow/Modeling">Modelling</a> site.
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| - | <p>
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| - | <table border>
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| - | <tr><b>Results for modelling, base on the single-cell models</b></tr>
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| - | <tr><td>Name of the molecule</td><td>Diffusion rate (mm<sup>2</sup>/min)</td><td>The critical concentration (molecule)</td><td>Achieving Effective Concentration* (min)</td><td>The attainable spatial resolution (mm)</td></tr>
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| - | <tr><td>Ranaspumin-2</td><td>1.68*10<sup>-4</sup></td><td>374</td><td>28</td><td>0.09</td></tr>
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| - | <tr><td>T4 Endolysin</td><td>1.52*10<sup>-4</sup></td><td>3000</td><td>52</td><td>~0.01</td></tr>
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| - | <tr><td>Latherin</td><td>1.38*10<sup>-4</sup></td><td>173</td><td>29</td><td>0.08</td></tr>
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| - | <tr><td>Colicin E2</td> <td>0.72*10<sup>-4</sup></td><td>1629</td><td>90</td><td>~0.01</td></tr>
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| - | </table>
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| - | *Due to the mode of action, for Colicin E2 and T4 Endolysin it is equal to the Critical Concentration and for Latherin and Ranaspumin-2 it is equal to 8x Critical Concentration
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| - | <p>Here there are the two equations we used to determinate diffusion of the molecules through a biofilm:
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| - | <p><center><img src="https://static.igem.org/mediawiki/2011/4/41/Equation1.jpg"></center>
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| - | <p align="right">Equation 1</p>
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| - | <p>
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| - | where:<br> P (r,t) – number of molecules at the certain distance after a certain time
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| - | <br>A – total number of molecules in the system
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| - | <br>r – distance from point of origin (mm)
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| - | <br>D – diffusion coefficient
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| - | <br>t – time (min)
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| - | <p><center><img src="https://static.igem.org/mediawiki/2011/3/38/Equation2.jpg"></center>
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| | | | |
| - | <p align="right">Equation 2</p>
| + | <td> |
| - | <p> | + | <h3>Tools for Pathway engineering</h3> |
| - | where:<br> P<sub>f</sub> (r,t) – Final number of molecules at given distance at given time
| + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/PathwayTools/Intro">Introduction</a></h5> |
| - | <br>P (r,t) – initial number of molecules according to Equation 1
| + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/PathwayTools/Pathways">Modular Pathways</a></h5> |
| - | <br>λ – rate of degradation (molecule/min)
| + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/Results/PromoterLibrary">Promoter Library</a></h5> |
| - | <br>t – time passed (min)
| + | <h5>- <a href="https://2011.igem.org/Team:Glasgow/Results/MCS">Multiple Cloning Site</a></h5> |
| | + | </br></br> |
| | + | </td> |
| | + | </tr> |
| | + | </table> |
| | </html> | | </html> |
Collated here are the results from numerous experiments that have been performed for the DISColi project.
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