Team:Glasgow/BiofilmResults

From 2011.igem.org

(Difference between revisions)
 
(12 intermediate revisions not shown)
Line 1: Line 1:
{{Team:Glasgow/Header}}
{{Team:Glasgow/Header}}
<html>
<html>
-
<h1> Properties of <i>P. Aeruginosa Biofilms</i></h1>
+
<h1>Results</h1>
-
<b>Aims</b>
+
<h6><a href="https://2011.igem.org/Team:Glasgow/Biofilm">Back to Biofilms</a></h6>
-
</br><p>
+
<h6><a href="https://2011.igem.org/Team:Glasgow/Results">Back to Results</a></h6>
-
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>
+
<p>The images below show a selection of stages of biofilm formation. Starting with Image 1 showing a lab strain of <i>E.coli</i>that has no fimbriae, and is not forming a biofilm.</p>
-
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.
+
<p>Image 2 shows an EM of <i>E.coli</i> Nissle 1917 in the early stages of biofilm formation. The fimbriae that allow the cells to cling to each other are clearly visible.</p>
 +
<p>Image 3 shows a Nissle biofilm in the later stages of formation, with the cells densely packed and the extracellular matrix that holds them together showing.</p>
 +
 
 +
<p>
 +
<div>
 +
<table align="centre">
 +
<tr>
 +
<td>
 +
<img src="http://farm7.static.flickr.com/6171/6170367511_51e5363dbd_m.jpg" width="230" height="170"/>
 +
<p><font size="1" color="grey"> Image 1: 15,000x EM of E.coli for comparison. </br>No fimbriae or EPS is visible. </br>(courtesy of Rocky Mountain Laboratories)</font></p>
 +
</td>
 +
<td>
 +
<img src="http://farm7.static.flickr.com/6156/6166741226_e4cfd217bd_m.jpg" /><p><font size="1" color="grey">Image 2: 10,000x SEM image of Nissle </br>showing the fimbriae</font></p>
 +
</td>
 +
<td>
 +
<img src="http://farm7.static.flickr.com/6177/6166139079_a35d6a5930_m.jpg" width="230" height="180" /><p><font size="1" color="grey">Image 3: SEM image of Nissle biofilm </br>showing the extracellular matrix</font></p>
 +
</td>
 +
</tr>
 +
<tr >
 +
<td>
 +
 
 +
</td>
 +
<td>
 +
<img src="http://farm7.static.flickr.com/6177/6170913398_6e217cc778_m.jpg"/>
 +
<p><font size="1" color="grey">Image 4: 1000x EM of P. aeruginosa biofilm, </br>showing its densely packed structure </br>(courtesy of Dan Walker, University of Glasgow)</font></p>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
</br>
</br>
 +
<p>The diagrams below show two 24-hour RFP Nissle biofilms compared to non-transformed Nissle and Nissle transformed with the puC19 vector. They show the distinct red fluorescence of RFP, as well as what appears to be innate green fluorescence of Nissle. Due to this property of <i>E. coli</i> Nissle, using GFP or YFP is not recommended. <p>
 +
<p>More details on these diagrams can be found in the lab book section.</p>
 +
<table>
<table>
<tr>
<tr>
<td>
<td>
-
<b>Method</b>
+
<img src="http://farm7.static.flickr.com/6156/6171223536_95ab16bc96_z.jpg">
-
<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>
+
<p><font size="1" color="grey"> Figure 1: Comparison of RFP E.coli Nissle biofilms to untransformed E.coli Nissle bioflims under light microscope and under excitatory and non-excitatory wavelengths</font></p>
-
<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>
+
-
</br></br>
+
</td>
</td>
 +
</tr>
 +
<tr>
<td>
<td>
-
<div align="center">
+
<p>
-
<img src="https://static.igem.org/mediawiki/2011/c/c7/Biofilmgraph.jpg" />
+
<tr>
-
</br>
+
<td>
-
</br>
+
<img src="http://farm7.static.flickr.com/6175/6170690387_75d453a871_z.jpg">
-
<img src="https://static.igem.org/mediawiki/2011/f/f5/Biofilmtable.jpg" />
+
<p><font size="1" color="grey"> Figure 2: Comparison of RFP E.coli Nissle biofilms to puC19 E.coli Nissle biofilms under light microscope and under excitatory and non-excitatory wavelengths</font></p>
-
<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 information for the time point hour 14 for the biofilm was not available.
+
-
</div>
+
-
</div>
+
</td>
</td>
</tr>
</tr>
</table>
</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>
+
 
-
</br>
+
<h2>Summary</h2>
-
<b>Results</b>
+
<p>We have inserted a new chassis into the registry with E. coli Nissle 1917 which we have also proven to be fully transformable at a rate of 300 colonies per microgram of DNA. We have supplied links to literature which details that the organism has been proved to be safe for use in humans.</p>
-
</br>
+
 
-
The number of cells that dispersed from the biofilm seemed to be proportional to the number of cells in the biofilm with a ratio of roughly 5 dispersed:1 in biofilm. Figure 1 shows the number of dispersed cells when compared to the number of cells in the biofilm in both a graph and a table.  
+
<p>Nissle 1917 has been shown to be backwardly compatible with a number of pre-existing biobricks, for example the RFP construct J04450 and the cells did not require a shuttle vector or codon optomisation before transformation. </p>
-
</br>
+
<p>Nissle produces 600 colonies per microgram of DNA in chemical tranformation </p>
-
<img src="https://static.igem.org/mediawiki/2011/f/f0/Biofilmtimeseries.jpg" width="100%" /></br>
+
<p>Nissle produces 1250 colonies per microgram of DNA in electroporation</p>
-
<b>Figure 2: <i>Pseudomonas aeruginosa</i> biofilm growth over time.</b> These photographs were taken after 1hr, 14hrs and 48hrs of biofilm growth. They were stained using a Grams stain method that is designed specifically to avoid sheer forces being applied to the delicate biofilm structure. Details of this method are included in the <a href="https://2011.igem.org/Team:Glasgow/Lab_Book"><i>Pseudomonas aeruginosa</i> biofilms</a>  lab book.
+
<p>We have also demonstrated that this chassis forms biofilms readily and carried out a time series experiment which shows that biofilm grows at similar speed to planktonic cell growth. <b>For these reasons we feel that the Nissle 1917 E. coli strain is well suited to biofilm investigations with BioBricks.</b></p>
-
</html>
+

Latest revision as of 04:10, 22 September 2011

Results

Back to Biofilms
Back to Results

The images below show a selection of stages of biofilm formation. Starting with Image 1 showing a lab strain of E.colithat has no fimbriae, and is not forming a biofilm.

Image 2 shows an EM of E.coli Nissle 1917 in the early stages of biofilm formation. The fimbriae that allow the cells to cling to each other are clearly visible.

Image 3 shows a Nissle biofilm in the later stages of formation, with the cells densely packed and the extracellular matrix that holds them together showing.

Image 1: 15,000x EM of E.coli for comparison.
No fimbriae or EPS is visible.
(courtesy of Rocky Mountain Laboratories)

Image 2: 10,000x SEM image of Nissle
showing the fimbriae

Image 3: SEM image of Nissle biofilm
showing the extracellular matrix

Image 4: 1000x EM of P. aeruginosa biofilm,
showing its densely packed structure
(courtesy of Dan Walker, University of Glasgow)

The diagrams below show two 24-hour RFP Nissle biofilms compared to non-transformed Nissle and Nissle transformed with the puC19 vector. They show the distinct red fluorescence of RFP, as well as what appears to be innate green fluorescence of Nissle. Due to this property of E. coli Nissle, using GFP or YFP is not recommended.

More details on these diagrams can be found in the lab book section.

Figure 1: Comparison of RFP E.coli Nissle biofilms to untransformed E.coli Nissle bioflims under light microscope and under excitatory and non-excitatory wavelengths

Figure 2: Comparison of RFP E.coli Nissle biofilms to puC19 E.coli Nissle biofilms under light microscope and under excitatory and non-excitatory wavelengths

Summary

We have inserted a new chassis into the registry with E. coli Nissle 1917 which we have also proven to be fully transformable at a rate of 300 colonies per microgram of DNA. We have supplied links to literature which details that the organism has been proved to be safe for use in humans.

Nissle 1917 has been shown to be backwardly compatible with a number of pre-existing biobricks, for example the RFP construct J04450 and the cells did not require a shuttle vector or codon optomisation before transformation.

Nissle produces 600 colonies per microgram of DNA in chemical tranformation

Nissle produces 1250 colonies per microgram of DNA in electroporation

We have also demonstrated that this chassis forms biofilms readily and carried out a time series experiment which shows that biofilm grows at similar speed to planktonic cell growth. For these reasons we feel that the Nissle 1917 E. coli strain is well suited to biofilm investigations with BioBricks.

Retrieved from "http://2011.igem.org/Team:Glasgow/BiofilmResults"