Team:Groningen/Project/Coating

From 2013.igem.org

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<h1>Coating</h1>
<h1>Coating</h1>
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<h2>Overview</h2>
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<h2>This is so awesome</h2>
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-
 
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<p>
<p>
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Coating the implant is one of the last steps of <a href="https://2013.igem.org/Team:Groningen/Navigation/Project">our plan</a>. Here is an overview of what we would have up to this point:
 +
<ul>
 +
<li><a href="https://2013.igem.org/Team:Groningen/Navigation/Construct">Backbone with IPTG inducable promoter</a> for tranformation of <i>Bacillus subtilis</i></li><br><li>
 +
<a href="https://2013.igem.org/Team:Groningen/Navigation/SilkAssemblyShop">Spider silk genes put together with various components</a> and separately put into backbones</li><br><li>
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<a href="https://2013.igem.org/Team:Groningen/Navigation/Motility">Our <i>Bacillus subtilis</i> immobilizes near heat</a></li>
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</ul>
 +
<br>
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With our engineered <i>B. subtilis</i> possessing all the desired genes and modifications, we can put it to work in our coating setup.
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</p>
 +
<br>
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<h2>Setup</h2>
 +
<p>
 +
Our setup consists of the following components:
 +
<br>
 +
<ul>
 +
<li>
 +
<u>A large, deep and circular bath</u></li>
 +
To contain enough volume for a large culture
 +
<br><br><li>
 +
<u>Suitable growth medium for our <i>B. subtilis</i> strain</u>
 +
Perhaps with agar
 +
</li><br><li>
 +
<u><a href="http://en.wikipedia.org/wiki/Water_aeration#Fine_bubble_aeration">Fine bubble aeration</a></u></li>
 +
Bacillus is a very aerobic bacterium. This system facilitates thorough and gentle aeration. The small bubbles will diffuse enough when released from below the deep bath.
 +
<br><br><li>
 +
<u>A heated implant hanging inside and in the centre of the bath</u></li>
 +
This is what we want to coat. As the implant is providing a temperature gradient, no heating of the medium is required, saving energy that would be spend on heating the enitre bath.
<br><br>
<br><br>
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Whole setup:(FRISO)<br>
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</ul>
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Spider Silk<br>
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<br>
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Localizing the cells near the implant<br>
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-
Secretion or lysis approaches<br>
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-
Binding to streptavidin coated implant<br>
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-
Polymerization and sterilization<br>
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<br><br>
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-
<a href="https://2013.igem.org/Team:Groningen/Project/secretion">Old page silk secretion</a>
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<br><br>
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</p>
</p>
<p>
<p>
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<div align="center">  <!--you can use left/right or center to align the image-->
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To give an overview of our plan, with each of its aspects:
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-
<ul>
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<li><a href="https://2013.igem.org/Team:Groningen/Navigation/Construct">Backbone is constructed for <i>Bacillus subtilis</i></a></li><br><li>
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-
<a href="https://2013.igem.org/Team:Groningen/Navigation/SilkAssemblyShop">Spider silk genes are put together with various components</a></li><br><li>
+
-
<a href="https://2013.igem.org/Team:Groningen/Navigation/Motility">Our <i>Bacillus subtilis</i> moves toward heat</a></li>
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-
<br>
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-
When we put it all together...
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-
<br><br>
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<div align="left">  <!--you can use left/right or center to align the image-->
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<table  id="layout" width=100%> <!--change the percentage to determine the size of your image-->
<table  id="layout" width=100%> <!--change the percentage to determine the size of your image-->
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<tr>
<tr>
<th>
<th>
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<b>The Coating GEMs</b>
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<b>Experimental setup for coating</b>
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</th>
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<th>
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<b><a href="https://static.igem.org/mediawiki/igem.org/d/d4/Final_experiment_setup-01.jpg">Possible Experimental Setup:</a></b>
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</th>
</th>
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</tr>
</tr>
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<tr>
<tr>
<td>
<td>
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<video controls="controls" width="100%"> 
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<a href="https://static.igem.org/mediawiki/igem.org/d/d4/Final_experiment_setup-01.jpg"><img src="https://static.igem.org/mediawiki/igem.org/d/d4/Final_experiment_setup-01.jpg" width="100%"></a>
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    <source src="https://static.igem.org/mediawiki/igem.org/d/d0/Animation-secretion_VP8.webm" type="video/webm">
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  </video>
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</td>
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<td>
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<img src="https://static.igem.org/mediawiki/igem.org/d/d4/Final_experiment_setup-01.jpg" width="100%">
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</td>
</td>
</tr>
</tr>
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<td>
<td>
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<font size="1">Figure 1:  A possible setup of our project </font>
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<font size="1">Figure 1:  A possible setup for coating </font>
</td>
</td>
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<td>
 
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<td>
</td>
</td>
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</table>
</table>
</div>
</div>
</p>
</p>
 +
<br>
<br>
 +
<h2>Remarks</h2>
 +
<p>
 +
Before going into the proposed procedures, it should be noted that this page covers aspects of our hypothetical situation.
 +
It is how we planned to proceed if the engineering of our <i>B. subtilis</i> would have been achieved earlier in our project.
 +
However, instead of assuming everything works as we intended, which would be the ideal situation, we have designed [u]alternative procedures[/u].
 +
These alternative procedures are adapted to the lack of a certain component, an assumption we make in our ideal situation, such as secretion of the spider silk protein.
 +
</p>
 +
<h2>Procedure 1 - Ideal situation</h2>
 +
<p>
 +
<br><u>Step 1</u><br>
 +
Initially the <i>B. subtilis</i> cells are grown in liquid medium (LB) at 37 °C until the culture is in the early exponential phase.
 +
This can be detected as the culture is rapidly increasing in number of cells, while the total number of cells is still relatively low.
 +
<br><br><u>Step 2</u><br>
 +
The cells are moved to the big volume of our setup, where The cells move around and grow further, though should still be in the exponential phase.
 +
Influenced by the temperature gradient most cells should lose their ability to move near the implant.
 +
This results in accumulation of cells in the heated area close to the implant.
 +
<br><br><u>Step 3</u><br>
 +
Expression of the spider silk protein is stimulated by addition of IPTG to the medium.
 +
The secreted spider silk will attach to the implant.
 +
<br><br><u>Step 4</u><br>
 +
...
 +
<br>
 +
</p>
 +
<h2>Procedure 2 - No silk secretion</h2>
 +
<p>
 +
<br><u>Step 1</u><br>
 +
Initially the <i>B. subtilis</i> cells are grown in liquid medium (LB) at 37 °C until the culture is in the early exponential phase.
 +
This can be detected as the culture is rapidly increasing in number of cells, while the total number of cells is still relatively low.
 +
<br><br><u>Step 2</u><br>
 +
The cells are moved to the big volume of our setup, where the cells move around and grow further, though should still be in the exponential phase.
 +
Influenced by the temperature gradient most cells should lose their ability to move near the implant.
 +
This results in accumulation of cells in the heated area close to the implant.
 +
<br><br><u>Step 3</u><br>
 +
Expression of the spider silk protein is stimulated by addition of IPTG to the medium.
 +
The secreted spider silk will attach to the implant.
 +
<br><br><u>Step 4</u><br>
 +
...
 +
<br>
 +
</p>
 +
 +
 +
<p>
 +
<br><br>
 +
Whole setup:(FRISO)<br>
 +
Spider Silk<br>
 +
Localizing the cells near the implant<br>
 +
Secretion or lysis approaches<br>
 +
Binding to streptavidin coated implant<br>
 +
Polymerization and sterilization<br>
 +
<br><br>
 +
<a href="https://2013.igem.org/Team:Groningen/Project/secretion">Old page silk secretion</a>
 +
<br><br>
 +
</p>
</html>
</html>

Revision as of 04:53, 3 October 2013

Coating

Overview

Coating the implant is one of the last steps of our plan. Here is an overview of what we would have up to this point:


With our engineered B. subtilis possessing all the desired genes and modifications, we can put it to work in our coating setup.


Setup

Our setup consists of the following components:

  • A large, deep and circular bath
    • To contain enough volume for a large culture

  • Suitable growth medium for our B. subtilis strain Perhaps with agar

  • Fine bubble aeration
    • Bacillus is a very aerobic bacterium. This system facilitates thorough and gentle aeration. The small bubbles will diffuse enough when released from below the deep bath.

  • A heated implant hanging inside and in the centre of the bath
    • This is what we want to coat. As the implant is providing a temperature gradient, no heating of the medium is required, saving energy that would be spend on heating the enitre bath.


Experimental setup for coating
Figure 1: A possible setup for coating


Remarks

Before going into the proposed procedures, it should be noted that this page covers aspects of our hypothetical situation. It is how we planned to proceed if the engineering of our B. subtilis would have been achieved earlier in our project. However, instead of assuming everything works as we intended, which would be the ideal situation, we have designed [u]alternative procedures[/u]. These alternative procedures are adapted to the lack of a certain component, an assumption we make in our ideal situation, such as secretion of the spider silk protein.

Procedure 1 - Ideal situation


Step 1
Initially the B. subtilis cells are grown in liquid medium (LB) at 37 °C until the culture is in the early exponential phase. This can be detected as the culture is rapidly increasing in number of cells, while the total number of cells is still relatively low.

Step 2
The cells are moved to the big volume of our setup, where The cells move around and grow further, though should still be in the exponential phase. Influenced by the temperature gradient most cells should lose their ability to move near the implant. This results in accumulation of cells in the heated area close to the implant.

Step 3
Expression of the spider silk protein is stimulated by addition of IPTG to the medium. The secreted spider silk will attach to the implant.

Step 4
...

Procedure 2 - No silk secretion


Step 1
Initially the B. subtilis cells are grown in liquid medium (LB) at 37 °C until the culture is in the early exponential phase. This can be detected as the culture is rapidly increasing in number of cells, while the total number of cells is still relatively low.

Step 2
The cells are moved to the big volume of our setup, where the cells move around and grow further, though should still be in the exponential phase. Influenced by the temperature gradient most cells should lose their ability to move near the implant. This results in accumulation of cells in the heated area close to the implant.

Step 3
Expression of the spider silk protein is stimulated by addition of IPTG to the medium. The secreted spider silk will attach to the implant.

Step 4
...



Whole setup:(FRISO)
Spider Silk
Localizing the cells near the implant
Secretion or lysis approaches
Binding to streptavidin coated implant
Polymerization and sterilization


Old page silk secretion

Retrieved from "http://2013.igem.org/Team:Groningen/Project/Coating"