Team:Groningen/Project/Coating

From 2013.igem.org

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<html>
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<h1>Coating</h1>
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<h1>The Future</h1>
<h2>Overview</h2>
<h2>Overview</h2>
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With our engineered <i>B. subtilis</i> possessing all the desired genes and modifications, we would be able to put it to work in our coating setup.
With our engineered <i>B. subtilis</i> possessing all the desired genes and modifications, we would be able to put it to work in our coating setup.
</p>
</p>
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<br>
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<h2>Schematic overview</h2>
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<div align="center">
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<table  id="layout" width=50%>
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<tr>
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<td>
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<img src="http://img545.imageshack.us/img545/3044/vn4r.png" width="100%">
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</td>
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</tr>
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<tr><td><b>The spider silk gene and the Pdes (cold sensing) promoter with CheY (movement) gene downstream of it are put in our backbone, and inserted into the <i>Bacillus subtilis</i> genome.</b></td></tr>
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<tr>
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<td>
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<img src="http://img546.imageshack.us/img546/3871/38gm.png" width="100%">
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</td>
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</tr>
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<tr><td><b>The transformed <i>B. subtilis</i> is then put in a bath with a heated implant</b></td></tr>
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<tr>
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<td>
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<img src="http://img18.imageshack.us/img18/8459/f55s.png" width="100%">
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</td>
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</tr>
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<tr><td><b>The secreted spider silk with strep-tag attaches to the implant its streptavidin coating. The coating is processed with evaporation, treated with kosmotropic ions and heated thoroughly</b></td></tr>
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<tr>
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<td>
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<img src="http://img534.imageshack.us/img534/8683/nn6u.png" width="60%">
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</td>
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</tr>
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<tr><td><b>The implant is now coated by the Coating GEMs and ready for use!</b></td></tr>
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</table>
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</div>
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<p>
<br>
<br>
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<ul>
<ul>
<li>
<li>
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<u>A large, deep and circular bath</u></li>
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<b>A large, deep and circular bath</b></li>
To contain enough volume for a large culture
To contain enough volume for a large culture
<br><br><li>
<br><br><li>
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<u>Suitable growth medium for our <i>B. subtilis</i> strain</u></li>
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<b>Suitable growth medium for our <i>B. subtilis</i> strain</b></li>
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Perhaps with agar
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Perhaps with agar to prevent turbulence caused by oxygenation.
<br><br><li>
<br><br><li>
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<u><a href="http://en.wikipedia.org/wiki/Water_aeration#Fine_bubble_aeration">Fine bubble aeration</a></u></li>
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<b><a href="http://en.wikipedia.org/wiki/Water_aeration#Fine_bubble_aeration">Fine bubble aeration</a></b></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.
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>
<br><br><li>
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<u>A heated implant hanging inside and in the centre of the bath</u></li>
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<b>A heated implant hanging inside and in the centre of the bath</b></li>
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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 entire bath.
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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 entire bath (Figure 1).
<br><br>
<br><br>
</ul>
</ul>
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<td>
<td>
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<font size="1">Figure 1:  A possible setup for coating </font>
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<font size="1">Figure 1:  A possible setup for our system </font>
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</td>
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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.
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><br>
</p><br>
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<h2>Procedure 1 - Ideal situation</h2>
<h2>Procedure 1 - Ideal situation</h2>
<p>
<p>
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<br><u>Step 1</u><br>
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<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.
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.
This can be detected as the culture is rapidly increasing in number of cells, while the total number of cells is still relatively low.
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Expression of the spider silk protein is stimulated by addition of IPTG to the medium.
Expression of the spider silk protein is stimulated by addition of IPTG to the medium.
The secreted spider silk will attach to the implant.
The secreted spider silk will attach to the implant.
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<br><br><u>Step 4</u><br>
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After the implant is fully coated, all medium is removed to let the spider silk proteins polymerize.
</p>
</p>
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<br>
<h2>Procedure 2 - No silk secretion</h2>
<h2>Procedure 2 - No silk secretion</h2>
<p>
<p>
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<br><u>Step 1</u><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.
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.
This can be detected as the culture is rapidly increasing in number of cells, while the total number of cells is still relatively low.
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Expression of the spider silk protein is stimulated by addition of IPTG to the medium.
Expression of the spider silk protein is stimulated by addition of IPTG to the medium.
The secreted spider silk will attach to the implant.
The secreted spider silk will attach to the implant.
 +
<br><br><u>Step 4</u><br>
 +
After the implant is fully coated, all medium is removed to let the spider silk proteins polymerize.
</p>
</p>
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<br>
-
 
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<h2>Procedure 3 - No heat motility</h2>
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<p>
<p>
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<br><br>
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<u>Step 1</u><br>
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Whole setup:(FRISO)<br>
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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.
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Spider Silk<br>
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This can be detected as the culture is rapidly increasing in number of cells, while the total number of cells is still relatively low.
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Localizing the cells near the implant<br>
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<br><br><u>Step 2</u><br>
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Secretion or lysis approaches<br>
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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.
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Binding to streptavidin coated implant<br>
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<br><br><u>Step 3</u><br>
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Polymerization and sterilization<br>
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Expression of the spider silk protein is stimulated by addition of IPTG to the medium.
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<br><br>
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<br><br><u>Step 4</u><br>
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<a href="https://2013.igem.org/Team:Groningen/Project/secretion">Old page silk secretion</a>
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The implant is swirled through the medium, because the implant is coated with streptavidin the silk with strep-tag will automatically attach itself to the implant.
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<br><br>
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<br><br><u>Step 5</u><br>
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After the implant is fully coated, all medium is removed to let the spider silk proteins polymerize.
</p>
</p>
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 +
</html>
</html>

Latest revision as of 03:52, 5 October 2013

The Future

Overview

Applying the Coating to the implant is the end phase of our project. 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 would be able to put it to work in our coating setup.


Schematic overview

The spider silk gene and the Pdes (cold sensing) promoter with CheY (movement) gene downstream of it are put in our backbone, and inserted into the Bacillus subtilis genome.
The transformed B. subtilis is then put in a bath with a heated implant
The secreted spider silk with strep-tag attaches to the implant its streptavidin coating. The coating is processed with evaporation, treated with kosmotropic ions and heated thoroughly
The implant is now coated by the Coating GEMs and ready for use!


Coating 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 to prevent turbulence caused by oxygenation.

  • 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 entire bath (Figure 1).


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


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 alternative procedures. 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
After the implant is fully coated, all medium is removed to let the spider silk proteins polymerize.


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
After the implant is fully coated, all medium is removed to let the spider silk proteins polymerize.


Procedure 3 - No heat motility

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.

Step 3
Expression of the spider silk protein is stimulated by addition of IPTG to the medium.

Step 4
The implant is swirled through the medium, because the implant is coated with streptavidin the silk with strep-tag will automatically attach itself to the implant.

Step 5
After the implant is fully coated, all medium is removed to let the spider silk proteins polymerize.

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