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Team:TU Munich/lab/results
From 2011.igem.org
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<h4>Distribution of cells and cell survival:</h4> | <h4>Distribution of cells and cell survival:</h4> | ||
<p>To make sure that the cells are distributed evenly within the matrix, the bacteria need to be added to the medium before gelling occurs. The gelling temperature of GELRITE depends on both the amount of GELRITE powder and the salt concentration of the used liquid. A 0.5 % GELRITE gel prepared with M9 medium solidifies at around 46 °C. This means that the cells that shall be immobilized must survive such temperatures for a few minutes. This is why we made some experiments with the heat resistant E. coli strain BH28 (kindly provided by Jeanette Winter). Fluorescence microscopy showed that the cells can survive the gelling process and produce GFP or RFP upon induction. The temperature seems to be the most critical factor regarding cell survival, as minimal media, with which the gel is prepared, provide enough nutrients for the bacteria.</p> | <p>To make sure that the cells are distributed evenly within the matrix, the bacteria need to be added to the medium before gelling occurs. The gelling temperature of GELRITE depends on both the amount of GELRITE powder and the salt concentration of the used liquid. A 0.5 % GELRITE gel prepared with M9 medium solidifies at around 46 °C. This means that the cells that shall be immobilized must survive such temperatures for a few minutes. This is why we made some experiments with the heat resistant E. coli strain BH28 (kindly provided by Jeanette Winter). Fluorescence microscopy showed that the cells can survive the gelling process and produce GFP or RFP upon induction. The temperature seems to be the most critical factor regarding cell survival, as minimal media, with which the gel is prepared, provide enough nutrients for the bacteria.</p> | ||
| - | <h2> | + | |
| + | <h2>Cloning of constructs</h2> | ||
| + | <p> | ||
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| + | Cloning of AND-Gate and reporter constructs were successful and plasmids have been verified via sequencing after every cloning step. Those sequences were entered into the registry. | ||
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| + | The cloning scheme for our <a href="https://static.igem.org/mediawiki/2011/d/d2/Klonierungsschema_Plasmid_1.jpg" rel="lightbox" title="Scheme Plasmid 1">AND-Gate</a> and <a href="https://static.igem.org/mediawiki/2011/3/3a/Klonierungsschema_Plasmid_2.jpg" rel="lightbox" title="Scheme Plasmid 2">reporter</a> constructs can be seen on our <a href="https://2011.igem.org/Team:TU_Munich/project/design" title="Design">Design</a> page. | ||
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| + | </p> | ||
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| + | <h2>Characterisation of parts</h2> | ||
<p></p> | <p></p> | ||
| - | < | + | <h3>Red Light Sensor</h3> |
<p></p> | <p></p> | ||
| - | < | + | <h3>Blue Light Sensor</h3> |
| + | <p></p> | ||
| + | <h3>Reporter Plasmid</h3> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 11:50, 21 September 2011
Results
Solid Matrix
Our idea of a 3D-printer made it necessary to immobilize our cells inside a three dimensional matrix. We figured that the matrix needed to have the following properties:
The idea to immobilize our bacteria in something like a solid growth medium (like agar-plates) was a step in the right direction, but standard LB-agar was not the solution. In the end, we chose GELRITE as a gelling agent, “a highly-purified, natural anionic heteropolysaccharide that forms rigid, brittle, agar-like gels” (source: gelrite spec sheet, see labbook/methods).
Clear substance with good transmission of light:
The matrix should be prepared with minimal media such as M9 instead of LB, because LB shows a strong absorption in the lower range of visible light (hence its yellow color). Minimal media are clear, which is a great advantage. The cell density in the matrix is also an important factor, because with increasing cell number the gel becomes blurry due to refraction of light (see diagrams).
Distribution of cells and cell survival:
To make sure that the cells are distributed evenly within the matrix, the bacteria need to be added to the medium before gelling occurs. The gelling temperature of GELRITE depends on both the amount of GELRITE powder and the salt concentration of the used liquid. A 0.5 % GELRITE gel prepared with M9 medium solidifies at around 46 °C. This means that the cells that shall be immobilized must survive such temperatures for a few minutes. This is why we made some experiments with the heat resistant E. coli strain BH28 (kindly provided by Jeanette Winter). Fluorescence microscopy showed that the cells can survive the gelling process and produce GFP or RFP upon induction. The temperature seems to be the most critical factor regarding cell survival, as minimal media, with which the gel is prepared, provide enough nutrients for the bacteria.
Cloning of constructs
Cloning of AND-Gate and reporter constructs were successful and plasmids have been verified via sequencing after every cloning step. Those sequences were entered into the registry. The cloning scheme for our AND-Gate and reporter constructs can be seen on our Design page.
Characterisation of parts
Red Light Sensor
Blue Light Sensor
Reporter Plasmid