Team:Groningen/Labwork/27 June 2013

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<h2>Mirjam</h2>
<h2>Mirjam</h2>
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<br/>Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using gel purification (Roche gel purification kit).
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Added 1 ul serva/100ml to the earlier made 0.8% agarose gel.
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<br/>FliZ  9,2 ng/ul
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<br/>LytB  8,2 ng/ul
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<br/>MotB  8,8 ng/ul
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<br/>EstA  8,8 ng/ul
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Because of the low concentrations a new PCR is done using the PCR products.
+
<p>Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using the Roche gel purification kit. Using nanodrop reveals the following concentrations:
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A PCR is done for some different silk constructs to start with. The following combinations are made (PCR mix preparation is done with the same protocol as described on 26-06-2013):
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<table border="1">
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<br/>1) signal sequence - N-terminal strep tag - silk
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  <tr>
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<br/>2) signal sequence - silk - C-terminal strep tag
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    <th>signal sequence</th>
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<br/>3) signal sequence - silk - no strep tag
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    <th>concentration (ng/&micro;l)</th>
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<br/>4) silk without tag
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  </tr>
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The following protocol is used:
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  <tr>
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<br/>98°C, 98°C, 50°C, 72°C, 72°C, 4°C
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    <td>FliZ</td>
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<br/>0:30  0:10  0:25  0:27  10:00 forever
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    <td align=center>9.2</td>
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  </tr>
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Added 1 ul serva/100 ml 0.8% agarose gel.
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  <tr>
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<br/>Gel run at 90V for 22 min at a 0.8% agarose gel.
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    <td>LytB</td>
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<br/>Gel imaging revealed that no bands where present. Therefore it is placed at a high concentration of Ethidium Bromide and it is examined that the bands appear. A big smear is seen for all the silk products with a bigger band around 200 bp. Because of these results it is decided to do a gradient PCR on the first combination from 55-75 degrees Celsius.
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    <td align=center>8.2</td>
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  </tr>
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  <tr>
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    <td>MotB</td>
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    <td align=center>8.8</td>
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  </tr>
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  <tr>
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    <td>EstA</td>
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    <td align=center>8.8</td>
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  </tr>
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</table>
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<p>Because of the low concentrations a new <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR</b></FONT></a> is done using the PCR products.
 +
 +
<p>A <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR</b></FONT></a> is done for some different silk constructs to start with. The following combinations are made:
 +
<ol>
 +
<li>signal sequence - N-terminal strep tag - silk</li>
 +
<li>signal sequence - silk - C-terminal strep tag</li>
 +
<li>signal sequence - silk - no strep tag</li>
 +
<li>silk without tag</li>
 +
</ol>
 +
<br>For every sample an annealing temperature of 50&deg;C is used, expecting a size of 900 bp.
 +
 +
<p>The samples are run over a 0.8% <a href="https://2013.igem.org/Team:Groningen/protocols/GelElectrophoresis"><FONT COLOR="black"><b>agarose gel</b></FONT></a>. This revealed that no bands are present at all. So the gel is stained with a high concentration of Ethidium Bromide and the bands appear. A big smear is seen for all the silk products, with a higher concentrated band around 200 bp. Because of this results it is decided to do a gradient PCR on the first combination from 55-75&deg;C.
 +
 +
<p>
<h2>Claudio</h2>
<h2>Claudio</h2>
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<br>Based on the following bibliography:
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The following bibliography summarizes the source of inspiration on which the heat-attracted bacteria sub-project is based on:
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<ul type="square">Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in Bacillus subtilis by L. M. Bredeston, D. Marciano, D. Albanesi, D. De Mendoza, and J. M. Delfino</ul>
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<ul type="square">
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<ul type="square">The three adaptation systems of Bacillus subtilis chemotaxis by Christopher V. Rao, George D. Glekas and George W. Ordal</ul>
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<li><i>Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in Bacillus subtilis</i> by L. M. Bredeston, D. Marciano, D. Albanesi, D. De Mendoza, and J. M. Delfino</li>
 +
<li><i>The three adaptation systems of Bacillus subtilis chemotaxis</i> by Christopher V. Rao, George D. Glekas and George W. Ordal</li>
 +
<li><i>Chemotaxis in Bacillus Sutilis: how bacteria monitor environmental signals</i> by Liam F. Garrity and George W. Ordal</li>
 +
<li><i>Regulation of Bacillus subtilis DesK thermosensor by lipids</i> by Mariana Martin and Diego De Mendoza</li>
 +
</ul>

Latest revision as of 17:01, 28 July 2013

Mirjam

Added 1 ul serva/100ml to the earlier made 0.8% agarose gel.

Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using the Roche gel purification kit. Using nanodrop reveals the following concentrations:

signal sequence concentration (ng/µl)
FliZ 9.2
LytB 8.2
MotB 8.8
EstA 8.8

Because of the low concentrations a new PCR is done using the PCR products.

A PCR is done for some different silk constructs to start with. The following combinations are made:

  1. signal sequence - N-terminal strep tag - silk
  2. signal sequence - silk - C-terminal strep tag
  3. signal sequence - silk - no strep tag
  4. silk without tag

For every sample an annealing temperature of 50°C is used, expecting a size of 900 bp.

The samples are run over a 0.8% agarose gel. This revealed that no bands are present at all. So the gel is stained with a high concentration of Ethidium Bromide and the bands appear. A big smear is seen for all the silk products, with a higher concentrated band around 200 bp. Because of this results it is decided to do a gradient PCR on the first combination from 55-75°C.

Claudio

The following bibliography summarizes the source of inspiration on which the heat-attracted bacteria sub-project is based on:
  • Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in Bacillus subtilis by L. M. Bredeston, D. Marciano, D. Albanesi, D. De Mendoza, and J. M. Delfino
  • The three adaptation systems of Bacillus subtilis chemotaxis by Christopher V. Rao, George D. Glekas and George W. Ordal
  • Chemotaxis in Bacillus Sutilis: how bacteria monitor environmental signals by Liam F. Garrity and George W. Ordal
  • Regulation of Bacillus subtilis DesK thermosensor by lipids by Mariana Martin and Diego De Mendoza

Retrieved from "http://2013.igem.org/Team:Groningen/Labwork/27_June_2013"