Difference between revisions of "Team:Freiburg/Notebook"

 
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<h1 class="sectionedit1"><a name="sporulation" id="sporulation">Sporulation</a></h1>
 
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        }
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<strong>Checklist:</strong><br/>
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<ul>
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<li class="level1"><div class="li"> Use the right Medium.</div>
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</li>
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<li class="level1"><div class="li"> Vortex cuvettes if you make a dilution</div>
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</li>
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<li class="level1"><div class="li"> Set Nanodrop to cuvettes</div>
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</ul>
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1. Overnight culture
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    <div class="color1">
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    <div class="img_menu">
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<li class="level1"><div class="li"> inoculate your culture in 5ml LB-Medium</div>
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        <h5 style="text-align: center"> Our Journals </h5>
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</li>
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        <center>
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<li class="level1"><div class="li"> let them grow over night at 37°C, 200rpm</div>
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            <div class="image">
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</li>
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                <a href="https://2016.igem.org/Team:Freiburg/NotebookCloning"> <img class="imga" src="https://static.igem.org/mediawiki/2016/1/14/T--Freiburg--CloningLab.png" alt="Smiley face"></a>
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</ul>
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<p>
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                <a href="https://2016.igem.org/Team:Freiburg/NotebookSpores"> <img class="imgb" src="https://static.igem.org/mediawiki/2016/9/93/T--Freiburg--SporesLab.png" alt="Smiley face"></a>
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<br/>
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2. Exponential growth
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                <a href="https://2016.igem.org/Team:Freiburg/NotebookProteins"> <img class="imgc" src="https://static.igem.org/mediawiki/2016/2/23/T--Freiburg--ProteinsLab.png" alt="Smiley face"></a>
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</p>
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<ul>
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<li class="level1"><div class="li"> meassure the OD600 of your overnight culture</div>
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</li>
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<li class="level1"><div class="li"> inoculate 2ml + Xml to measure OD (personal preference) LB-Medium to OD600=0,1 from overnight culture </div>
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</li>
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<li class="level1"><div class="li"> let the cells grow to an OD600=0,8 (37°C, 200rpm)</div>
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</li>
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</ul>
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<p>
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                <a href="https://2016.igem.org/Team:Freiburg/NotebookTargeting"><img class="imgd" src="https://static.igem.org/mediawiki/2016/f/f4/T--Freiburg--TargetingLab.png" alt="Smiley face"></a>
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3. Sporulation
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</p>
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<ul>
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<li class="level1"><div class="li"> centrifuge the 2 ml of cells at 13.000 rpm for 1 minute</div>
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</li>
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<li class="level1"><div class="li"> wash the pellet with PBS</div>
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</li>
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<li class="level1"><div class="li"> resuspend the pellet in 1 ml DSM (Difco Sporulation Medium)<a href="/igem2016/doku.php?id=labprotocols:media" class="wikilink1" title="labprotocols:media">Culture medium</a></div>
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</li>
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<li class="level1"><div class="li"> let the cells grow (24h,37°C 200rpm)</div>
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</li>
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</ul>
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<p>
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                <a href="https://2016.igem.org/Team:Freiburg/NotebookDelivery"><img class="imge" src="https://static.igem.org/mediawiki/2016/4/4b/T--Freiburg--DeliveryLab.png" alt="Smiley face"></a>
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4. Lysozym treatment
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</p>
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<ul>
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<li class="level1"><div class="li"> dilute the cells 6:1 in lysozym solution (15mg/μl)</div>
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</li>
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<li class="level1"><div class="li"> incubate for 1 hour at room temperature</div>
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</li>
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<li class="level1"><div class="li"> wash 6 times with 1x PBS</div>
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</li>
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</ul>
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<p>
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            </div>
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5. Count the spores (Neubauer-counting chamber, usually dilute in BPS 1 to 100)<br/>
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        </center>
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    </div>
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  </div>
  
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<a href="/igem2016/lib/exe/fetch.php?media=labprotocols:sorecountingb54.png" class="media" title="labprotocols:sorecountingb54.png"><img src="/igem2016/lib/exe/fetch.php?w=400&amp;media=labprotocols:sorecountingb54.png" class="media" alt="" width="400" /></a><br/>
 
  
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6. Make aliquots (around 100million spores per aliquot)
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</p>
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<div class="color8">
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<div class="para_center_20">
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<span style="color:#e8a126">I) Cloning</span>  <br><br>
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The construction of fusion genes involves the assembly of genes for passenger proteins and anchoring motifs. Both had to be fused while avoiding unnecessary cloning scars. We generated and provided all integration vectors for the transformation of B. subtilis. <br>
 +
To keep an overview of the cloned constructs every plasmid was assigned to an ID: pIG16_000. All used oligos were assigned to an ID as well: oIG16_000.<br>
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The complete list of the resulting bacterial strains and oligos can be found in the attached tables. The spore coat proteins cotZ, cotG, cotB and cgeA were amplified from the genome of B. subtilis 168. <br>The anti-GFP nanobody and the GST were amplified from plasmids provided by Dr. Nicole Gensch and Dr. Maximilian Ulbrich. For the cloning strategy see Project - <a target="_blank" href='https://2016.igem.org/Team:Freiburg/Goals_Approach' >Approach</a>.
  
 
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<h1 class="sectionedit2"><a name="induktion_of_sporulation" id="induktion_of_sporulation">Induction of sporulation</a></h1>
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<div class="level1">
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<div class="color8">
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<a href="https://www.researchgate.net/post/How_to_induce_sporulation_of_Bacillus_spp_by_heating_or_any_other_simple_technique" class="urlextern" target="_Blank" title="https://www.researchgate.net/post/How_to_induce_sporulation_of_Bacillus_spp_by_heating_or_any_other_simple_technique"  rel="nofollow">https://www.researchgate.net/post/How_to_induce_sporulation_of_Bacillus_spp_by_heating_or_any_other_simple_technique</a><br/>
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<div class="para_center_20">
  
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<a href="https://www.researchgate.net/post/How_to_sporulate_the_bacteria_or_how_to_induce_the_sporulation_in_bacteria_especially_for_Bacillus_spp" class="urlextern" target="_Blank" title="https://www.researchgate.net/post/How_to_sporulate_the_bacteria_or_how_to_induce_the_sporulation_in_bacteria_especially_for_Bacillus_spp"  rel="nofollow">https://www.researchgate.net/post/How_to_sporulate_the_bacteria_or_how_to_induce_the_sporulation_in_bacteria_especially_for_Bacillus_spp</a><br/>
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<span style="color:#e8a126">II) Bacillus subtilis</span><a name="spores"></a><br><br>
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Bacillus subtilis was made competent, transformation with the  constructs, selected, cultivated and of course sporulated aka the Nanocillus was made.  <br><br>
  
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<a href="/igem2016/doku.php?id=tag:workwithbs_labprotocols" class="wikilink1" title="tag:workwithbs_labprotocols" rel="tag">WorkwithBS labprotocols</a>
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<span style="color:#e8a126">III) Expression analysis</span><a name="proteins"></a><br><br>
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</span></div>
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Verification of the expression of the constructs is important to confirm the successful transformation into Bacillus subtilis. <br>The used methods include SDS-PAGEs, Western Blots and flow cytometry analyses.
 +
Besides the confirmation of expression, flow cytometry is also used to confirm the binding of GFP to the aGFP-nanobody that is shown on the spores.
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 +
 
 +
<br><br>
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<span style="color:#e8a126">IV) Targeting</span><a name="targeting"></a><br><br>
 +
 
 +
The targeting is one of the main requirements the spores have to fulfill. An adhesion-assay was conducted to verify the binding of the spores to a desired target. <br><br>
 +
 
 +
 
 +
<span style="color:#e8a126">V) Delivery</span><a name="delivery"></a><br><br>
 +
The focus of this group lays on the confirmation of the enzymatic activity of our spores. <br>To show that the GST is correctly expressed by the spores a GST-assay is adjusted to the use in a plate reader. <br>Another application of the spores would be for a (anti-dandruff) shampoo, so this is tested by washing a GFP-coated surface with different detergents and our construct and measuring the fluorescence.
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{{Freiburg/Footer}}

Latest revision as of 21:02, 18 October 2016

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Our Journals
Smiley face Smiley face Smiley face Smiley face Smiley face
I) Cloning

The construction of fusion genes involves the assembly of genes for passenger proteins and anchoring motifs. Both had to be fused while avoiding unnecessary cloning scars. We generated and provided all integration vectors for the transformation of B. subtilis.
To keep an overview of the cloned constructs every plasmid was assigned to an ID: pIG16_000. All used oligos were assigned to an ID as well: oIG16_000.
The complete list of the resulting bacterial strains and oligos can be found in the attached tables. The spore coat proteins cotZ, cotG, cotB and cgeA were amplified from the genome of B. subtilis 168.
The anti-GFP nanobody and the GST were amplified from plasmids provided by Dr. Nicole Gensch and Dr. Maximilian Ulbrich. For the cloning strategy see Project - Approach.
II) Bacillus subtilis

Bacillus subtilis was made competent, transformation with the constructs, selected, cultivated and of course sporulated aka the Nanocillus was made.

III) Expression analysis

Verification of the expression of the constructs is important to confirm the successful transformation into Bacillus subtilis.
The used methods include SDS-PAGEs, Western Blots and flow cytometry analyses. Besides the confirmation of expression, flow cytometry is also used to confirm the binding of GFP to the aGFP-nanobody that is shown on the spores.

IV) Targeting

The targeting is one of the main requirements the spores have to fulfill. An adhesion-assay was conducted to verify the binding of the spores to a desired target.

V) Delivery

The focus of this group lays on the confirmation of the enzymatic activity of our spores.
To show that the GST is correctly expressed by the spores a GST-assay is adjusted to the use in a plate reader.
Another application of the spores would be for a (anti-dandruff) shampoo, so this is tested by washing a GFP-coated surface with different detergents and our construct and measuring the fluorescence.

Posted by: iGEM Freiburg

Nanocillus - 'cause spore is more!