Difference between revisions of "Team:Freiburg/Notebook"

 
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         <h5 style="text-align: center"> Our Protocols </h5>
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         <h5 style="text-align: center"> Our Journals </h5>
 
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<span style="color:#e8a126">I) Cloning</span>  <br><br>
 
<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.  
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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>
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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.
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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. 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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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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<span style="color:#e8a126">II) Bacillus subtilis</span><a name="spores"></a><br><br>
 
<span style="color:#e8a126">II) Bacillus subtilis</span><a name="spores"></a><br><br>
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Lab group 2 is responsible for all the basic work with our model organism <i>Bacillus subtilis</i>. We are looking after making <i>Bacillus subtilis</i> competent, the transformation of our constructs, the selection, cultivation and of course the sporulation aka the making of our nanocillus. We basically provide the groundwork so everyone else can keep on working. <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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<span style="color:#e8a126">III)Expression analysis</span><a name="proteins"></a><br><br>
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<span style="color:#e8a126">III) Expression analysis</span><a name="proteins"></a><br><br>
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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.
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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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<span style="color:#e8a126">IV) Targeting</span><a name="targeting"></a><br><br>
 
<span style="color:#e8a126">IV) Targeting</span><a name="targeting"></a><br><br>
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The mission of lab group 4 is to verify that our spores are able to bind on a target structure.  We validate that the spores are exposing the nanobody on their surface by binding GFP. The GFP binding is verified by fluorescence microscopy and FACS anylysis. <br><br>
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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>
 
<span style="color:#e8a126">V) Delivery</span><a name="delivery"></a><br><br>
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Focus of lab group 5 is the confirmation of the enzymatic activity of our spores. To show that the GST is correctly expressed by the spores we use a GST-assay adjusted to the use in a plate reader. Another application of our spores would be for a (anti-dandruff) shampoo, so we test this by washing a GFP-coated surface with different detergents and our construct and measuring the fluorescence.<br><br>
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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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Latest revision as of 21:02, 18 October 2016

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Our Journals
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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!