Difference between revisions of "Team:Freiburg/Notebook"
D.Steinberg (Talk | contribs) |
|||
| Line 61: | Line 61: | ||
<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> | ||
| − | + | Bacillus subtilis was made competent, transformation with the constructs, selected , cultivated and of course sporulated aka the Nanocillus was made. <br><br> | |
Revision as of 17:21, 15 October 2016
Our Protocols
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.
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 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.
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.
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 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.
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.
