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Team:Hannover/Results/GFP
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<h1>Results / GFP Photos</h1><p class="text">To avoid cytotoxic effects on the plant caused by the heavy metals we decided to place T4MBP to the cell exterior. With a secretion signal (Expa4) the T4MBP is directed to the extracellular space. The protein is tethered to the cell surface by using a cellulose binding domain (CBD). Via the cellulose binding domain the protein is attached to the cell wall.</p> | <h1>Results / GFP Photos</h1><p class="text">To avoid cytotoxic effects on the plant caused by the heavy metals we decided to place T4MBP to the cell exterior. With a secretion signal (Expa4) the T4MBP is directed to the extracellular space. The protein is tethered to the cell surface by using a cellulose binding domain (CBD). Via the cellulose binding domain the protein is attached to the cell wall.</p> | ||
| - | <h2>Labwork</h2><p class="text"><ul><li>CBD fused with mGFP using<a href="" target="_blank"> our EMP protocol</a></li><ol><li>we used pEarleyGate with the sequenz of mGFP5 as a template to generate our megaprimer</li><li>we used our GenArt Vector (with the sequenz of | + | <h2>Labwork</h2><p class="text"><ul><li>CBD fused with mGFP using<a href="" target="_blank"> our EMP protocol</a></li><ol><li>we used pEarleyGate with the sequenz of mGFP5 as a template to generate our megaprimer</li><li>we used our GenArt Vector (with the sequenz of the EXPA4 signal peptide followed by the T4MBP, a tag and the CBD) as the target plasmid</li></ol><li>cloned the Expa~GFP~CBD into our modified pORE_E3_2x35S </li></ul></p> |
<h2>Results</h2> | <h2>Results</h2> | ||
<center><table><tr><td><a href="https://static.igem.org/mediawiki/2014/6/6b/Hannover_20141004_Figure_confocal_replicate1.png" data-lightbox="galery3" data-title="Fig. 3: confocal detection of fluorescence from transformed plant cells (replicate 1) | <center><table><tr><td><a href="https://static.igem.org/mediawiki/2014/6/6b/Hannover_20141004_Figure_confocal_replicate1.png" data-lightbox="galery3" data-title="Fig. 3: confocal detection of fluorescence from transformed plant cells (replicate 1) | ||
Revision as of 12:52, 12 October 2014
Results / GFP Photos
To avoid cytotoxic effects on the plant caused by the heavy metals we decided to place T4MBP to the cell exterior. With a secretion signal (Expa4) the T4MBP is directed to the extracellular space. The protein is tethered to the cell surface by using a cellulose binding domain (CBD). Via the cellulose binding domain the protein is attached to the cell wall.
Labwork
- CBD fused with mGFP using our EMP protocol
- we used pEarleyGate with the sequenz of mGFP5 as a template to generate our megaprimer
- we used our GenArt Vector (with the sequenz of the EXPA4 signal peptide followed by the T4MBP, a tag and the CBD) as the target plasmid
- cloned the Expa~GFP~CBD into our modified pORE_E3_2x35S
Results
 |
Fig. 3: confocal detection of fluorescence from transformed plant cells (replicate 1) Transformed cells of Nicotiana tabacum were analyzed via confocal microscopy. Colum A-I: red channel, shows chlorophyll’s autofluorescence which shows the chloroplasts. Colum B-J: green channel showing GFP. Colum C-K: red and green channel merged. Colum D-L: pseudo transmission detection. Lane A-D shows a transformed cell with raw GFP. Lane E-H shows a plasmamembranemarker (Nelson et al. 2007). Lane I-L shows the detection of the analyzed construct: Expa4-GFP-CBD. |
