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From 2014.igem.org

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
1. we used pEarleyGate with the sequenz of mGFP5 as a template to generate our megaprimer
2. 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.