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From 2014.igem.org

Results / GFP Photos

Detection of GFP was done to prove relevant aspects of our project. We constructed a protein which is needed to be secreted by plant cell and being attached to the cell wall. At the C-terminus of the protein we placed a cellulose binding domain. This domain should be attached to the cellulose rich cell wall of plant cell. Therefor we built a secretion signal (signal peptide) fused it to GFP (middle of the protein) and the cellulose binding domain (C-terminus). Via confocal microscopy we analyzed the subcellular localization of GFP to prove the secretion and cellulose binding of the protein. This is a frequently used procedure to characterize the subcellular localization of proteins in eukaryotes (Cutler et al. 2000).

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 sequence 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. 1 (left): and Fig 2 (right): confocal detection of fluorescence from transformed plant cells (replicate 2)Transformed cells of Nicotiana tabacum were analyzed via confocal microscopy. Column A-I: red channel, shows chlorophyll’s autofluorescence which shows the chloroplasts. Column B-J: green channel showing GFP. Column C-K: red and green channel merged. Column D-H: 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-K shows the detection of the analyzed construct: Expa4-GFP-CBD.

Figure 1 shows detected fluorescence of transformed Nicotiana tabacum cells. A GFP control without protein fusion is shown in lane A-D. The signal is specific for a cytosolic protein, which is indicated by chloroplast surrounded with GFP-Signal. In contrast the plasmamembranemarker (E-H) shows a specific signal at the periphery. The signal doesn’t surround the chloroplast, which shows that it is located at the cell cover and not in the cytoplasm. Because it’s a marker, it’s location is already known. The analyzed construct is shown in I-K. In contrast to cytoplasmic GFP, the signal appears to be in the exterior. The signal appears to diffuse between the cells, at the cell wall. This indicates that the secretion signal and the tethering at the cell wall via CBD might work.

1. Cutler, S. R.; Ehrhardt, D. W.; Griffitts, J. S.; Somerville, C. R. (2000): Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. In Proceedings of the National Academy of Sciences 97 (7), pp. 3718–3723. DOI: 10.1073/pnas.97.7.3718.