After getting the production and preparation of the protoplasts and the bacteria right we incubated them together in a solution of 0.8M and 0.6M Mannitol ( for onion and tobacco respectively) in MgM-MES buffer with a PH of 5. samples from the solutions were taken after 2hours, 3 hours and the day after(approx 18hours). To evalute the samples we used a fluorescent microscope. The results seem to be negative or inconclusive.

Samples incubated with the strains that didn’t have the signal sequence showed bright fluorescence(of both GFP and Mcherry) but no apparent secretion into protoplasts, although some cell membranes appeared to have a greater fluorescence than their surroundings, it was too inconclusive for making any qualitative assumptions. Some of the protoplasts appeared to be full of bacteria in which case the protoplasts had probably burst, forming a “bag-like” structureand filled with bacteria.

Figure 2 and 3 Birght field vs fluorescence filter showing tobacco proplasts forming “bag-lige” structures filled with GFP.

Samples incubated with the strains containing the signal sequence showed a very weak fluorescence(of both GFP and Mcherry) and no clear fluorescence around or inside the protoplasts. We hoped to see some proteins inside the protoplasts and a greater fluorescence around the protoplasts.

We can therefore conclude that the preliminary results from the injection assay using onion and tobacco protoplasts, were inconclusive. It could be that the strains containing the signal sequence attached to the protoplasts and injected protein into them but the fluorescence emitted was to weak to observe it using a fluorescence microscope. it could also be that the signal sequence somehow disrupts the production of the proteins, perhaps during folding. Furthermore, it could simply be due to the fact that the injectisome does not recognize and bind to the membrane of the protoplasts.

Team:UCopenhagen/Results

Overview of results for the different experiments

Protoplast experiment with plants