Overview of results for the different experiments

Protoplast experiment with plants

Figure 1 Onion protoplasts

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.

Egg membrane experiment

In the first experiment 2 samples were taken, 20h after filling the chambers and after 47h. The bacterial load of lower chamber solution was tested by plating it on agarose gel plate with added chloramphenicol and arabinose. If the colonies appeared the following day and they were red in appearance (due to mCherry) that meant that the membrane was breached and lower chamber compromised.

As a control, a setup with LB media in both chambers was used.

Unfortunately all plates showed red colonies of different sizes the following days, meaning we got false positive results. Red colour came from produced mCherry protein (after induction with arabinose), since we were working with mCherry-expressing E coli in this experiment.

Figure 7: Presence of red violet colour meant that bacteria had indeed breached the membrane.

Troubleshooting

Few more tests were performed similar to the experiment described above. Increase of fluorescence was always connected with breaching of the membrane, however there were a few experiments where sterility of the collection chamber remained uncompromised, but also no increase in fluorescence was detected.

The strain used in that experiment was I+,S-,C+ meaning that it had the injectisome and chaperone but lacked the secretion signal. It was noted that the overnight culture turned red after incubation in 1% (w/w) arabinose already after 12 hours. However this strain was unable to secrete the protein through membrane - we speculated that this may be due to the lack of secretion signal.

To test this theory the only logical thing to do would have been to try the strain with secretion signal but the strain I+S+C+ didn't show enough fluorescence in overnight culture to even start the experiment. We believed that signal sequence was somehow disrupting the folding process of mCherry protein and therefore tried growing bacteria at lower temperature (28°C instead of 37°C as in the first experiments). Bacteria was allowed to grow for 5 days yet it still didn't produce enough of the fluorescent protein.

The same issue was encountered when trying to repeat the experiment with E coli producing sfGFP (superfolder green fluorescent protein). The strain without secretion signal produced enough fluorescent protein for experiments, yet the one with secretion signal produced protein below detectable levels for fluorescent plate reader. As the strains without secretin signal failed to secrete the fluorescent protein across the membrane and the strains with secretion signal failed to produce enough of it the experiments were eventually discontinued.

Conclusions

After initial difficulties with the "hardware" i.e. leaky membranes, the manufacturing process was eventually perfected to produce a reasonable number of leak free membrane systems of required quality. The protocols were also eventually optimized at least concerning the incubation time, volumes and similar, but the problem with signal interfering with the folding of the protein remained unsolved.

Nevertheless, had the issue with folding of the protein (with secretion signal) been solved the developed setup would be the best possible option to test whether the whole idea of secreting the proteins across membrane worked. Instead of egg yolk membrane, different substitutes could be used, either using membranes from nature or biomimetic membranes.

An interesting thing to investigate however would be how long can a membrane be exposed to bacteria before being damaged.