Figure 2: Description of the experiment. In time intervals a sample was taken from the collection chamber. Fluorescence was measured to detect if protein was being secreted (mCherry), while plating was performed to check whether bacteria crossed the membrane. Since our strain was resistant to Chloramphenicol, we used agarose plates with added Chloramphenicol. This way only bacterial strains with resistance to this antibiotic could grow on the plates. In order to prove that the colonies on the plate were indeed the strain we were working with arabinose was added since it induces production of mCherry protein in our strain. If colonies turned red then we can safely assume that the bacteria has somehow breached the membrane and that detected increase in fluorescence in collection chamber was indeed a false positive result.

Technical details

The following experiment has probably not yet been done. The experiment therefore required a great deal of improvisation, flexibility and imagination, not mentioning the number of trials before optimizing the protocols.

Strains used

For preliminary experiments the following strains of SIEC (Synthetic injector E. coli) bacteria were used:

All had chaperone and mCherry as a reporter protein.

Legend:

• I = injectisome, (+) means functional injectisome, while (-) means the plasmid is lacking promoter in front DNA that in encoding injectisome.
• S = secretion signal, a sequence of 20 amino acids which is believed to provide selectivity when it comes to secretion via injectosome.
• C = chaperone. In this experiment all strains had chaperone. It is believed that they facilitate unfolding of the protein prior to injecting, however we tried to validate this in some other experiments.

Egg yolk extraction

In contrary to popular belief - it is actually possible to extract the membrane separating yolk from the white. However, some practice is needed beforehand and a specialized tools which are not part of the standard laboratory inventory.

First step is to separate the white of the egg, which can be done by simply cracking the egg and carefully pouring the white of the egg out. Once the intact yolk is obtained it should be submerged into water to allow for easier manipulation afterwards.

Next, the membrane should be carefully cut - there are many ways that this can be done, but the best results are usually obtained with sharp scissors and a pincet. First a hole should be made into yolk carefully to serve as a starting point for cutting. The hole should be ideally on the top of the yolk to prevent excessive spilling which quickly clouds the water and makes any further operations much more difficult.

Starting from this hole a line can be cut in any direction. The easiest way seems to be by making (almost) 180° circle cut from the side and allowing for yolk to "open" and spill to the opposite side. It is worth noting that movements should be relatively slow and gentle, since we don't want to spill the yolk and cloud the water before finishing the cutting.

Once the desired cut is made the yolk will spill out. Careful exchange of water either with syringe or any other method will gradually return the visibility in the media to initial levels. The purification should be done gently, though, as membrane may very well rupture or be flushed out with exiting water. It is advisable to hold membrane gently with pincers where membrane is the strongest i.e. on the "poles" or on "chalaza", the white membrane structure on the "poles" of the egg yolk. Chalaza can be seen on the figure 3.

Figure 3: A submerged egg yolk with visible white membrane structure (chalaza).

After some purification relatively pure membranes can be obtained. White like sheet can be manipulated using pincers or any similarly shaped tool with no sharp edges.

The membrane should be cut to appropriate size (smaller is better).

Figure 4: Purified membrane in a petri dish.

"Fixation" of the membrane

After several attempts a glue was found to be a better alternative to wax. The membrane had to be held tightly against the bottom of the chamber covering the holes, so that no liquid can pass between the membrane and the chamber bottom. Since any mechanical pressure system i.e. with rubber bands pressing against the membrane would inevitably result in its penetration, a glue was used instead. Dropping melted wax seemed to be a better solution at first, since it allowed for a very fine manipulation, but after it hardened it tended to separate from the base of the chamber thus destroying its purpose. The following figure (figure 5) illustrates the crude design of the chamber:

Figure 5: A schematics of the "hardware". Into plastic cups the holes were made, which were later covered with membrane.

Tests for quality - leakiness

Each membrane was tested for leakines. Some water was placed in the chamber and a paper tissue was placed below. Upon any signs of water on the tissue the chamber was discarded.

Final setup

In the final setup two chambers were separated by glued egg yolk membrane with different orientations. Initially the outer side of the membrane (that was originally facing the white of the egg) was facing the medium with bacteria.

The overnight culture of needed strain was incubated in 1% arabinose (w/w%) and 1mM IPTG for 2 hours prior to being poured over membrane (In next experiments this time was increased to an overnight incubation). Approximately 1 mL of overnight culture was poured into upper chamber while in collection chamber there was LB media (3-4mL) with antibiotic Chloramphenicol. Previous experiments characterizing the pBAD arabinose promoter suggested that 1% arabinose was optimal for our setup.

Figure 6: As can be seen from the pictures improvisation was an important part of the experiment. Two plastic cups were cut and on the upper one holes were made with heated needle. After gluing the egg yolk membrane and testing for leakage, the setup was placed on the petri dish to minimize further contamination. In the presented picture the violet red colour comes from the mCherry fluorescent protein that was produced by strain having functional injectosome and chaperone but no secretion signal sequence.

Results

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.

Experiments with biomimetic membrane

1. Introduction

The membrane is an important component of our protein production system as it separated the bacterial culture from the protein collection chamber. In the final protein production chamber the membrane would be an artificial biomimetic membrane. In this experiment, we investigated the use of a biomimetic membrane using supported lipid bilayers and liposomes.

2. Aim

The first aim of this part of our project was to investigate if the SIEC strain (i.e. the E. coli strain expressing the injectosome) would bind to a biomimetic membrane and secrete proteins through it.

The second aim was to investigate if the secretion signal, Map20, (biobrick ID) would increase the amount of protein secreted into liposomes.

3. Membrane composition

A mixture containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) was used for the supported lipid bilayer and liposomes in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl). The ratio of the membrane components was inspired from Chatterjee and colleagues [1] who showed that EspD, which is required for formation pores in the host membrane, spontaneously bound to lipid vesicles with this lipid composition. This lipid composition is also similar to the composition of the outer leaflet of a mammalian cell plasma membrane [2]. The dye Atto655 and biotin were also included in the lipid mixtures. Atto655 was included to enable detection of the lipid bilayers and liposome by fluorescence microscopy.

References

[1] Chatterjee, A., Caballero-Franco, C., Bakker, D., Totten, S., Jardim, A. (2015) Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD. J Biol Chem. 290 (42) pp. 25579-25594.

[2] Zachowski, A. (1993) Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem. J. 294, pp. 1-14.