Protoplast experiment with plants

Aim

The aim of the experiment was to check, using onion and tobacco protoplasts and E. Coli bacteria expressing GFP, Mcherry and the injectisome, if the injectisome(T3SS) was successful in injecting proteins across a membrane.

Background and Introduction

The Idea

The inspiration for this project came from the 2011 Hokkaido iGEM team and their "injection assay using onion cells". We wanted to replicate the experiment to show that the Injectisome served its function, a proof of concept of sorts. First we were focused on using onion cells but in the process of the experiment some problems arose which led us to look to other options. We decided on that the other option should be tobacco cells.

Bacteria and reporter proteins

The E. coli strain we wanted the proof of concept for contained the injectisome, secretion signal and a reporter protein(GFP and Mcherry). We wanted to use Mcherry for the assay on the onion and GFP for the assay on the tobacco due to ease of analysis of results in a fluorescent microscope. IPTG induces the formation of the injectisome and arabinose induces the reporter protein.

Protoplasts

Tobacco protoplasts and onion protoplasts differ quite a bit. Onion cells are large and without any chlorophyll but tobacco cells are small and contain chlorophyll. Protoplasts are in essence plant cells without a cell wall. To make our protoplasts, the cells with an intact cell wall, have to be incubated with a solution with cell wall degrading enzymes, an optimal osmotic pressure and PH value, so the cells don't shrink and/or burst. The optimization of the production and handling of the protoplasts was one of the biggest barriers to overcome, during the experimental process.

The reason for making the protoplast in the first place is that a plant cell with an intact cell wall might pose a problem to the attachment of the injectisome to the cell and the injection of the protein into the cell.

Figure 1 showing experimental overview

Methods, process and results

Intro

We used the protocol from the 2011 Hokkaido iGEM team as a start point and tried to follow their protocol as best as possible. When that didn't give useful results other than burst cells, flabs of apparent onion remnants and fluorescent bacteria floating about, we had to optimise several aspects of the protocol to our needs.

Protoplast production and preperation

After trying effortlessly attaching onion cell sheets with agarose to a slide glass and incubating with an MgM-MES buffer(ph 5) with 0.4M Sorbitol and cell wall degrading enzymes to obtain protoplasts, we got the idea of making tobacco protoplasts instead and keep the onion to the side.

We then tried incubating a few pieces of a tobacco leafs (downside-down without their cuticle) in a culture plate with the same solution mentioned above. Looking at a sample of the solution after a few hours of incubation revealed burst protoplasts and other plant materials. the production of onion protoplast was tried again on glass slides and revealed no apparent degradation of the cell wall, it looked like the cells had shrunk.

After mostly getting burst or shrunk cells or protoplasts we decided to look at the osmolarity. It was decided to try out different concentrations of Mannitol in an MgM-MES buffer of PH 5. This time the onion cell sheets were treated the same way as the tobacco cells. both were put in a cell wall degrading enzyme solution in an MgM-MES buffer with different concentrations of Mannitol in a culture plate. We observed the solutions in a microscope after incubating the onion and tobacco cells for a couple of hours. This time we successfully produced protoplasts, both onion and tobacco. We decided from quantitative observations in the microscope to use 0.8M Mannitol for the onion cell sheets and 0.6M Mannitol for the tobacco leaves.

Figure 2 showing experimental setup of culture plates with plant samples and enzyme buffer

Bacteria preperation

We mostly followed the Hokkaido 2011 team's protocol in preparation of our bacteria. We incubated them overnight and induced them with IPTG and arabinose. the next day the bacteria were washed and resuspended, in the solution mentioned above(The solution differs in Osmolarity between onion cells and tobacco cells). To check that our bacteria actually emit fluorescence we looked at them in a fluorescence microscope. In the microscope we saw clearly that our strains expressing Mcherry and GFP without a signal sequence gave of clear and bright fluorescence but our strains which contained the signal sequence showed much weaker fluorescence.

Introduction: Egg membrane experiment

The final product of PharMARSy would be a device that allows for growth of bacteria in one chamber and secretion of pure protein into another. Chambers would be divided by a biomimetic membrane, which should allow for bacteria to "dock"on it and use injectosome for secretion, yet it should also be robust to prevent bacteria from crossing across to other chamber. Having bacteria residues or living bacteria in collection chamber would be very dangerous for patient (could result in sepsis) if no additional purification steps are implemented.

It is thus quite evident that membrane is probably one of the most important component of our final product.

However as we couldn't obtain satisfactory biomimetic membrane we were forced to improvise and look into what nature could offer us. One of the possibilities was to use egg yolk membrane - the biggest membrane we could obtain and robust enough for experimenting.

Figure 1: Schematic of the device on which membrane was tested.

Experiment: Hypothesis and setup

The goal was to prove that only the secreted proteins can pass the membrane and not the bacteria. We believed that bacteria would recognize the phospholipid bilayer characteristic for eukaryotic cells and secrete the protein via injectosome.

Detection of increase in fluorescence from samples taken from collection chamber would mean a successful demonstration of our idea. However the solution in collection chamber has to remain sterile, otherwise we would get false positive result. (i.e. bacteria could pass to collection chamber and start producing protein which increases the fluorescence).

The following illustration provides the somewhat simpler explanation of the setup of the experiment .

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.