Testing for different buffer conditions

In order to test the feasibility of our project, we wanted to simulate the periplasmic conditions of our chassis, S. cerevisiae, and investigated the growth rates of different bacteria which we considered as promising endosymbionts. This was done by measuring the growth rates of those on purified yeast lysate with a microplate reader (Fig. 1) and by performing flow cytometry (Fig. 2).

These experiments showed promising results since several microorganisms were able to grow at rates similar to our controls in corresponding media. This led us to the improvement of the buffer conditions for the PEG mediated fusion of the cells.

All steps of the PEG fusion protocol are performed in liquid media. Therefore, it is crucial to achieve a maximal efficiency for spheroplast formation and regeneration for establishing a dependency and a production pathway.

Due to the osmotic instability of yeast spheroplasts, buffers containing 1M sorbitol showed to be most suitable to minimize cell lysis. This applies for the spheroplasting medium as well, which is composed out of 1x YNB, 2% glucose, 1x amino acids, 50 mM Hepes-KOH, pH 7.2 and 1 M sorbitol. This buffer was chosen for further procedures since it showed to be the most stable efficiency, even though the spheroplasting efficiency is lower than in 1M sorbitol.

However, the results from microscopy do not take cell lysis into account. Therefore, we also measured the optical density of yeast cells during zymolyase digestion in distilled water (Fig. 4). Since spheroplasts will undergo lysis in water, a decrease of the OD shows efficient digestion of the cell wall. By comparing the percentage of decrease in optical density to the ratio of intact cells and spheroplasts the percentage of lysed cells can be obtained.

These measurements show an efficiency for spheroplast conversion of about 50 % with a minimum of 5U Zymolyase for 5mL spheroplasts in 1M sorbitol with about 50% cell lysis. We decided to use the spheroplasting medium for further experiments, since no fluctuation regarding the detectable cell number could be obtained.

Similar experiments have been performed to check the regeneration efficiency of spheroplasts after PEG treatment. We figured that the spheroplasting medium does not meet the requirements for regeneration due to a lack of essential nutrients. The most obvious approach, using 1M sorbitol for osmotic stability in YPD instead of water, showed promising results (Fig. 5). Unfortunately, we were unable to obtain suitable microscopy images for cell counting. This was a result of occurring cell aggregates after adding PEG to the cell suspension. Hence, division and therefore growth of regenerated cells cannot be taken into account.

As expected, the regeneration medium containing 1M sorbitol in YPD showed to be most effective, since it contains all essential nutrients for yeast growth as well as an osmotically stabilizing concentration of sorbitol.

Optimization and verification of PEG-mediated cell fusion

An optimization of buffer conditions enabled us to test several variations of the originally described PEG protocols for bacterial uptake into yeast spheroplasts [1,2].

As initial setting we performed an uptake as described first [1], using calcofluor-white stained S. cerevisiae and E. coli carrying pFAB3677, a plasmid, which encodes for the mTangerine reporter protein (Fig. 6, Fig. 7).

These results were rather unsatisfying because the location of the bacteria cannot be distinguished. It is not clear whether they are inside of yeast or attached to the surface. In order to determine this, we switched from wild type CEN.PK-117 to a modified yeast constitutively expressiong mTurquoise in the cytoplasm.

This gave better results (not shown), even though the exact localization of the bacteria still remained unclear. Additionally, the cells showed to be extremely unstable which made fixation for proper microscope images rather difficult.

To obtain better microscopy images for showing a clear uptake of the bacteria, we optimized the PEG protocol further, starting with an optimization described previously [2]. Microscopy with this new attempt showed an improvement for fluorescent microscopy images (Fig. 8, Fig. 9), yet with still unclear localization. Although this has not been certain evidence, the qualitative improvement helped us define a final and optimized uptake protocol (see supplementary information). Even though only minor changes have been made to the already described optimized protocol, improvements towards the regeneration, with respect to further applications regarding dependency and production, were achieved.

To obtain certain evidence regarding the uptake and location we performed confocal microscopy. Through this we were able to make a 3D model by adding a third dimension through high resolution z-stacks (Fig. 10, Fig. 11).

This proves the uptake of bacteria into yeast spheroplasts. Comparable models could be obtained for different bacteria using PEGs of different molecular weights, respectively (Fig. 12, Fig. 13). Even though this proves the uptake, the optimization of uptake conditions was determined by trial-and-error without the possibility of obtaining data for exact quantification of protocol variations.

To obtain data regarding the quantity of uptake we performed flow cytometry directly after the applying the fusion protocol. As a control, besides E. coli and wild type yeast as well as spheroplasts, we performed the PEG fusion protocol omitting the cell wall digestion. Unfortunately, the flow cytometer could not distinguish between taken up bacteria and those that are attached to the host cell surface through the adhesive properties of PEG (Fig. 14). Hence, an exact quantification of the protocol still remains unclear.