Team:Marburg/PEG Method

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Fusion of microorganisms

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Different approaches to create artificial endosymbiosis have been described previously. The used methods vary depending on the host organism, including microinjection, invasion or phagocytosis for mammalian cells and a polyethylene glycol (PEG) -mediated fusion of vesicle enclosed microorganisms with yeast spheroplasts [1][2]. Even though the uptake was successful, these methods have not been utilized to date.

In order to realize our idea of a mostly self-sustainable production we had to decide for an applicable uptake mechanism of the symbiont into the host organism. Using mainly S. cerevisiae and E. coli, we chose the aforementioned PEG-induced uptake due to its easy applicability for different yeast strains without further engineering.

Figure 1
Figure 1. Overview scheme for the single steps using the PEG mediated fusion protocol.

The procedure, which has been described before [2][3], has been adapted and optimized for regeneration of the S. cerevisiae cell wall in liquid media. After initial growth of S. cerevisiae host cells and desired endosymbionts to a mid-logarithmic culture, the cell wall of S. cerevisiae is digested using Zymolyase, an enzyme which cleaves the 1,3-ß-glucane bonds of the yeast’s cell wall [4]. Therefore, the yeast cells are only enclosed by their lipid membrane. Additionally, this step can be universally applied to most yeast strains since they do not vary much in cell wall components rather than in the ratio of these. The digestion of the cell wall is a necessary requirement for the next crucial step: enclosure of the chosen endosymbiont by PEG liposomes.

For the formation of liposomes under aqueous conditions, PEG of different molecular weights, ranging from 3,350 to 20,000 kDa has been used for E. coli. The molecular weight of utilized PEG can be adapted according to the size of the endosymbiont since as it correlates directly with the diameter of formed liposomes [5].

After mixture and gentle centrifugation of the cells, PEG can be added directly to the pellet. This step should be repeated several times with a dilution of the PEG concentration at every step. Next, the cells can be completely resuspended in a defined medium which enables the yeast cells to regenerate their cell wall. In order to select only those cells which have fused, a suitable selection pressure (for example a carbon source dependency for yeast and a low pH for E. coli) needs to be established.

Literature

  1. [1] Agapakis, Christina M., et al. "Towards a synthetic chloroplast." PLoS One 6.4 (2011): e18877.
  2. [2] Yamada, Takashi, and Kenji Sakaguchi. "Polyethylene glycol-induced uptake of bacteria into yeast protoplasts." Agricultural and Biological Chemistry 45.10 (1981): 2301-2309.
  3. [3] Guerra-Tschuschke, I., I. Martin, and M. T. Gonzalez. "Polyethylene glycol-induced internalization of bacteria into fungal protoplasts: electron microscopic study and optimization of experimental conditions." Applied and environmental microbiology 57.5 (1991): 1516-1522.
  4. [4] Pastor, FI Javier, et al. "Structure of the Saccharomyces cerevisiae cell wall: mannoproteins released by zymolyase and their contribution to wall architecture." Biochimica et Biophysica Acta (BBA)-General Subjects 802.2 (1984): 292-300.
  5. [5] Boni, Lawrence, et al. "Preparation of large liposomes by infusion into PEG." U.S. Patent Application No. 09/999,191.