Bacillus subtilis When we first came up with the idea of B. fruity, we immediatly thought that B. subtilis was the perfect chassis for a possible marketable application:
  1. Bacillus subtilis sporulates and can be stored in an inactive state;
  2. Bacillus subtilis is not pathogenic and therefore can be used safely for food applications.
Bacillus subtilis would be the perfect chassis for a fruit-ripening household product that exploit ethylene (or MeSA) production upon spores activation. We designed a B. fruity home edition based on this principle.
To achieve this goal we started working with EFE, an ethylene forming enzyme from Pseudomas syringae pv. phaseolicola (BBa_K1065002), which were inserted into pSBBs0K-Pspac (IPGT inducible) and pSBBs4S-Pxyl (xylose inducible) These two bio-brick plasmids were designed for B. subtilis by the iGEM 2012 LMU Munich team (please note that we used the new functional versions of these plasmids that were kindly provided by LMU Munich). Cloning of BBa_K1065204 The integrative plasmid pXyl was digested prior transformation in minimal media and the correct integration of the insert into B. subtilis genome was confirmed by threonine assay.
Figure 1: transformation of BBa_1065204 in B. subtilis. Transformation of the integrative vector pXyl carrying the EFE gene was achieved by digestion of the plasmid with ScaI to obtain a linear DNA (left panel) which was then transformed into B. subtilis 168 cultured in minimal medium. Correct integration was confirmed by threonine test: cells that carry the insert in the proper position become auxotrophic and can no longer grow in the absence of threonine. Toxicity assay We measured the optical density of cells induced and non induced for both constructs.
Figure 2: B. subtilis 168 cells transformed with BBa_K1065204 or BBa_K1065203 were grown until an OD of 0.9 and then split in two samples before induction. Cells were induced with 1% xylose for BBa_K1065204 and 0.5 mM of IPTG for BBa_K1065203. In both cases the induced samples (blue) grow slightly slower than the controls (red). Sporulation assay Spores were obtained growing the transformed B. subtilis 168 cells in DSM medium, subjecting them to a heat shock at 60 °C and plating them on a preheated glass slide. Spores were visualized at the microscope. Ethylene detection Ethylene production was tested by Gas Chromatography as we previously did for BBa_K1065001. The experiment was performed both from cultures started from fresh plates and from dry spores.
We did not observe any production of ethylene after 4 hours, nor after overnight induction.
At this point we are not able to confirm that EFE was correctly expressed under these conditions but, surprisingly, induced culture had a strong smell of methane and mercapto compounds. The presence of sulfur compounds was confirmed by exposing the culture to lead acetate paper strips . Hydrogen sulfide and other mercapto compounds react with lead-acetate to form lead(II) sulfate, a black insoluble precipitate that darkens the white strip. Figure 4: detection of sulfur compounds. B. subtilis 168 cells non transformed, transformed with BBa_K1065203 and transformed with BBa_K1065204 were grown until O.D. 0.9 was reached. At this O.D. one sample was then supplemented with 1% xylose or with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples showed a darker strip indicating the presence of sulfur compounds. Non transformed cells supplemented with the inducer did not show that precipitate. Future directions For future experiments and improvement of the system we have identified additional potential drawbacks, including:
  • pXyl could be inhibited by glucose although the threonine test confirmed the correct insertion of the vector;
  • the acquisition of pSpac could not be confirmed by colony PCR yet; even if the growth of colonies in the presence of the antibiotic indicates that the episomal vector carrying EFE gene is present;
  • at present the expression of the EFE gene has not been demonstrated. We plan to perform an additional real-time PCR experiment (to assess transcription).