Team:UNITN-Trento/Project/Bacillus

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 it can be stored in a inactive state;
2. Bacillus subtilis is not pathogenic;
3. we have always worked only with E. coli and we thought to try out how it is to employ a new organism. It’s been really challenging but we got it!

Bacillus subtilis would be the perfect chassis for a fruit-ripening household product, that exploit ethylene (or MeSA) production upon spores activation. We have designed a B. fruity home edition that exploits this principle.
To achieve this goal we started working with EFE, a ethylene forming enzyme from Pseudomas Syringae pv. phaseolicola (BBa_K1065002), which were inserted into pSBBs0K-Pspac (IPGT inducible) and pSBBs4S-Pxyl (xylose inducible), two biobrick plasmids designed for B. subtilis by the iGEM 2012 LMU Munich team (please note that we used a new functional version of these plasmids, that were sent to us from LMU Munich). Cloning of BBa_K1065203 The integrative plasmid pXyl was digested prior transformation in minimal media and the correct integration of the insert into B. subtilis genome was confirmed with the threonine assay. Figure 1: transformation of BBa_1065203 in B. subtilis. Transformation of the integrative vector pXyl carrying the EFE gene was achieved by digesting the plasmid with ScaI to obtain a linear DNA (left panel) which was then transformed into B. subtilis 168 using minimal medium. Correct integration was confirmed with the threonince test: cells that carry the insert in the proper position become auxotrophic and can not longer grow in the absence of threonine. Toxicity assay We then measured the optical density of cells induced and non induced for both contructs. Figure 2: B. subtilis str.168 cells transformed with BBa_K1065203 or BBa_K1065204 were grown until an OD=0.9 and then splitted in two samples before induction. Cells were induced with 1% xylose for BBa_K1065203 and 0.5 mM of IPTG for BBa_K1065204. In both cases the induced samples grow slightly slower than the controls. Sporulation assay Spores were obtained by growing the transformed B. subtilis str.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.