Investigation of social interactions in Bacillus subtilis population between wild type and mutant in gene amyE

V magistrski nalogi smo želeli vzpostaviti kompeticijski modelni sistem pri bakteriji Bacillus subtilis, kjer je divji tip producent zunajceličnega encima ?-amilaze, medtem ko sev z mutacijo v genu amyE encima ne proizvaja in v kokulturi z divjim tipom uporablja produkte ?-amilaze. Z gojenjem na gojišču s škrobom kot edinim virom ogljika smo ustvarili pogoje, kjer je produkcija encima ?-amilaze nujno potrebna za rast kulture. Raziskave interakcije smo se lotili na dva načina: v stresani kulturi in na trdnem gojišču. Iz poskusov s stresanimi, tekočimi kulturami smo želeli raziskati ali je strošek produkcije ?-amilaze zadosten. Ugotavljali smo ali pride do kolapsa populacije kokulture divjega tipa in mutante. Rezultati različnih poskusov nakazujejo na nizko metabolno ceno produkcijo ?-amilaze, zato ne opazimo razlik v razmerjih med sevi v kokulturah in v nobenem izmed poskusov ni prišlo do kolapsa kokulture po gojenju 100 generacij. Ker stresani pogoji niso pogosti v naravnih okoljih, smo se odločili socialno interakcijo preučiti tudi na trdnem gojišču. Ugotovili smo, da z daljšanjem medsebojne razdalje nacepitve med divjim tipom in sevom z mutacijo v genu amyE, divji tip pridobi prednost, zelo verjetno zaradi omejene difuzije in preferenčnega pridobivanja hranil. Zanimalo nas je tudi, kaj se zgodi s sevom z mutacijo v genu amyE, ko biofilm kokulture divjega tipa in mutante propade in se ustvari na svežem trdnem gojišču nov biofilm. Rezultati kažejo, da ima v novih biofilmih divji tip prednost pred mutanto, saj se ob precepitvi na sveže gojišče razdalja med divjim tipom in mutanto poveča in tako mutanta izgubi dostop do hranil.In this work we designed a competition model system for bacterium Bacillus subtilis where the wild type is the producer of extracellular enzyme α-amylase, while the non-producing amyE mutant strain exploits products of enzyme when grown in coculture with the wild type. By growing the two strains in the growth medium with starch as the sole carbon source we created the conditions where the production of enzyme α-amylase was essential for the microbial growth. In order to research the microbial social interactions we used two methods: growth in shaken liquid culture and on a solid growth medium. By using both shaken culture and solid medium we wanted to explore whether the metabolic cost of the production of α-amylase is sufficient to be observed as a lower cell concentration or whether the collapse of the population occurs when wild type and mutant strain are simultaneously inoculated. The results of our experiments suggest that the metabolic price of the production of α-amylase is low, which explains why we did not observe the difference in strain ratios of co-cultures. We also did not observe the collapse of co-cultures when grown for 100 generations in any of the experiments. As shaken conditions in nature are uncommon, we decided to extend the research on a solid growth medium. The results suggest that wild type gains advantage when we increased the distance between wild type and mutant strain, likely due to the limited diffusion and preferential nutrient acquisition. To further explore this phenomenon we incoulated fresh solid growth medium with the incolum of the of the old biofilm co-culture. Our results show the advantage of wild type to the mutant strain, as the distances between the cells at the time of the inculation on the fresh medium were now large enough to make α-amylase partially private