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Naravne izolate Bacillus subtilis lahko uvrščamo v sorodstvene skupine na podlagi testa rojenja, pri katerem se filogenetsko visoko sorodni (»kin«) sevi združujejo, manj sorodni (»non-kin«) sevi pa tvorijo ob srečanju mejno linijo. Ta pojav različnega vedenja do manj sorodnih imenujemo sorodstveno razlikovanje ali sorodstvena diskriminacija. V magistrski nalogi smo raziskovali kako se sorodni (»kin«) in nesorodni (»non-kin«) sevi prostorsko razporedijo znotraj kolonije. Seve smo označili s konstitutivno izraženimi fluorescenčnimi proteini (YFP in mKate) in pri različnih razmerjih in redčitvah dveh sevov opazovali njihovo prostorsko razporeditev v koloniji z lupo z vgrajenimi fluorescenčnimi filtri. Poleg tega smo kvantificirali število celic posameznega seva v koloniji s štetjem kolonij (CFU) na plošči. Ugotovili smo, da sev PS-216 zavira rast seva PS-218 in PS-196. Za vsako izbrano kombinacijo sevov smo izračunali relativni fitnes. Ugotovili smo, da je fitnes skladen s slikami kolonij. Pri nesorodni kombinaciji je en sev prevladal nad drugim, pri sorodnih kombinacijah pa smo zaznali oba seva. Pri nizki gostoti celic, kjer imajo celice dovolj prostora in hranil, so imeli vsi sevi ne glede na izbrano kombinacijo fitnes okoli ena. Z večanjem gostote celic obeh sevov se vrednost fitnesa bolj odmika od vrednosti ena. Pri nesorodni kombinaciji je vrednost fitnesa zmagovalnega seva (PS-216) večja od ena, seva, ki zgubita v kombinaciji (PS-196 in PS-218) pa imata fitnes blizu vrednosti nič. Opazili smo, da imajo sevi, označeni s reporterskim proteinom YFP, boljši fitnes od sevov, označenih z reporterskim proteinom mKate. Ugotavljali smo tudi vpliv gostote celic na prostorsko razporeditev posameznega seva znotraj kolonije. Preučili smo vpliv razmerij med sevi in časa inkubacije na rast kolonije. Spreminjanje razmerja v prid enega seva je povečalo številčnost celic seva znotraj kolonije. Čas inkubacije vpliva samo na velikost kolonije, ne pa na končno razporeditev sevov znotraj kolonije. Natural isolates of Bacillus subtilis can be classified as related groups on the basis of a swarming test in which phylogenetically highly related (»kin«) strains are combined, and less-related (»non-kin«) strains form a borderline at the meeting. This phenomenon of different behaviors to less-related ones is called kin differentiation or kin discrimination. In the master's thesis, we investigated how the related (»kin«) and non-related (»non kin«) strains spread spatially within the colony. The strains were labeled with constitutively expressed fluorescence proteins (YFP and mKate). In different proportions and dilutions of the two strains, their spatial distribution in the colony was observed by magnifier with fluorescence filters. In addition, we quantified the number of cells in each colony with colony count (CFU) on plate. We found that PS-216 strain inhibits the growth of strain PS-218 and PS-196. Relative fitness was calculated for each selected combination of strains. We found that the fitness is consistent with colony images. In an unrelated combination, one strain prevailed over the other, and in both related combinations we detected both strains. At low cell density, where cells have enough space and nutrients, all strains had, regardless of the chosen fitness combination, about one. By increasing the density of the cells of the two strains, the value of fitness starts to move from one. In an unrelated combination, the fitness value of the winning strain (PS- 216) are greater than one, the strains that lose in combination (PS-196 and PS-218) have a fitness near zero. We observed that strains labeled with the YFP reporter protein have a better fitness than strains labeled with reporter protein mKate. We also detected the influence of cell density on the spatial distribution of a single strain within the colony. We studied the influence of strains between the strains and incubation times on colony growth. The change in the ratio in favor of one strain has increased the number of cells within the colony. The incubation time affects only the size of the colony, but not the final distribution of strains within the colony. |
