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Kvantifikacija odziva biosenzorja Bacillus subtilis na signalni peptid ComX
Pandur, Žiga (Author), Mandić Mulec, Ines (Mentor) More about this mentor... This link opens in a new window, Dogša, Iztok (Co-mentor)

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Abstract
V magistrskem delu smo spremljali odziv bakterije Bacillus subtilis PS-216 na signalni peptid ComX, ki sproži odziv na kvorum (zadostno število) celic. Optimizirali smo protokol za pripravo rekombinatnega peptida ComX v bakteriji Escherichia coli ED367 in ga uspeli izboljšati tako, da smo kulturo najprej namnožili do visoke celične gostote in nato v naslednji stopnji inducirali sintezo ComX z IPTG. Tako pripravljen ComX shranjen v pufru SS in BSA je bil relativno stabilen pri temperaturi 4 °C in razpolovna doba ComX je bila 100 dni. Odziv surfaktinskega promotorja (PsrfAA) na ComX smo spremljali s pomočjo reporterskega gena yfp ali gena, ki kodira -galaktozidazo. Primerjali smo odziv na ComX dveh biosenzorjev (BM1456 in BM1455), ki ne proizvajata signala in ki smo jima dodali ComX: biosenzor 1 je imel gen comQ prekinjen z antibiotičnim označevalcem; biosenzor 2 je imel gen comQ prekinjen s tehnologijo brez označevalca. Naši rezultati so potrdili, da je biosenzor 1 bolj občutljiv na ComX (k= 2,7 ± 0,2) kot biosenzor 2 (k=3,5 ± 0,2). Izboljšali smo tudi metodo za kvantitativno analizo odziva biosenzorja na ComX s konfokalno mikroskopijo na nivoju posamezne celice. Rezultati so pokazali, da se B. subtilis odziva šele po treh urah izpostavljenosti ComX, ko je populacija v eksponentni fazi rasti, odziv se poveča na prehodu v stacionarno fazo (po 4 urah) do maksimalne jakosti in je primerljiv odzivu v stacionari fazi rasti (po 6h urah inkubacije).

Language:Slovenian
Keywords:Bacillus subtilis, komunikacija med celicami, zaznavanje kvoruma, signalni peptid, ComX, mikrobiološke metode, laserska konfokalna mikroskopija
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2018
Publisher:[Ž. Pandur]
UDC:579.22/.26:579.852.1:543.456
COBISS.SI-ID:4918392 This link opens in a new window
Views:964
Downloads:483
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Secondary language

Language:English
Title:Quantification analysis of biosensor Bacillus subtilis on ComX signal peptide
Abstract:
We studied a quorum sensing (QS) response to the ComX signaling peptide in Bacillus subtilis PS-216. First of all, we optimized the protocol for synthesis of signal peptide ComX from Escherichia coli ED367 to reach bigger yield. Bacteria were firstly incubated in rich medium to reach high cell density and then followed IPTG induction of cell culture in minimal medium. Furthermore, we also checked stability of pure ComX signal peptide over time stored in SS buffer with BSA at 4 °C. ComX signal peptide in buffer was relatively stable (DT50~100 days). To study the response of B. subtilis PS-216 to ComX, we used biosensors that do not produce the peptide and carry a fluorescence reporter (yfp) fused to the surfactin promoter (PsrfAA), known to be under the QS control. We used two different mutant strains B. subtilis PS-216 to compare quorum sensing response. One strain had inactivation of comQ gene with antibiotic marker (BM1456), the other strain had deletion of comQ gene with markerless technology (BM1455). Our results show oversensitivity of biosensor 1, BM1456 (k= 2,7 ± 0,2) to biosensor 2, BM1455 (k=3,5 ± 0,2). Cell response to ComX was measured at 3 different incubation times – 3 h, 4 h, and 6 h. We observed cell response to ComX after 3 h incubation (exponential growth phase), with stronger response to ComX at late growth phase (4 h) and at stationary phase (6 h).

Keywords:Bacillus subtilis, intracellular communication, quorum sensing, signal peptide, ComX, microbiological methods, laser confocal microscopy

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