Izražanje genov za sideroforje bakterije Escherichia coli na ravni posameznih celic

Trajkova, Marija (author), Žgur Bertok, Darja (mentor)

Postavili smo hipotezo, da se promotorja genov entC, za sintezo sideroforja enterobaktin in ybtA, za sintezo sideroforja, jersiniabaktin, izražata heterogeno oz., da manjši del bakterijske populacije izraža oba gena. V ta namen smo pripravili genski fuziji promotorja entC z genom gfp in promotorja ybtA z genom mCherry ter analizirali njuno izražanje. Genski fuziji entC-gfp in ybtA-mCherry smo konstruirali v vektorju pBAC, in ga s pomočjo transformacije vstavili v sev MG1655. Slednjega smo gojili v različnih pogojih, ter aktivnost promotorjev genov entC in ybtA analizirali s fluorescentno mikroskopijo. Pokazali smo, da se pri bakteriji Escherichia coli, sevu MG1655 obe fuziji ne izražata heterogeno, temveč homogeno v vseh celicah populacije. Aktivnost promotorja za sintezo entC smo zaznali pri celicah gojenih na minimalnem trdnem gojišču M9 z dodanim 0,25 mM 2,2-dipiridilom in glukozo, pH 7, pri 37 °C, medtem ko je bil promotor gena ybtA aktiven, v celicah, gojenih na minimalnem trdnem gojišču M9 z dodanim 0,25 mM 2,2-dipiridilom in glicerolom, pH 7, pri 30 °C. Z rastjo na hranilnem LB gojišču bakterija Escherichia coli pridobi dovolj železa, zato sinteza sideroforjev za privzem železa ni potrebna.

• Language: Slovenian
• Keywords: Escherichia coli, sideroforji, jersiniabaktin, enterobaktin, genske fuzije, heterogeno izražanje, razdelitev metabolnega dela
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: M. Trajkova]
• Year: 2019
• PID: 20.500.12556/RUL-109784
• UDC: 579.25:579.842.1/.2:577.2
• COBISS.SI-ID: 5094520
• Publication date in RUL: 08.09.2019

Secondary language

• Language: English
• Title: Expression of genes for Escherichia coli siderophores on the level of individual cells

Abstract

We hypothesized that promoters of genes entC and ybtA, for production of the siderophores enterobactin and yersiniabactin, respectively, are expressed heterogenously and that a small part of a bacterial population expresses both genes. To this end we prepared gene fusions of entC and ybtA promoters with the promoterless gfp and mCherry genes, respectively. The gene fusions were constructed on the pBAC vector and transformed into strain MG1655. Activity of the entC and ybtA promoters was analysed in bacterial cells grown under a number of conditions using fluorescent microsopy. We demonstrated that in E. coli strain MG1655, both fusions were not expressed heterogenuosly in a population of genetically identical cells. Activity of the entC promoter was detected in bacterial cells grown on solid M9 minimal medium with 0.25 mM 2.2-dipyridyl and glucose, pH 7 at 37 °C while the ybtA promoter was active in bacteria grown in M9 minimal medium, with 0.25 mM 2.2-dipyridyl and glycerol, pH 7 at 30 °C. We also conclude that when grown on rich LB medium, E. coli obtains enough iron that synthesis of siderophores for iron uptake is not required.

• Keywords: Escherichia coli, siderophores, yersiniabactin, enterobactin, fusion genes, phenotypic heterogeneity, division of labour