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Vrednotenje izbranih promotorjev bakterije Streptomyces rimosus s pomočjo poročevalskega gena xylE
Buzar, Ilija (Author), Petković, Hrvoje (Mentor) More about this mentor... This link opens in a new window, Paš, Maja (Co-mentor)

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Abstract
Streptomyces rimosus je ena izmed najbolj raziskanih industrijsko pomembnih streptomicet. Znana je predvsem kot proizvajalka oksitetraciklina. Za optimizacijo metabolnih poti z namenom izboljševanja donosa sekundarnih metabolitov je pomembno poznavanje regulacije izražanja genov vpletenih v te biosintezne poti. Pot etilmalonil-CoA omogoča bakteriji rast na acetatu kot edinemu viru ogljika, geni za to pot pa se skupaj z genom tetR nahajajo v operonu emc. Pri nekaterih bakterijah je znana tudi vloga regulatornega proteina TetR v tej poti, saj regulira izražanje gena ccr. V obsegu magistrske naloge smo ovrednotili vlogo tetR, ki se nahaja v emc operonu. Gen tetR smo prekomerno izrazili in testirali ali ima acetat vpliv na izražanje tega gena. V prvem delu naloge smo ovrednotili aktivnosti promotorjev PermE*, PoxyI, Phs in Ptg. Homolog gena tetR smo konstruirali tako, da smo tetR postavili pod promotor PermE*, ki se je izkazal za najmočnejšega. Nato smo tako konstruiran gen tetR klonirali v integrativni vektor pAB15. Le-tega smo nato transformirali v seve S. rimosus, v katerih se pred poročevalskim genom xylE z zapisom za encim katehol 2,3-dioksigenazo že v kromosomu nahajajo promotorske regije Pemc iz bakterij vrst Streptomyces rimosus in Streptomyces tsukobaensis. Transformirane celice smo gojili v gojiščih TSB in GOTC in s pomočjo poročevalskega gena xylE vrednotili vpliv regulatornega proteina TetR in dodanega acetata v obeh gojiščih. V obeh gojiščih nismo zaznali razlike med aktivnostjo promotorjev pri sevih ki so vsebovali Pemc brez regulatornega proteina TetR, aktivnostjo promotorjev Pemc s kloniranim genom tetR. Opazili pa smo rahlo pozitivno povečanje aktivnosti promotorjev Pemc s kloniranim genom tetR in dodatkom acetata v gojišču, vendar razlike niso bile statistično značilne.

Language:Slovenian
Keywords:aktinomicete, streptomicete, Streptomyces rimosus, EMC operon, plazmidi, tetR
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2017
Publisher:[I. Buzar]
UDC:602.6:602.3:579.873
COBISS.SI-ID:4849272 Link is opened in a new window
Views:527
Downloads:522
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Secondary language

Language:English
Title:Evaluation of selected promotors from bacteria Streptomyces rimosus with xylE reporter gene
Abstract:
Streptomyces rimosus is one of the most researched industrially important streptomycetes. It is known primarily as the manufacturer of oxytetracycline. In order to optimize the metabolic pathway to raise the yields of secondary metabolites, it is essential to know how to regulate the genes involved in these pathways. The ethylmalonyl-CoA pathway allows bacteria to grow on acetate as the sole carbon source, and the genes for this pathway together with the tetR gene are found in the emc operon. The role of regulatory protein TetR in this pathway is in some bacteria already known, as it regulates expression of the ccr gene. In the scope of the master's thesis we tested the role of tetR, which is located in the emc operon. We overexpressed tetR and tested whether acetate has an effect on the expression of this gene. In the first part of the thesis we evaluated the activities of PermE*, PoxyI, Phs and Ptg promoters. The tetR gene under the control of PermE* promoter showed the highes expression. Next, we cloned the tetR gene into the integrative vector pAB15 and transformed it into S. rimosus carrying xylE reporter gene under the control of Pemc from the bacteria Streptomyces rimosus and Streptomyces tsukobaensis. Transformed cells were grown in TSB and GOTC medium. We investigated the influence of TetR on Pemc expression in the presence and absence of acetate in the media. No difference was observed in the activity of Pemc in the presence and absence of TetR except for the slight positive increase in the activity of Pemc promoters in strains with tetR that were grown with acetate. However, this diference was not statistically significant.

Keywords:actinomycetes, streptomycetes, Streptomyces rimosus, EMC operon, plasmids, tetR

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