Isolation and use of Prevotella ruminicola TC18 plasmid pTC18 in Escherichia coli-P. ruminicola shuttle vector construction

Accetto, Tomaž (author)

A cryptic plasmid of approximately 3 kilobases named pTC18 was discovered in a ruminal Prevotella ruminicola TC18 strain and cloned into Escherichia coli. Based on pTC18, several shuttle vectors, containing Prevotella/Bacteroides tetQ selection marker and E. coli vector pUC19 inserted at two different positions in pTC18 were constructed. The shuttle vectors, protected with HaeIII methylase against the P. ruminicola 23 restriction were electroporated into P. ruminicola. Despite numerous attempts a tetracycline resistant recombinant strain 23 was not obtained. The possible causes for electroporation failure are discussed.

• Language: English
• Keywords: microbiology, anaerobic bacteria, Prevotella ruminicola, shuttler vector, rumen
• Work type: Not categorized
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Year: 2009
• Number of pages: Str. 143-146
• Numbering: Letn.94, št. 2
• PID: 20.500.12556/RUL-324
• UDC: 579
• ISSN on article: 1581-9175
• COBISS.SI-ID: 2546824
• Publication date in RUL: 11.07.2014

Record is a part of a journal

• Title: Acta agriculturae Slovenica
• Shortened title: Acta agric. Slov.
• Publisher: Biotehniška fakulteta
• ISSN: 1581-9175
• COBISS.SI-ID: 213840640

Secondary language

• Language: Slovenian

Abstract

V vampnem sevu Prevotella ruminicola TC18 smo odkrili 3 kilobazne pare dolgo plazmidno DNA, jo poimenovali pTC18 in klonirali v Escherichia coli. Na njeni osnovi smo razvili več različic prenosljivega plazmida, ki je poleg pTC18 vseboval še selekcijski marker tetQ iz sevov rodu Bacteroides in plazmidni vektor E. coli pUC19. Prenosljive vektorje smo s HaeIII metilazo zaščitili proti restrikciji v P. ruminicola 23 in jih nato poskusili vnesti v P. ruminicola 23 z elektrotransformacijo. Kljub mnogim poskusom nismo uspeli pridobiti proti tetraciklinu odpornih sevov P. ruminicola 23.

• Keywords: mikrobiologija, anaerobne bakterije, Prevotella rumiciola, prenosljivi vektor, vamp