A recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila

doi:10.1186/1471-2180-7-12

BMC Microbiology

Volume 7

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Bockau U
Rave A
Herrmann L
Hartmann MW

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Methodology article

A recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila

Thomas Weide¹ , Ulrike Bockau²^,3 , Angelika Rave² , Lutz Herrmann²^,4 and Marcus WW Hartmann²

¹Universitaetskliniken Muenster (UKM), Abteilung für Molekulare Nephrologie, Domagkstr. 3a, D-48149 Muenster, Germany

²Cilian AG, Johann-Krane-Weg 42, D-48149 Muenster, Germany

³Institut für allgemeine Zoologie und Genetik, Universitaet Muenster, Schloßplatz 5, D-48149 Muenster, Germany

⁴Provendis GmbH, Eppinghofer Str. 50, 48468 Muelheim an der Ruhr, Germany

author email corresponding author email

BMC Microbiology 2007, 7:12doi:10.1186/1471-2180-7-12


Published:	1 March 2007

Additional files

Additional File 1:

Construction of the first donor plasmid. A: Scheme of the used modular artificial cassette K42. It allows the substitution of promoter, gene of interest and terminator sequences without losing the flanking loxP sites. B: The "floxed" artificial sequence was inserted into a pCRTOPO backbone, lacking the ampicillin resistance gene. In the next steps a chloramphenicol resistance and a sacB counter-selection cassette were added. This basic donor plasmid was used to replace the EYFP cDNA (see figure 5).

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Additional File 2:

Recombination and fragmentation of large AT-rich plasmids. This figure illustrates the undesired recombination events that lead to fragmented plasmids during the standard cloning procedure (ligation, transformation, selection and propagation) in E. coli. M: marker; 1 kb ladder (generuler, MBI Fermentas, 1–8: Analyzed clones; B/I: backbone DNA (8.4 kb) and insert DNA (ca. 1.2 kb) that was used for the ligation reaction.

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