Abstract
We have constructed two plasmids that can be used for cloning as templates for PCR- -based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418) and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.
Highlights
Plasmid-mediated antibiotic resistance is commonly used as selection criterion for genetically engineered bacteria and fungi due to its efficiency and the availability of the respective drugs from commercial sources [1,2]
Construct structure Here we report the genetic organization and restriction map of two novel, multidrug resistant marker plasmids: pMM (5581 bp) (Fig. 1) and pMM-CEN (7556 bp) (Fig. 2)
These plasmid constructs contain four important genetic elements placed on the same DNA molecule: a dominant resistance gene for ampicillin, kanamycin and geneticin (G418 in yeast), hygromycin [11], and an ARS-CEN4 DNA module providing stable maintenance in the yeast cells
Summary
Plasmid-mediated antibiotic resistance is commonly used as selection criterion for genetically engineered bacteria and fungi due to its efficiency and the availability of the respective drugs from commercial sources [1,2]. A strict selection in combination with stable maintenance will be very advantageous when using shuttle plasmids as shuttle vectors for cloning purposes in yeast For this reason, we decided to develop a plasmid that is highly selective for both expression purposes as well as for the preparation of functional analysis constructs, allowing a substantial decrease in unwanted background resistance. We decided to develop a plasmid that is highly selective for both expression purposes as well as for the preparation of functional analysis constructs, allowing a substantial decrease in unwanted background resistance This has been achieved by combining the coding sequences for antibiotic resistance to three widely available drugs: ampicillin, kanamycin (geneticin in yeast) and hygromycin B, in a specially engineered order.
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