Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning

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.

Keywords: DNA manipulation; functional analysis; gene knock-out; multi-marker vectors; multiple-antibiotic resistance; yeast.

Fig. 1

pMM vector map with unique restriction sites. This construct contains functional cassettes conferring resistance to hygromycin (Hyg^R), kanamycin (Kan^R), and ampicillin (Amp^R). Both, kanamycin and hygromycin are under control of the translation elongation factor (TEF) promoter and terminator. Numbers in brachets represent the position of the restriction sites

Fig. 2

pMM-CEN vector map with unique restriction sites. This vector can be used as a shuttle vector due to the presence of CEN4 and ARS elements providing stable maintenance in the buddying yeast. Analogously to the previous vector, this construct confers resistance to kanamycin (Kan^R) and hygromycin (Hyg^R) thanks to the cassettes present within its structure. Moreover, for selection in the bacterial hosts, ampicillin might be used due to the presence of the Amp^R gene. TEF=translation elongation factor. Numbers in brachets represent the position of the restriction sites

Fig. 3

Determination of minimum inibitory concentration (MIC) values with pMM-containing cells on each of the different concentrations of drugs. E. coli DH5µ strains were used for the experiments with the appropriate plasmids: pFA6a-kanMX4 for kanamycin, pUC18 for ampicillin and pAG32 for hygromycin B resistance. The spots represent growth of different dilutions of a cell culture starting with approx. 10³ cells/mL from left to right. Amp=ampicillin, Hyg=hygromycin, Kan=kanamycin, wt=wild type strains

Fig. 4

At the top of the figure, the PCR amplification of the cassette carrying the Kan^R-Hyg^R genes using the pMM template is shown. The primers used, named F (forward) and R (reverse; sequences in Table 1), are drawn in the figure as thick black arrows. At the bottom, the scheme of the translocation between chromosomes XV and IX is represented. The primers used to check the correct integration and their relative position are indicated by thin arrows with sequence numbers. The two break lines on chromosome XIV indicate that the drawing of the chromosomes is not proportional

Fig. 5

a) Colony PCR verification of translocants obtained with Kan^R-Hyg^R cassette: lane 1A: ADH1 band obtained on San1 strain. Lane 1B: PCR product obtained from San1 strain carrying the translocated chromosome. The upper band reveals integration of the cassette into chromosome XV, while the lower band is the ADH1 wild type (wt) band. Analogously, lanes 2A and 2B contain colony PCR products on the wt and translocant strain for the second translocation point (SUC2 on chromosome IX); b) CHEF gel electrophoresis of translocation involving chromosomes XV and IX. Lane 1: wt San1 strain, lane 2: San1 translocant; c) and d) Southern hybridization with DIG probe specific for kanamycin and hygromycin respectively, providing direct evidence of translocation cassette integration into the buddying yeast genome