Rapid conversion of replicating and integrating Saccharomyces cerevisiae plasmid vectors via Cre recombinase

Abstract

Plasmid shuttle vectors capable of replication in both Saccharomyces cerevisiae and Escherichia coli and optimized for controlled modification in vitro and in vivo are a key resource supporting yeast as a premier system for genetics research and synthetic biology. We have engineered a series of yeast shuttle vectors optimized for efficient insertion, removal, and substitution of plasmid yeast replication loci, allowing generation of a complete set of integrating, low copy and high copy plasmids via predictable operations as an alternative to traditional subcloning. We demonstrate the utility of this system through modification of replication loci via Cre recombinase, both in vitro and in vivo, and restriction endonuclease treatments.

Keywords: Cre recombinase; genetic engineering; homologous recombination; molecular cloning; plasmid; replication; shuttle vector; yeast.

Figure 1

Functional maps for pDN5xx and pDN6xx series of low-copy and high-copy vectors. (A) Maps displaying consistent architectural features and specific functional differences of pDN5xx and pDN6xx families. Selected restriction enzyme cut sites and LoxP sequences flanking replication loci are displayed. (B) MCS in focus, displaying nucleotide sequence of single strand (template strand for lacZα, β-galactosidase) and unique restriction enzyme cut sites. Subscript and superscript numerals with each enzyme indicate capacity for enzyme to cut yeast selectable marker loci as indicated in legend. CEN/ARSH4, low copy yeast replication locus containing CEN6 centromeric sequence and ARSH4 ARS. 2 µ, yeast high copy replication locus including STB partitioning locus. Amp^R, ampicillin resistance gene (β-lactamase). ori, high copy E. coli origin or replication. f1 ori, f1 bacteriophage origin of replication. Plasmid loci depicted at approximate scale. Full plasmid sequences and annotated maps are available in supplementary materials.

Figure 2

Conversion of replicating, episomal vector to integrating vector via Cre recombinase. (A) Plasmid maps of low copy replicating (pJM1) and integrating (pJM3) plasmids, including relevant endonuclease enzyme cut sites and predicted restriction fragment product sizes. Expression of GFP-CPS1 is driven by the PRC1 promoter. (B) Agarose gel electrophoretic analysis of restriction digest products derived from pre-Cre-treated plasmid pJM1 and post-Cre-treated plasmid pJM3 candidates A and B. Note that 1243 and 1242 bp fragments predicted from EcoRV-PvuI double digests of pJM3 appear as a single band. Undigested and uncut plasmids show high molecular weight bands representing supercoiled, nicked, and catenated circular DNA whose gel migration should not be compared to linear ladder size standards. Unlabeled DNA ladder bands are of length halfway between neighboring labeled bands. (C) Agarose gel electrophoretic analysis of “mapping” PCRs to confirm chromosomal integration of pJM3. pJM3 was digested with SacII to integrate at chromosomal PRC1 promoter in strains SEY6210 and MBY3, generating strains JMY1 and JMY2 respectively. Chromosomal integrants were selected on media lacking uracil. Genomic DNA was extracted from candidate colonies to serve as PCR template. Upstream and downstream primer sets both employ a primer that anneals in the integrating plasmid and another that anneals in the neighboring chromosomal DNA, producing expected PCR products of 728 and 782 bp, respectively. Negative control samples used unmodified SEY6210 genomic DNA as template. (D) Fluorescence microscopy of FM 4-64-labeled, logarithmic phase yeast expressing chromosomally integrated pJM3. Cells were cultured in nonselective media prior to imaging. Scale bar = 1 µm.

Figure 3

Cre-mediated removal of 2 µ replication locus in vivo and in vitro. (A,B) Agarose gel electrophoretic confirmation of removal of 2 µ replication locus after passage of pDN624 (A) or pDN626 (B) through Cre-expressing bacterial strain N2114Sm. Each candidate represents a unique plasmid isolated from a single N2114Sm colony and subsequently transformed into and re-isolated from TOP10F’ cells. SmaI-cut (linearized) pDN624 produces predicted bands of 5693 bp and 4303 bp before and after removal of 2 µ locus, respectively. SmaI-cut (linearized) pDN626 produces predicted bands of 5792 bp and 4402 bp before and after removal of 2 µ locus, respectively. (C) Agarose gel electrophoretic confirmation of removal of 2 µ replication loci after in vitro treatment of pDN624 and pDN626 with Cre recombinase. Cre-treated samples represent polyclonal populations that include both unmodified (5693 bp for pDN624; 5792 bp for pDN626) and modified plasmids (4303 bp for pDN624; 4402 bp for pDN626). (D) Sanger DNA sequencing of Cre-treated pDN624 plasmid candidates in panels A and C confirming absence of 2 µ locus and remainder of a single LoxP site. Polyclonal in vitro Cre-treated pDN624 sample was transformed into TOP10F’ cells and plasmid candidates were purified from single colonies. Fifteen (15) in vitro candidates were pre-screened by restriction analysis to confirm linearized plasmid length consistent with removal of 2 µ locus, yielding four (4) candidates for sequencing. Unlabeled DNA ladder bands are of length halfway between neighboring labeled bands.

Figure 4

Example workflow to generate low copy, high copy, and integrating plasmids from a common precursor. (A) Workflow schematic representing modification of original low copy replicating vector by insertion of a PCR product at MCS containing SacI cut site, followed by removal of original replication locus and replacement with high copy replication locus. (B) Agarose gel electrophoresis and restriction enzyme analysis of plasmids resulting from demonstrated workflow. Observed AatII restriction fragments conform to predicted sizes: 6174 bp and 563 bp for pDN366; 6174 bp for pDN370; and 6174 bp and 1369 bp for pDN369. (C) Agarose gel electrophoretic analysis of efficiency of removal of CEN/ARSH4 replication locus and replacement with 2 µ replication locus. All samples shown were digested with AatII to linearize vector (no replication locus) or cut on either side of replication locus. * and ^ symbols indicate failed pDN369 candidates. All plasmid samples presented for analysis are monoclonal plasmid populations purified from transformed bacteria grown from single cell colony isolates. Unlabeled DNA ladder bands are of length halfway between neighboring labeled bands.