Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones

Abstract

The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome

Figure 1

The pBAC/oriV vector permitting its single-copy (SC) maintenance and, alternatively, its conditional, tightly regulated DNA amplification. This new derivative of the pBeloBAC11 vector preserves most of its original specific features, including the plasmid F-derived SC maintenance system based on the oriS–repE–parABC genes (see Kim et al. 1996), but was equipped with a second origin of DNA replication, oriV, from the broad-host-range plasmid RK2 (Stalker et al. 1981). We cloned the NotI-less oriV at the HpaI or XhoI sites, but for reasons not fully anticipated, the TrfA–oriV-directed DNA amplification (see Figs. 2 and 3) was the highest for oriV in the XhoI site. Four derivatives of pBAC/oriV have been constructed: (1) The pBeloBAC11/SceI/oriV (pJW408 = pBAC/oriV/SceI) vector with the I-SceI recognition site at the HpaI site of pBAC/oriV. (2) The pTrueBlue-BAC2/oriV (pJW406) vector, with oriV at the XhoI site. This vector features dark-blue colonies (darker than for original BACs and similarly dark as for pIndigoBAC/oriV; see below), thus allowing more accurate blue/white colony screening. It is based on pTrueBlue-BAC2 (Genomics One 1999 Catalog), which contains four additional cloning sites, as compared with pBeloBAC11, all within the specially constructed lacZα segment (Slilaty and Lebel 1998). (3) The pTrueBlue-BAC2/oriV/SceI (pJW419) vector with the I-SceI recognition site cloned into the Eco47III site of pTrueBlue-BAC2/oriV. (4) The pIndigoBAC/oriV (pJW550) vector with oriV at the XhoI site. This vector features enhanced, dark-blue-color colony screening and is based on pIndigoBAC-5 (Epicentre 2001 Catalog). Details of the construction of pBAC/oriV and their derivatives are described in Methods. [NotI] Inactivated NotI site.

Figure 2

Construction of four DH10B-based hosts carrying a tightly regulated trfA gene that supplies, but only upon induction, the TrfA replication protein. (A) A representation of an integration plasmid and of a fragment of the host genome with the attB site for site-specific recombination. Four integration plasmids carrying four various trfA copy-up mutations have been constructed (see Table 1), as described in Methods. Each integration plasmid carries a cassette consisting of araC–P_BAD fused to the specific trfA gene copy-up mutant. All integration plasmids have (1) an easily removable NotI-flanked ori of plasmid pBR322, and (2) the attP_λ site for site-specific integration into attB of the DH10B host genome, as shown below the plasmid drawing. (B) A diagram of the genomic segment of the host upon recombination of the trfA-integration plasmid. Such hosts permit conditional, tightly regulated synthesis of the TrfA protein. Experimental details on Int-mediated integration of the four plasmids into the DH10B host strains (Table 2) are described in Methods. TT1 represents the t1 and t2 terminators (both clockwise) from rrnB; TT2 represents the t_L3 (clockwise) and t_L1 (anticlockwise) terminators of phage λ.

Figure 3

Maintenance and amplification of the pBAC/oriV vector: effects of the host, glucose, and L-arabinose-induced synthesis of the TrfA protein. The DH10B host and its derivative JW366, containing the araC–P_BAD–trfA203 cassette at the λ attB site (Table 2), were transformed either with pBeloBAC11 (BAC) or pBAC/oriV (see Table 1). Transformants were grown in the Luria-Bertani medium (LB), LB + 0.2% D-glucose (G) or LB + 0.01% L-arabinose (A). After 5 h of growth, a 4.5-mL volume of each culture was centrifuged and the DNA was prepared using Wizard columns (Promega). All lanes (0.8% agarose gel) show two NcoI fragments of plasmid pBeloBAC11, either without (lanes 1, 3–5) or with inserted oriV (pBAC/oriV in lanes 2, 6–8). Successful DNA amplification is seen only in lane 8, whereas lanes 1–7 represent various controls. (Lane 1) pBeloBAC11 in the DH10B host grown in LB; (lane 2) pBAC/oriV in the DH10B host grown in LB; (lanes 3–5) pBeloBAC11 in the JW366 host grown in LB, LB + 0.2% G or LB + 0.01% A, respectively; (lanes 6–8) pBAC/oriV in the JW366 host grown in LB, LB + 0.2% G or LB + 0.01% A, respectively. Whereas 0.2% G reduces the plasmid number to one per cell (lane 7 vs. 6), induction with A amplifies DNA up to 100-fold (lane 8 vs. 6). The induced high-copy (HC) replication of pBAC/oriV provides an ample amount of vector DNA for construction of libraries.

Figure 4

Maintenance and amplification of pBAC/oriV that carries DNA inserts of various length. (A) Comparison of amplification of pBAC/oriV vector, with or without a 20-kb insert. After 5 h of growth, cells from the 4.5-mL volume of the culture were collected, and DNA was phenol-extracted, precipitated with 70% ethanol, digested with NcoI (lanes 1–3) or SalI (lanes 4–6), and run on an 0.8% agarose gel. (Lanes 1–3) Strain JW371 carrying pBAC/oriV grown in the LB medium (LB), LB + 0.2% D-glucose (G) or LB + 0.01% L-arabinose (A), respectively (two bands are analogous to those in Fig. 3); (lanes 4–6) strain JW378 carrying pBAC/oriV with the 20-kb insert grown, respectively, in LB, LB + 0.2% G or LB + 0.01% A. (B) Assessment of amplification by diluting of the amplified DNA of pBAC/oriV clones containing 40-kb or 80-kb inserts. Growth conditions, DNA analysis after SalI digestion, and abbreviations are as described for A and in Methods. Numbers below the lanes indicate the fold of DNA dilution prior to SalI digestion (results were similar for dilutions made after digestions and are not shown here). (Lane 1) Uninduced strain JW389 carrying pBAC/oriV with the 40-kb insert grown in LB + G; (lanes 2–5) induced strain JW389 grown in LB + A; (lane 6) uninduced strain JW390 carrying pBAC/oriV with the 80-kb insert grown in LB + G; (lanes 7–10) induced strain JW390 grown in LB + A. The DNA in lanes 1, 2, 6, and 7 is undiluted. In lanes 3–5 and 8–10, the DNA was diluted, as specified below the lanes.

Figure 5

Effects of L-arabinose, D-glucose, and D-fucose on the amplification of the pBAC/oriV with a 20-kb insert. (A) Induction by L-arabinose (A). Strain JW378 (pBAC/oriV + 20-kb insert) was grown in LB medium (LB) supplemented with various concentrations of A. Induction, DNA extraction, and digestion were performed as described in Methods and in the legend to Figure 4A (lanes 4–6). (Lane 1) No inducer present in LB; (lanes 2–5) LB + 0.01% A. (Lane 2) An undiluted DNA sample was run; (lanes 3–5) DNA samples were diluted 5-, 10-, or 20-fold, respectively, prior to the SalI digestion; (lanes 6–9) LB supplemented with 0.001%, 0.0002%, 0.00015%, or 0.0001% A, respectively. By comparing the DNA bands in lanes 1 and 5, we estimate that DNA amplification was ∼80-fold. (B) Inhibition of amplification by D-glucose (G). Strain, experimental design, and abbreviations are as in description of A. (Lane 1) No A added, LB + 0.2% G; (lanes 2–8) LB was supplemented with 0.01% A and with 0.2, 0.18, 0.16, 0.14, 0.12, 0.1, or 0.05% G, respectively; (lane 9) LB supplemented only with 0.01% A. (C) Inhibition of amplification by D-fucose (F). Strain, experimental panel design, and abbreviations are as in description of A. (Lane 1) LB + 0.2% G only; (lanes 2–7) LB + 0.01% A, supplemented with none, 0.0001%, 0.001%, 0.01%, 0.1%, or 0.5% F, respectively.

Figure 5

Effects of L-arabinose, D-glucose, and D-fucose on the amplification of the pBAC/oriV with a 20-kb insert. (A) Induction by L-arabinose (A). Strain JW378 (pBAC/oriV + 20-kb insert) was grown in LB medium (LB) supplemented with various concentrations of A. Induction, DNA extraction, and digestion were performed as described in Methods and in the legend to Figure 4A (lanes 4–6). (Lane 1) No inducer present in LB; (lanes 2–5) LB + 0.01% A. (Lane 2) An undiluted DNA sample was run; (lanes 3–5) DNA samples were diluted 5-, 10-, or 20-fold, respectively, prior to the SalI digestion; (lanes 6–9) LB supplemented with 0.001%, 0.0002%, 0.00015%, or 0.0001% A, respectively. By comparing the DNA bands in lanes 1 and 5, we estimate that DNA amplification was ∼80-fold. (B) Inhibition of amplification by D-glucose (G). Strain, experimental design, and abbreviations are as in description of A. (Lane 1) No A added, LB + 0.2% G; (lanes 2–8) LB was supplemented with 0.01% A and with 0.2, 0.18, 0.16, 0.14, 0.12, 0.1, or 0.05% G, respectively; (lane 9) LB supplemented only with 0.01% A. (C) Inhibition of amplification by D-fucose (F). Strain, experimental panel design, and abbreviations are as in description of A. (Lane 1) LB + 0.2% G only; (lanes 2–7) LB + 0.01% A, supplemented with none, 0.0001%, 0.001%, 0.01%, 0.1%, or 0.5% F, respectively.

Figure 5

Effects of L-arabinose, D-glucose, and D-fucose on the amplification of the pBAC/oriV with a 20-kb insert. (A) Induction by L-arabinose (A). Strain JW378 (pBAC/oriV + 20-kb insert) was grown in LB medium (LB) supplemented with various concentrations of A. Induction, DNA extraction, and digestion were performed as described in Methods and in the legend to Figure 4A (lanes 4–6). (Lane 1) No inducer present in LB; (lanes 2–5) LB + 0.01% A. (Lane 2) An undiluted DNA sample was run; (lanes 3–5) DNA samples were diluted 5-, 10-, or 20-fold, respectively, prior to the SalI digestion; (lanes 6–9) LB supplemented with 0.001%, 0.0002%, 0.00015%, or 0.0001% A, respectively. By comparing the DNA bands in lanes 1 and 5, we estimate that DNA amplification was ∼80-fold. (B) Inhibition of amplification by D-glucose (G). Strain, experimental design, and abbreviations are as in description of A. (Lane 1) No A added, LB + 0.2% G; (lanes 2–8) LB was supplemented with 0.01% A and with 0.2, 0.18, 0.16, 0.14, 0.12, 0.1, or 0.05% G, respectively; (lane 9) LB supplemented only with 0.01% A. (C) Inhibition of amplification by D-fucose (F). Strain, experimental panel design, and abbreviations are as in description of A. (Lane 1) LB + 0.2% G only; (lanes 2–7) LB + 0.01% A, supplemented with none, 0.0001%, 0.001%, 0.01%, 0.1%, or 0.5% F, respectively.

Figure 6

Effect of copy-up mutations in the trfA gene on DNA amplification. All host strains carry the same pBAC/oriV plasmid with a 108-kb DNA insert (pCG275). Growth conditions and the induction of TrfA synthesis by L-arabinose (A) are described in Methods. DNA samples prepared by phenol extraction and ethanol precipitation were digested with SmaI and run on a 0.6% agarose gel. (Lanes 1,3,5,7) LB medium (LB); (lanes 2,4,6,8) LB + 0.01% A. (Lanes 1,2) Strain JW439 containing the trfA254 mutation; (lanes 3,4) strain JW499 containing the trfA173 mutation; (lanes 5,6) strain JW500 containing the trfA171 mutation; (lanes 7,8) strain JW501 containing the trfA250 mutation. Amplifications (lanes 2,6,8) of plasmid + 108-kb insert are estimated to be up to 30-fold.

Figure 7

Amplification of the DNA of pBAC/oriV clones carrying (A,C) 108-kb or (B) 122-kb inserts of foreign DNA, when propagated in the DH10B host and in two commercial hosts, GeneHogs (Invitrogen) and Stbl2 (Life Technologies, presently Invitrogen). All three host strains contain the araC–P_BAD–trfA254 cassette at their attB_λ site. Growth conditions and induction of TrfA synthesis by L-arabinose (A) are described in Methods. DNA samples prepared by phenol extraction and ethanol precipitation were digested with SalI (A,C) or with SmaI (B) and run on a 0.6% agarose gel. (A) Amplification of pBAC/oriV carrying a 108-kb insert (pCG275) in the JW427 host (trfA254; see JW439 in Table 2). (Lane 1) LB medium (LB); (lane 2) LB + 0.01% A. (B) Amplification of pBAC/oriV carrying a 122-kb insert (pCG274) in JW480 (GeneHogs trfA254). (Lane 1) LB; (lane 2) LB + 0.01% A. (C) Amplification of pBAC/oriV carrying a 108-kb insert (pCG275) in JW526 (Stbl2 trfA254). (Lane 1) LB; (lanes 2–6) LB + 0.01% A. (Lanes 3–6) DNA preparations were diluted 1/2, 1/4, 1/10, and 1/20, respectively. Comparison of lanes 1 and 6 indicates an ∼30-fold amplification of the plasmid with the 108-kb DNA insert.