Assembly of large genomic segments in artificial chromosomes by homologous recombination in Escherichia coli - PubMed (original) (raw)

Assembly of large genomic segments in artificial chromosomes by homologous recombination in Escherichia coli

M Sosio et al. Nucleic Acids Res. 2001.

Abstract

We developed a method for the reconstruction of a 100 kb DNA fragment into a bacterial artificial chromosome (BAC). The procedure makes use of iterative rounds of homologous recombination in Escherichia coli. Smaller, overlapping fragments of cloned DNA, such as cosmid clones, are required. They are transferred first into a temperature-sensitive replicon and then into the BAC of choice. We demonstrated the usefulness of this procedure by assembling a 90 kb genomic segment into an E.coli-STREPTOMYCES: artificial chromosome (ESAC). Using this procedure, ESACs are easy to handle and remarkably more stable than the starting cosmids.

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Figures

Figure 1

Figure 1

Starting genomic segment and constructs required for assembly. (A) The 90 kb P.rosea genomic fragment and the three cosmids pRP16, pRP31 and pRP58. Fragments A_–_D are color-coded. (B) The in vitro generated plasmids pMAB1, pMBC1, pMCD1 and pPAD1. (C) Scheme of cointegrate formation and resolution between each cosmid and the cognate ts construct. The interrupted empty bars designate the genomic segments comprised between fragments A and B, B and C, and C and D. Chloramphenicol and kanamycin are abbreviated to Cm and Km, respectively. cat, aph and tet represent Cm, Km and tetracycline resistance genes, respectively; ts, the temperature-sensitive replication origin. _Dra_I sites in pPAD1 are indicated as D.

Figure 2

Figure 2

Strategy for assembly into an ESAC. (A) A genomic segment, covered by three overlapping cosmids, with the positions of fragments A_–_D. (B) Schematic of the recombination steps. The first crossover event between pMAB2 and pPAD1 leads to formation of the cointegrate (after recombination via fragment A), while the second crossover event (via fragment B) leads to the resolved cointegrate. The starting pPAC-S1 derivative is then enlarged in a step-wise fashion by cointegrate formation and resolution with the appropriate ts plasmids. Fragments A_–_D are color-coded as in Figure 1. Abbreviations are as in Figure 1.

Figure 3

Figure 3

Isolation and resolution of a cointegrate. (A) Selection for the pMCD1::pRP58 cointegrate at 44°C (non-permissive temperature) and resolution at 30°C to yield pMCD2. The antibiotics used for selection are indicated. _Bam_HI sites are indicated by bars. Other symbols and abbreviations are as in Figure 1. (B) Analysis of cointegrates and resolved cointegrates. The _Bam_HI profiles of pMCD2 (lane 1), of three independent pMCD1::pRP58 cointegrates (lanes 2–4) and of pRP58 (lanes 5–6) are shown. M, molecular weight marker, with relevant sizes (in kb) on the left.

Figure 4

Figure 4

Analysis of the growing ESACs. (A) Pulse field gel electrophoresis analysis of ESACs after _Dra_I digestion: pPAC-S1 (lane 1), pPAD1 (lane 2), PAD2 (lane 3), PAD4 (lane 4) and PAD6 (lane 5). M, molecular weight markers, with relevant sizes (in kb) on the left. Running conditions: 1% agarose gel in 0.5× TBE, 6–9 V/cm for 20 h at 14°C. (B) Comparisons of the _Bam_HI profiles. Lanes 1–6 contain pPAD1, PAD2, PAD3, PAD4, PAD5 and PAD6, respectively; pRP16, pRP31 and pRP58 are in lanes 7–9, respectively. M, molecular weight marker, with relevant sizes (in kb) on the left.

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