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. 2008 Aug;36(14):e84.
doi: 10.1093/nar/gkn359. Epub 2008 Jun 21.

Rapid and efficient construction of markerless deletions in the Escherichia coli genome

Byung Jo Yu  1 , Kui Hyeon Kang, Jun Hyoung Lee, Bong Hyun Sung, Mi Sun Kim, Sun Chang Kim
Affiliations

Rapid and efficient construction of markerless deletions in the Escherichia coli genome

Byung Jo Yu et al. Nucleic Acids Res. 2008 Aug.

Abstract

We have developed an improved and rapid genomic engineering procedure for the construction of custom-designed microorganisms. This method, which can be performed in 2 days, permits restructuring of the Escherichia coli genome via markerless deletion of selected genomic regions. The deletion process was mediated by a special plasmid, pREDI, which carries two independent inducible promoters: (i) an arabinose-inducible promoter that drives expression of lambda-Red recombination proteins, which carry out the replacement of a target genomic region with a marker-containing linear DNA cassette, and (ii) a rhamnose-inducible promoter that drives expression of I-SceI endonuclease, which stimulates deletion of the introduced marker by double-strand breakage-mediated intramolecular recombination. This genomic deletion was performed successively with only one plasmid, pREDI, simply by changing the carbon source in the bacterial growth medium from arabinose to rhamnose. The efficiencies of targeted region replacement and deletion of the inserted linear DNA cassette were nearly 70 and 100%, respectively. This rapid and efficient procedure can be adapted for use in generating a variety of genome modifications.

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Figures

Figure 1.
Figure 1.
Description of rapid markerless deletion with pREDI. (A) Plasmid pREDI provides (i) arabinose-inducible (promoter = ParaB) λ-Red recombinase functions [gam (γ), bet (β), and exo] necessary for the replacement of a target genomic region with a linear DNA cassette, and (ii) rhamnose-inducible (promoter = PrhaB) I-SceI expression required for DSB-mediated markerless deletion. (B) Schematic representation of markerless deletion system with pREDI. To delete the E. coli chromosomal targeted region between homology boxes A and C, a linear DNA cassette containing a positive selective marker (CmR), a negative selective marker (sacB), a I-SceI endonuclease recognition site (S) and three homology boxes (A–C) was generated by recombinant PCR using pSCI and the E. coli genome as templates (refer to Materials and methods section). The linear DNA cassette was electroporated into pREDI-containing E. coli cells, where the cassette could replace a target genomic segment with the help of the λ-Red proteins (Red proteins) encoded by pREDI. Next, to remove the introduced selection markers, expression of the pREDI-encoded I-SceI endonuclease was induced by changing the carbon source in the media from 10 mM arabinose to 10 mM rhamnose. As a result, the chromosome was cleaved at the I-SceI endonuclease recognition site (S) present on the integrated DNA cassette, inducing the DSB repair function. Then, the DSB-mediated intramolecular recombination between the two homology arms (box C) resulted in the removal of the inserted deletion cassette, producing a clean, markerless deletion.
Figure 2.
Figure 2.
Simultaneous deletion of two nonadjacent genomic targeted regions. To simultaneously delete two separate genomic regions (b0980–b1052 and b1137–b1168), we constructed two linear DNA cassettes: (i) b0979-b1053-CmR-sacB-I-SceI-b1052 (C1), for deletion of the first target genomic region (b0980–b1052), and (ii) b1136-b1169-KmR-sacB-I-SceI-b1168 (K1), for deletion of the second target genomic region (b1137–b1168) (refer to Materials and methods section). (A) The b0980–b1052 genomic region was replaced with deletion cassette C1, generating E. coli deletion strain Δb0980–b1052::C1. Then, the b1137–b1168 genomic region was replaced with the deletion cassette K1, producing E. coli deletion strain Δb0980–b1052::C1 Δb1137–b1168::K1. The subsequent expression of the I-SceI endonuclease in the double-replaced strain resulted in the simultaneous removal of the integrated DNA cassettes, generating the E. coli Δb0980–b1052 Δb1137–b1168 markerless double-deletion strain. (B) Markerless deletion of the two targeted regions was confirmed by PCR using three pairs of primers (If1/Ir1, If2/Ir2 and MD1/2) specific to both ends of the targeted regions and two pairs of primers (b1014F/R and b1150F/R) specific to the internal genes of each targeted region. PCR primers are indicated with arrows in (A). For the sequences of the PCR primers, see Material and methods section. Lane 1 shows the multiplex-PCR results obtained with the E. coli MG1655 wild-type genome and specific primers If1/Ir1 and If2/Ir2. Lanes 2 and 3 display PCR products obtained with the E. coli Δb0980–b1052::C1 genome and the E. coli Δb0980–b1052::C1 Δb1137–b1168::K1 genome and specific primers If1/Ir1 and If2/Ir2, respectively. Lanes 4 and 5 show PCR results obtained with the E. coli Δb0980–b1052 Δb1137–b1168 genome using primers If1/MD1 and If2/MD2 after simultaneous markerless deletions were carried out. Lanes 6 and 7 indicate multiplex PCR results obtained with the E. coli MG1655 wild-type genome and the E. coli Δb0980–b1052 Δb1137–b1168 genome and two pairs of primers specific to the internal sites (genes) of two deletion regions (internal genes selected for primers: b1014 and b1150, respectively). M indicates the lane containing DNA size markers.
Figure 3.
Figure 3.
Deletion of an E. coli genomic region that contains an essential gene. Deletion of the E. coli target genomic region that contained the essential gene argS was performed with a pREDI-containing strain of E. coli. (A) To delete the b1867–b1901 targeted region, that contained the essential gene argS (b1876) (which encodes arginyl-tRNA synthetase), we generated the linear DNA cassette b1866-argS-b1902-CmR-sacB-I-SceI-b1901 (E1), which carried the argS gene, and used this reagent to replace the selected genomic targeted region with the deletion cassette E1. Markerless deletion of the introduced selection markers was carried out as described in Figure 2. (B) Correct replacement of the genomic targeted region and complete removal of the inserted deletion cassette (E1) were confirmed by PCR using a pair of primers (IE1-f and IE1-r) specific to the ends of E1, and two pairs of primers (b1871F/R and b1897F/R) specific to the internal genes flanking argS. All PCR primers are indicated with arrows in (A). For the sequences of the PCR primers, see Material and methods section. Lanes 1 and 2 show PCR products obtained with the E. coli MG1655 wild-type genome and the E. coli Δb1867–b1901::E1 genome, respectively, using specific primers IE1-f and IE1-r. Lane 3 displays PCR products obtained with the E. coli Δb1867–b1901::argS genome and pair of primers IE1-f and MD3. Lanes 4 and 5 show the multiplex-PCR products obtained with the E. coli MG1655 wild-type genome and the E. coli Δb1867–b1901::argS genome, respectively, using two pairs of primers (b1871F/R and b1897F/R) specific to the internal sites (genes b1871 and b1897, respectively) of the deleted genomic region. M indicates the lane containing DNA size markers.
Figure 4.
Figure 4.
Deletion of an E. coli genomic region that contains two essential genes. We performed deletion of an E. coli targeted region (b3130–b3163) that contained two nonadjacent essential genes, yraL (b3146) and yhbV (b3159). (A) The targeted genomic region b3130–b3163 was replaced with a linear DNA cassette, b3129-yraL-yhbV-b3164-KmR-sacB-I-SceI-b3163 (E2). Markerless deletion of the introduced selection markers was carried out as described in Figure 2. (B) Correct replacement of the target genomic region and complete removal of E2 were confirmed by PCR using a pair of primers (IE2-f and IE2-r) specific to the ends of E2 and three pairs of primers (b3138F/R, b3152F/R and b3162F/R) specific to the internal genes located in the targeted region. All PCR primers are indicated with arrows in (A). For the sequences of the PCR primers, see Material and methods section. Lanes 1 and 2 indicate PCR products obtained with the E. coli MG1655 genome and the E. coli Δb3130–b3163::E2 genome, respectively, and primers IE2-f and IE2-r. Lane 3 shows a PCR product obtained with the E. coli Δb3130–b3163::yraL yhbV genome and a primers IE2-f and MD4. Lanes 4, 6 and 8 indicate PCR products obtained with the E. coli MG1655 wild-type genome and three pairs of primers (b3138F/R, b3152F/R and b3162F/R) that were specific to internal genes in the targeted region (b3138, b3152 and b3162, respectively). Lanes 5, 7 and 9 also indicate PCR products obtained with the E. coli Δb3130–b3163::yraL yhbV genome and the same primers as in lanes 4, 6 and 8. M indicates the lane that contains the DNA size markers.
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