A place for everything: chromosomal integration of large constructs

Abstract

We have developed an easy, reliable two-step method for the insertion of large DNA fragments into any desired location in the E. coli chromosome. The method is based on the recombineering of a small (∼1.3 kbp) "Landing Pad" into the chromosome at the insertion site, to which the large construct is subsequently delivered via I-SceI endonuclease excision from a donor plasmid. To demonstrate the power of this method, we here show the insertion of a fragment containing the entire lac operon (∼9 kbp) into four predefined novel locations in the E. coli chromosome, a feat not possible with existing technologies. In addition, the chromosomal breaks induced by landing pad excision provide sufficient selective pressure that positive selection by antibiotics is unnecessary, making precise, exact insertion without extraneous sequence possible.

Keywords: chromosome modification; exact integration; homologous recombination; markerless integration; recombineering.

Figure 1

Insertion of the complete lac operon at four chromosomal locations. (A) Plasmids used in the insertion protocol. For the landing pad plasmid pTKS/CS and the donor plasmid pTKIP, red and green squares indicate the 25 bp LP regions and I-SceI restriction sites respectively. (B) The lac operon was deleted from its original location in the E. coli chromosome (green triangle) and re-inserted near the gene loci indicated in the diagram by grey circles. (C) Agarose gel of PCR products resulting from amplification using primers binding in the chromosomal regions flanking each insertion location. A negative control (−) before insertion and a positive sample (+) after integration at each locus is shown. The black arrow highlights the resulting ∼9 kbp PCR fragment indicating insertion of the operon. (D) Structure of the insertion fragment consisting of the lac operon genes lacI, lacZ, lacY and lacA, as well as their promoters and terminators. A kanamycin resistance gene, neo, was also included in this fragment for easy selection of integrants. The 25 bp Landing Pad regions (LPs) are indicated by red rectangles. The binding locations of primers in the chromosomal flanking regions used for verification of the insertion are indicated by black arrows.

Figure 2

Necessary modifications and strategy for exact chromosomal integration. (A) Modifications required for exact integration. (1) 50 bp sequences flanking the desired insertion site are identified [Flanking Region 1 (FR1) and Flanking Region 2 (FR2), black squares]. (2) Primers containing FR1 and FR2 sequences are used to amplify the landing pad from plasmid pTKS/CS, containing the tetA gene and I-SceI restriction sites (green squares). (3) The insertion fragment of the donor plasmid is amplified with primers bearing FR1 and FR2, as well as I-SceI restriction sites. This product and the donor plasmid backbone are digested with I-SceI, purified, and ligated together. (B) Strategy for exact integration. (1) The modified landing pad is recombineered into the integration site. (2) The cells are transformed with the modified donor plasmid, pTKIP. I-SceI expression is induced (green circles) and the insertion fragment is excised from the donor backbone. λ-Red expression (red circle) is induced, incorporating the fragment into the FR sites. (3) The temperature sensitive helper plasmid pTKRED is eliminated from the cell by growth at non-permissive temperature.