Small genetic elements conferring resistance to antibiotics are widely used for strain construction in bacteria. Here, we describe a gentamicin resistance cassette with properties optimized for efficient gene replacement in Escherichia coli. This cassette offers advantages in versatility and ease of use over previously described versions.
The Red recombination system of bacteriophage λ, in the absence of other components of the phage lytic cycle, promotes efficient homologous recombination between the chromosome and linear DNAs in some bacteria (1,2,3,4). We and others have generally found that minimal antibiotic resistance cassettes, consisting of one or two genes from a transposon along with closely linked regulatory sequences, work well as selectable chromosomal markers in such crosses. Table 1 shows combinations of priming sequences and templates, which we have used for gene replacements (for an example, see Reference (5). A colony of a bacterial strain bearing the transposon can serve as template in PCR with primers that amplify the cassette and have 40-base flanks targeting the cassette to a site in the chromosome of a recipient. The PCR product in each case is small (1–2 kb) and is introduced via electroporation into a Red-expressing recipient strain, in which it generates a recombinant that can be easily selected. That is, the recombinant forms a reasonably large colony on a plate of LB agar containing a high enough concentration of an antibiotic to suppress colony formation by 108 nonrecombinants, following overnight incubation at 37°C. Cassettes of this type conferring resistance to chloramphenicol, kanamycin, and tetracycline have been used widely. The bla gene of Tn3 also works well for this purpose, but is perhaps less useful due to its widespread use as a selectable marker (ampicillin resistance) on plasmid cloning vectors.
The aadA1 (spectinomycin/streptomycin resistance) gene of Tn21 and the aacC1 (gentamicin resistance) gene of Tn1696 have been developed as useful markers for other applications (6,7,8,9). We tested the suitability of cassettes, containing them for Red-mediated gene replacement, and found that both worked well. However, the gentamicin cassette suffered from two disadvantages. First, we found that cassettes lacking Tn1696 sequences downstream from the aacC1 gene conferred only weak gentamicin resistance in single copy in the chromosome. Second, the upstream regulatory sequences of aadA1 and aacC1 are nearly identical. A strain bearing aadA1 would thus contain a homology target for Red-mediated recombination involving aacC1. Both of these drawbacks also apply to the small gentamicin resistance cassette described previously by Schweizer (9).
To circumvent the disadvantages of the natural gentamicin cassette, we constructed a synthetic operon containing the aacC1 gene (Figure 1A). The aacC1 coding sequence was fused to an artificial ribosome binding sequence designed according to the scheme of Gold and Stormo (10). This construct was fused to a number of different non-E. coli promoters and inserted in the E. coli chromosome upstream of lacZ, replacing lacI and its promoter, as well as Plac and Olac. The resulting strains were tested for expression of gentamicin resistance and β-galactosidase production. The promoter producing what we deemed to be the optimum combination of effective resistance with moderate β-galactosidase was a variant of the synthetic promoter CP6 described by Jensen and Hammer (11).
Use of the gentamicin cassette is described in Protocol 1. Insertions in or replacements of the galK, lacZ, lamB, dinB, and sbcCD genes have been made with the gentamicin cassette. In all cases, correct recombinant formation was verified by PCR with outside primers (data not shown; and Kenan Murphy, personal communication). The sequence of the cassette has been deposited with GenBank® (accession no. DQ530421).
We thank Yanxia Guo, Candice Evans, Amy Silverio, and Solimar Diaz-Cintron for technical assistance and Kenan Murphy for communication of unpublished results. This research was supported by the National Science Foundation.
The authors declare no competing interests.