In this report we describe transposon-directed base-exchange mutagenesis (TDEM), an efficient and controllable method for introducing a mutation into a gene. Each round of TDEM can remove up to 11 base pairs from a randomly selected site within the target gene and replace them with any length of DNA of predetermined sequence. Therefore, the number of bases to be deleted and inserted can be independently regulated providing greater versatility than existing methods of transposon-based mutagenesis. Subsequently, multiple rounds of mutagenesis will provide a diverse mutant library that contains multiple mutations throughout the gene. Additionally, we developed a simple frame-checking procedure that eliminates nonfunctional mutants containing frameshifts or stop codons. As a proof of principle, we used TDEM to generate mutant lacZα lacking α-complementation activity and recovered active revertants using a second round of TDEM. Furthermore, a single round of TDEM yielded unique, inactive mutants of ccdB.

Introduction

Many different methods of mutagenesis have been developed to alter protein function or activity (1,2). UV (3,4,5), chemical mutagenesis (6,7), or site-directed mutagenesis (8) have traditionally been used to introduce mutations. Current mutagenesis methods can be categorized into two groups: controlled or random mutagenesis (9).

PCR-based site-directed mutagenesis (10,11) is the method of choice for controlled mutagenesis. Alanine (12,13,14,15,16) or cysteine scanning mutagenesis (17,18,19) has been widely used to investigate protein function or structure. Additionally, mutator bacterial strains (20,21,22,23), gene shuffling (24), and error-prone PCR (epPCR) (25) have been widely used for random mutagenesis. While these methods have been used for many studies, they have several disadvantages (9,26). Site-directed mutagenesis can be costly and labor-intensive, as different mutations require the use of different oligo-nucleotides, PCR, and cloning. Although random mutagenesis methods can provide mutant libraries using simple procedures, the rate and the site of mutations cannot be controlled. Therefore, there is a risk of high wild-type background and if a gene contains multiple mutations, each mutation must be individually analyzed to identify the amino acid change(s) responsible for the mutant phenotype (24). Although the random insertion and deletion (RID) method was developed to surmount these shortcomings, its use is limited to those who are technically sophisticated (27). As an alternative approach, transposons have recently been used for pentapeptide scanning (28) and linker scanning mutagenesis (29), but these methods are also limited and not appropriate for protein evolution because they only insert extra sequences into the target gene.

With an optimal mutagenesis method, it should be possible to (i) control the number of mutations per round of mutagenesis and the sequence to be inserted or substituted, (ii) have low wild-type background, and (iii) have flexibility so that different types of mutations—including insertions, deletions, or substitutions—can be obtained. None of the currently available mutagenesis methods satisfies all of these requirements. In order to achieve these characteristics, we developed transposon-directed base-exchange mutagenesis (TDEM), which can substitute up to 11 bases of the target gene with a predetermined sequence of any length into a site selected by the random integration of a transposon (30,31). TDEM introduces a mutation into a single site of the target gene so that the diversity of the mutant library can be easily evaluated. Furthermore, because it can substitute ≥3 bases, TDEM can be used for amino acid scanning mutagenesis using up to 3 consecutive amino acids providing a novel approach for directed evolution.

As a proof of principle, we used TDEM to substitute 3 random bases of the target genes lacZα (α fragment of β-galactosidase) and ccdB (controller of cell division protein B) with three bases determined by the mutation insert (MI). In both target genes, we showed that (i) one round of TDEM generates a mutation at a single site with no wild-type background, (ii) TDEM can be used to generate a predetermined 3-base substitution at a random site, and (iii) repeated rounds of TDEM accumulate mutations within the gene. Therefore, TDEM exhibited characteristics of both controlled and random mutagenesis. Thus, we propose that the use of TDEM will dramatically facilitate structural studies and directed evolution of proteins.

Materials and methods

Plasmid constructs and transposons

The pCompact-Kana vector was constructed by ligating an origin of replication with kanamycin resistance gene (Supplementary Figure 1 and Supplementary Table 1). All inserts were cloned into pCompact-Kana, which had been amplified by PCR with primers Kan-up and Ori-lo (Supplementary Table 1) to make a linear plasmid. The detailed methods are in the Supplementary Materials.

Transposition

For TDEM of lacZα (150 bp), transposition was carried out using commercial Tn7 transposase (TnsABC^* transposase; New England Biolabs, Ipswitch, MA, USA) and Mu-transposase (Finnzymes, Espoo, Finland) according to the manufacturers’ protocol. The mixture of Tn7 and Mu transposition reactions was used in order to increase the spectrum of possible mutations. For TDEM of ccdB (300 bp), N-terminally deleted MuA prepared in the laboratory was used in the modified buffers that altered site preference (A: 25 mM Tris-Cl (pH 7.0), 5 mM MgCl₂, 0.05% (v/v) Triton X-100, 10 mM NaCl and 40% (v/v) glycerol; B: same as A without NaCl; C: same as A with 15% (v/v) DMSO; and D: same as A with 10% (v/v) glycerol) (32) and transposition reactions carried out using these buffers were mixed. Tn7 was not used for ccdB mutagenesis because it showed batch to batch variation in enzyme activity. Following transposition, the DNA mixture was electrotransformed into Escherichia coli. The transformants were inoculated into LB broth containing 50 µg/mL kanamycin and 3 µg/mL chloramphenicol and cultured overnight at 37°C with shaking (Excella E24, New Brunswick Scientific, Edison, NJ, USA). All transposition reactions in this study yielded >20,000 transposition events. The plasmid DNA from the pool was harvested and digested with the restriction enzymes that flank the target gene: NcoI/AgeI (New England Biolabs) for lacZα or BsaI (New England Biolabs) for ccdB. The target gene carrying the transposon was purified from agarose gels after electrophoresis, ligated back into the parental vector digested with compatible restriction enzymes and electrotransformed again yielding ~1×107 colonies. This step eliminated plasmids having the transposon in the vector sequence and resulted in a plasmid population with the transposon only in the target gene.