High-efficiency Scarless Genetic Modification Method in

Escherichia coli without Counterselection

INTRODUCTION

Genetic modifications of bacteria chromosomes are important for both cognitive and applied research.
In this study, a new, two-plasmid method for unmarked genetic modification of E. coli was established. The method uses ?-Red recombination and I-SceI cleavage. E. coli endogenous sequences were used as substrates for recombination and no additional segment was introduced into genome. The new method was successfully used for gene deletion and insertion, seamless gene deletion, site-directed mutagenesis, and gene replacement. The new method could be used to modify both nonessential and essential genes.

RESULTS

The scarless genetic modification strategy was based on a simple two-plasmid procedure using ?-Red recombination and endonuclease I-SceI cleavage. A fragment with I-SceI recognition sites was inserted into the target site using ?-Red recombination and positively selected by conventional ?-Red PCR targeting. I-SceI cleaved both the donor

plasmid and the chromosome. Incorporation of the insertion fragment was enhanced by the expression of ?-Red enzymes and led to integration of the donor fragment and scarless genetic modification. Generally, the strategy took about 1.5-2 weeks, with donor plasmid construction and ?-Red PCR targeting procedure performed simultaneously. We used this strategy in both the K-12 strain MG1655 and the B strain BL21(DE3). This strategy should be applicable in any E. coli strains that can be modified by site-specific ?-Red PCR targeting recombination.

Figure 1. Diagram of this method.

A: Antibiotic cassette fragment with I-SceI recognition sites integrated at a target via ? Red -mediated recombination creating an intermediate strain. B: Intermediate strain with mutation(s) and I-SceI recognition sites was transformed with donor plasmid. Expression of I-SceI induced by L-arabinose or IPTG (isopropyl
?-D-1-thiogalactopyranoside). I-SceI recognition sites in donor plasmid and chromosome were cleaved. Integration of donor fragment was mediated by ? Red recombination.
Three groups of plasmids were constructed to facilitate this method: (1) bifunctional (?-Red and I-SceI) helper plasmids, constructed from the ?-Red helper plasmid pKD46; (2) templates plasmids, as PCR templates to amplify apramycin-resistance or spectinomycin-resistance genes flanked by I-SceI recognition sites; (3) modular vectors for donor plasmids, consisted of a pBluescript II KS (-) backbone, MCS, and two I-SceI recognition sites
An apramycin-resistance gene cassette flanked by I-SceI recognition sites was integrated into the cadA locus by ?-Red PCR targeting to obtain an intermediate strain. Positive clones were screened by PCR. PCR products were digested with I-SceI, to check for functionality of the inserted I-SceI restriction sites.Truncated cadA segments with I-SceI recognition sites was cloned into donor plasmid. The donor plasmid was transformed into the intermediate strain for the final recombination step. In the recombination step, ?-Red enzymes, then I-SceI were induced, leading to cleavage of both the donor plasmid and the chromosome. Once DSBs were generated by I-SceI, recombination between the resulting donor fragment and the DSB chromosomal locus was induced by ?-Red enzymes. After
growth in IPTG or L-rhahnose to induce I-SceI expression with L-arabinose to induce expression of ?-Red enzymes, most surviving cells that were apramycin sensitive were confirmed as having the desired deletion.
Process and results for knock-in of the gdhA gene were similar to those for knocking out cadA. To demonstrate the repeatability of the protocol, we inserted the gdhA gene into the chromosome of a cadA-disrupted E. coli strain, following the procedures described above. Similar modification efficiency was obtained.
To determine if the new method could be used for seamless deletion of a selected gene, we deleted the pepD gene following a procedure similar to the process described above. An apramycin-resistance gene cassette flanked by I-SceI recognition sites was integrated into the pepD locus, replacing 600 bp in the pepD ORF.To construct a donor plasmid, upstream and downstream fragments for the pepD gene were joined and subcloned into pKSI-I. The fusion fragment was suitable for seamless deletion of pepD. The apramycin-resistance gene fragment and remaining 1.0 kb pepD fragments were replaced by â??nothingâ?. The 1.6 kb pepD ORF was seamlessly deleted from the ATG start codon to the TAA terminator codon
To determine if the new method could be used to modify an essential gene locus, we used the method to modify the metK gene. metK could not be directly deleted, but mutations can be made in its coding region. The metK gene can be replaced by a Rickettsia SAM transporter gene and the resulting cells take up extracellular SAM and survive with SAM in the medium. The intermediate strain A10 was constructed with two resistance genes with 2 pairs of I-SceI sites inserted into the loci next to metK. A donor plasmid was constructed with
mutagenesis fragments containing metK sequences with three synonymous mutations or SAM transporter gene. The plasmid was transformed into the intermediate strain A10 with the helper plasmid. The wild-type metK gene and the two resistance genes were removed from the chromosome by the I-SceI endonuclease. Subsequently, the mutation fragment or SAM transporter gene from the donor plasmid replaced the wild-type fragment via homologous recombination, introducing modifications into the metK ORF without scar fragments.
For curing plasmids, donor plasmids are efficiently cured by I-SceI cleavage, and helper plasmids can be cured by 42°C growth.
For recombination for modification and dominant marker recycling, IPTG induction of the trc promoter to stimulate I-SceI expression gave recombination frequencies as high as >80%, in our exprements. Preparatory steps followed an established ?-Red PCR targeting method. Integration frequencies were greater than 50%, depending on the size of the homologous regions and the targeted genomic locus.

DISCUSSION

Our method could be a simple and easy way to move mutations from one strain into another. For example, generating a strain with a desired phenotype generated by random mutagenesis or evolution require introducing every mutation into a wild-type strain to analyze the effect on function. Such mutations can be identified by high-throughput genome sequencing and confirmed by PCR and dideoxy chain-termination sequencing. Donor
plasmids could be constructed from the PCR products. A resistance gene cassette and I-SceI recognition sites could be integrated into the gene locus of the recipient strain by ?-Red PCR targeting. The fragment from the donor plasmid harboring the mutation could be integrated into the genome of recipient strains to replace the wild type allele.
Another advantage of our new method is that different modifications could be performed on a single intermediate strain. By changing the donor plasmid, a target gene of interest could be modified including site-directed mutatagenesis, complete deletion, or replacement by alleles or reporter genes. Any methods could be used to construct the donor plasmid, including traditional restriction cloning, site-specific mutagenesis, Gilbson assembly or artificial whole gene synthesis. The only requirement is inclusion of upstream and downstream homologous regions and I-SceI recognition sites. According to our experiments, homologous regions in 300~500bp length could benefit both plasmid construction and recombination efficiency. The designed sequence on the donor plasmid will be the exact sequence introduced in the final strains.
Our data demonstrated that the new method presented here are a useful tool for unmarked genetic modification of E. coli. This effective method can be used with both essential and nonessential genes modification, and will benefit basic and applied genetic research.
This work was supported by The National Basic Research Program (973 Program) of
China (2014CB745100, 2011CBA00800).