One of the basic methodologies of modern biology is the introduction of exogenous genes and gene networks into cells to measure their properties, for example the qualitative effects of gene expression on phenotype or quantitative characteristics of regulation of a gene network. One of the standard methods for delivery and expression of exogenous genes in bacteria is molecular cloning of the genes of interest into a multi-copy plasmid or bacterial artificial chromosome (BAC), followed by transformation of this construct into the cell. After transformation, the plasmid is maintained by inclusion of a selection marker, usually genes encoding proteins conferring antibiotic resistance.
In many cases gene delivery via plasmid is sufficient for the purpose at hand. However, for some applications expression from multi-copy plasmids is not ideal, and it would be far more desirable to incorporate the construct directly into the host cell's chromosome. Currently, there are two primary technologies for chromosomal insertion in E. coli
and phage-derived methods.6,7
However, both of these methods have significant drawbacks.
Recombineering is very easy to perform and can target constructs to any desired site in the chromosome. However, the efficiency of insertion at the targeted location drops precipitously as the size of the construct increases.8
Several labs report routinely performing recombineering of fragments up to 3–4 kbp.9–12
We have found recombineering of constructs this size or larger to be prohibitively difficult due to nonspecific recombination of fragments into sites other than the target,8
making identification and isolation of correct integrants a time consuming and tedious task. Nonspecific integration can be mitigated through the use of counterselection methods using e.g., galK2
however the low efficiency of integration still makes this a non-trivial endeavor and success is by no means guaranteed.
The remaining possibility for insertion is methods which apply phage-derived mechanisms.6
These methods have the benefit of being able to reliably deliver much larger DNA fragments into the chromosome than recombineering. The downside is reduced flexibility. It is only possible to incorporate DNA into endogenous phage attachment sites already present in the chromosome. Furthermore, targeting different attachment sites requires the tedious construction of entirely new constructs, one for each insertion location specific to that particular phage attachment site.
To circumvent these limitations, we have developed a new standardized method for the integration of large constructs into the E. coli
Our system combines the advantages of recombineering and phage-based methods: the ability to insert very large constructs, and the ability to target the insertion specifically to any location in the chromosome. The system is very easy to use and effective, while also being extremely flexible.
The system uses three plasmids, shown in , and the strategy (modified to illustrate exact integration—see below) is shown in . The helper plasmid, pTKRED, carries a gene encoding the homing endonuclease I-SceI, as well as genes encoding the recombinogenic λ-Red proteins3
and RecA. The Landing Pad plasmid pTKS/CS carries a tetracycline resistance gene, tetA
, flanked by I-SceI restriction sites and novel 25 bp sequences we refer to as “Landing Pad Regions”, or LPs. These LPs were designed to be unique to the E. coli
genome, and ultimately serve as the recombination target for insertion of the large construct. This construct, encompassing the LPs, I-SceI recognitions sites, and tetA
gene, is amplified by PCR and recombineered using standard methods5
into the desired insertion site.
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 (more ...)
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 (more ...)
Next, a donor plasmid, pTKIP, is transformed into the cell. This plasmid carries the insertion fragment, which is flanked on either side by LPs and I-SceI restriction sites. Upon induction of I-SceI and λ-Red expression from the helper plasmid, both the insertion fragment and the Landing Pad are excised from their host molecules, and λ-Red mediates homologous recombination between the fragment and chromosomal LP regions. The fragment is therefore integrated into the chromosome, repairing the chromosomal breaks induced by excision of the landing pad. The helper plasmid pTKRED, which contains a temperature sensitive pSC101 replication origin, can then be cured from the cell by growth at nonpermissive temperature. The end result is a bacterial strain in which the construct has been successfully inserted into the desired location in the chromosome.
As a demonstration, we have used this method to integrate the entire lac operon, including the genes lacI, lacZ, lacY and lacA, as well as their promoters and terminators, into the four novel locations in the E. coli chromosome indicated in . The insertion was performed once in a given strain, generating a set of bacterial strains with the lac operon integrated into one of the four different chromosomal positions. An agarose gel of PCR products verifying insertion into the indicated sites is shown in . To simplify selection of successful integrants, the fragment also contained the kanamycin resistance gene neo (see ), yielding a fragment size of ~9 kbp. As far as we are aware, site specific insertion of such large constructs is impossible with any other existing technology.