The ability to engineer plasmid constructs containing genetic elements of arbitrary complexity has transformed biology. The use of engineered plasmids has become ubiquitous as a way to controllably express and study genes and gene networks, and has proven to be an indispensable tool for determining the function of many gene products. While proving extraordinarily useful, problems with copy number, DNA size and stability often arise. For example, plasmids and bacterial artificial chromosomes (BACs) are generally maintained in multiple copies, with copy numbers ranging over several orders of magnitude (1–1000), depending on the replication origin. Average plasmid copy numbers can also be affected by the growth state of the cell (1
), and even when maintained at constant growth conditions, cell-to-cell plasmid copy number fluctuations can be substantial (2
). While the systematic variation of average plasmid copy number and copy number fluctuations have been studied extensively for a few systems, for the majority of plasmid replication origins it is unknown how copy number depends on the growth state of the cell, and how much cell to cell variation there is. This can lead to problems with the interpretation of experimental data, for example, in measurements of noise in gene expression (3
), because the magnitude and effects of these fluctuations are almost completely unknown.
Thus, it is advantageous to incorporate constructs directly into the chromosome where the construct can be stably maintained without the need for antibiotic selection. While the position of the insertion relative to the replication origin can still lead to cell-to-cell copy number variability because of multiple replication forks, this variability is systematic, well understood (4
), and can be corrected for or exploited.
Unfortunately, it remains difficult to insert large DNA segments into the Escherichia coli
chromosome. Currently, there are two main approaches to chromosomal integration: recombineering (5–11
) and phage-derived methods (12
). Recombineering is highly effective and easy to use, involving the expression of λ-Red enzymes in order to promote site-specific homologous recombination between the chromosome and a small linear polymerase chain reaction (PCR) fragment containing the desired sequence. By amplifying the linear fragment using primers, which contain 40–50-bp flanking regions homologous to the sequence of the desired insertion site, recombineering allows great flexibility in designing and choosing the chromosomal location and orientation. In addition, once the construct has been created, it can be inserted into various locations by designing new primers with the appropriate homology regions. Despite these advantages, recombineering in E. coli
suffers from several shortcomings. For large fragments, it becomes increasingly difficult to generate PCR product in sufficient quantity, and the increased size of these fragments makes transformation and integration significantly less efficient. As a representative example, the number of recombinants we obtain when deleting lacZ
with progressively larger PCR fragments bearing 50-bp homology extensions is illustrated in (6
). Other laboratories have reported the successful and reliable integration of fragments up to ~3.5 kb (13–16
). In addition, integration efficiency can be enhanced by another order of magnitude by including homology regions 1 kb or larger (7
). However, this generally requires the engineering of plasmid constructs bearing unique homology arms for each individual insertion fragment or location. Further restricting this approach is the general requirement to include an antibiotic marker on the inserted fragment to allow for the selection of successful integrants, occupying valuable real estate on the recombinant fragment. Because of these limitations, the insertion of large fragments into specific sites on the chromosome remains a non-trivial task.
Figure 1. Representative recombineering efficiency as a function of insert size. 1000–4500-bp inserts containing the neo gene and bearing 50-bp flanking homology regions were inserted into the lacZ gene of strain K-12 MG1655 pTKRED via the method of Datsenko (more ...)
An alternative approach uses phage-integration systems to facilitate the insertion of synthetic constructs into the chromosome (12
). Here, the donor plasmid contains a phage-specific attachment site (attP
), which, when transformed into a host cell expressing the appropriate phage integrase enzyme, is integrated into complementary phage attB
attachment sites in the chromosome. These phage-based systems have many advantages: they are highly efficient (17
), and, in some instances, when the appropriate phage xis
enzyme is expressed, the constructs can also be easily removed (12
). Perhaps the greatest advantage of the phage-based systems is that there is effectively no limit to the size of the fragment that can be inserted at the attachment site. However, these approaches also have many disadvantages. Chief among these is the requirement for unique constructs to insert the same fragment into multiple different locations, since for each desired insertion location a new construct must be made bearing the required phage attP
site. In addition, as the phage systems currently in widespread use in E. coli
utilize endogenous chromosomal attachment sites, flexibility in choosing the insertion location is drastically reduced.
Recently, several groups have exploited the fact that double-strand DNA breaks stimulate in vivo
recombination, thereby facilitating the high-throughput construction of plasmid libraries [MAGIC (18
)], the subcloning of large fragments into BACs [ALFIRE (19
)], or the recombination of short DNA fragments to introduce or repair mutations within the chromosome [gene gorging (20
)]. These techniques utilize the yeast mitochondrial homing endonuclease I-SceI to introduce double strand breaks in the donor and/or recipient DNA molecule to enhance site-specific recombination. As the large 18-bp I-SceI recognition site does not exist naturally within the E. coli
chromosome, introduction and cleavage of the recognition site at the desired location enhances site-specific recombination by several orders of magnitude (18
) without any additional chromosomal damage.
Here, we describe a method for the chromosomal insertion of constructs that circumvents the limitations on insert size and location described above. To accomplish this, the cell is first transformed with a helper plasmid, pTKRED, harboring genes encoding the λ-Red enzymes, I-SceI endonuclease, and RecA. λ-Red enzymes expressed from the helper plasmid are used to recombineer a small (1.3 kb) ‘landing pad’, a tetracycline resistance gene (tetA) flanked by I-SceI recognition sites and 25-bp landing pad regions, into the desired location in the chromosome. After tetracycline selection for successful landing pad integrants, the cell is transformed with a donor plasmid carrying the desired insertion fragment; this fragment is excised by I-SceI and incorporated into the landing pad via recombination at the landing pad regions. In this manner, very large constructs can be inserted at any desired location within the chromosome. After successful integration, the I-SceI recognition sites in both the landing pad and the inserted fragment are eliminated, allowing successive applications of this protocol without modification of the landing pad regions. The entire procedure, from start to verified product, takes ~1.5–2 weeks. This method has proven to be very easy to use and highly successful, allowing us to insert large (7 kb) fragments into several chromosomal locations without a single failure.