An easy and versatile 2-step protocol for targeted modification and subcloning of DNA from bacterial artificial chromosomes using non-commercial plasmids
© Hartwich et al; licensee BioMed Central Ltd. 2011
Received: 14 October 2011
Accepted: 20 March 2012
Published: 20 March 2012
Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs). This approach requires modification and subcloning of BAC-DNA by recombineering technologies in Escherichia coli. Most current protocols rely on commercial kits or isolation of BACs, their transfer between different host strains, and their restriction.
In this report we present a 2-step protocol for efficient modification and subcloning of DNA from bacterial artificial chromosomes using the non-commercial plasmids pKM208 and pTP223, distributed from addgene.com. A targeting cassette was successfully integrated into a BAC and 42 kb of this construct were subcloned. Both a plasmid-derived substrate with longer homology arms and a PCR-generated substrate with short homology arms (50 bp) were used for recombination. pKM208 and pTP223 contain all required genes for recombineering, but differ in their antibiotic resistance genes. This makes the system independent of the selection markers on the DNA molecules targeted for recombination.
The time and cost saving protocol presented here compares favorably to currently used systems. Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb) fragments in vivo without restriction and ligation. Furthermore, both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains.
Bacterial artificial chromosomes (BACs) are low copy plasmids based on the F-plasmid of E. coli. They can carry up to 300 kb of foreign DNA. The large insert size has made BACs an important source for the generation of transgenic organisms, because they carry entire genes including their regulatory elements. This can be used to express foreign genes in a spatiotemporal pattern, which mimics endogenous gene expression [1–4]. BACs are propagated in recombination deficient E. coli strains to achieve high clonal stability and low rate of chimerism.
Methods to introduce genetic modifications into BACs such as insertions, deletions, or point mutations are primarily based on homologous recombination between the BAC and targeting molecules and do not require restriction endonucleases or DNA ligases. The most popular approach for the in vivo manipulation of DNA molecules is called recombinogenic engineering or simply recombineering [5, 6]. Two main recombineering systems are currently employed. One system is based on the proteins RecE and RecT from the prophage Rac . The second system makes use of Redα, Redβ, and Redγ, encoded by the λ phage Red operon . The exonucleases RecE and Redα generate ssDNA overhangs, to which the DNA annealing proteins RecT and Redβ bind. The interaction between RecE and RecT or their functional analogues Redα and Redβ is required to initiate recombination between short homologous regions. Finally, Redγ prevents the RecBCD-dependent degradation of the targeting DNA molecule .
Recombineering systems have been used to engineer the chromosome of E. coli[7, 8, 10–12], the chromosome of other bacterial pathogens such as Serratia, Shigella, Yersenia, Salmonella and Pseudomonas[13–17], and to manipulate BACs [18, 19]. For BAC manipulation, two major systems exist. One is the commercially available Red/ET® system from Gene bridges. The second one consists of a publically available system developed by Copeland and coworkers , and requires isolation and transformation of BACs into different host strains.
Materials and methods
Transformation of BAC hosts with Red-expressing plasmids
A 20 ml culture from a single BAC carrying host colony was grown to an OD600 nm of 0.5 in the presence of chloramphenicol (Cm) (12.5 μg/ml) on a shaker at 37°C. After centrifugation at 6,000 rpm for 10 min, cells were resuspended in 20 ml precooled deionized water. After another round of centrifugation, cells were resuspended in 50 μl precooled deionized water and electroporated with 100 ng of pKM208 (; addgene.com, plasmid 13077) or pTP223 (; addgene.com, plasmid 13263) using a Gene Pulser (Bio-Rad Laboratories, Munich, Germany). Thereafter, pKM208 electroporated cells were incubated in 1 ml LB broth for 1 h at 30°C to account for the temperature-sensitive origin of replication, whereas pTP223 containing cells were incubated at 37°C. After plating, the selective LB-agar plates were incubated at the appropriate temperatures (30°C or 37°C).
Preparation of electrocompetent cells for recombination experiments
BAC hosts carrying pKM208 were grown at 30°C in 50 ml LB broth, containing Cm (12.5 μg/ml) and Ap (100 μg/ml), whereas BAC hosts carrying pTP223 were grown at 37°C in the presence of Cm (12.5 μg/ml), kanamycin (Km) (25 μg/ml) and Tc (10 μg/ml). After they reached an OD600 nm of 0.2 - 0.3, cultures were split into two 25 ml cultures. One culture was supplemented with IPTG to a final concentration of 1 mM, whereas the other culture served as a negative control. After 1 h of shaking, cells containing pKM208 were heat-shocked for 15 min at 42°C and kept on ice to stimulate subsequent gene replacement . This step was omitted for cultures containing pTP223. After centrifugation of 20 ml culture at 6,000 rpm for 10 min, cells were resuspended in 20 ml precooled deionized water. This step was repeated once. Finally, cells were resuspended in 50 μl precooled deionized water and kept on ice until use.
Generation and of the plasmid-derived targeting cassette for modifying the BAC
The plasmid pMath5-CreERT2-Neo was constructed by cloning a PCR product containing the CreERT 2 -Neo from pl451-CreERT2 into pMECA . This CreERT 2 -Neo cassette was flanked by two homology arms derived from the Math5 gene of the BAC RP23-328P3. After validation by sequencing, a 5,107 bp long fragment, containing CreERT 2 -Neo and flanking 708 bp and 372 bp long homology arms, was released by restriction with Dra I (MBI Fermentas, St. Leon-Rot, Germany) and gel-purified using peqGOLD Gel Extraction Kit (Peqlab, Erlangen, Germany).
PCR based generation of a pBR322-derived targeting cassette for subcloning
To generate the targeting cassette for subcloning, a PCR was performed with Phusion-Polymerase (New England Biolabs, Frankfurt am Main, Germany) using pBR322 as template and the primers pBR322-Math5-R 5'-TGTCTCCCACAGTACCTAACATAGGATCTTACAGAGTAGACACACATGAT GTTTAAACGATACGCGAGCGAACGTG-3' and pBR322-Math5-L 5'-CCTGCAGCACAGCCTCCAACAGTTTTCAGTCTACTCATCTTTCCTAGTAT GTTTAAACTTAGACGTCAGGTGGCAC-3' (an Mss I site is underlined, Math5 homology regions are in italics). After Dpn I (MBI Fermentas) restriction, the PCR product was gel-purified using peqGOLD Gel Extraction Kit (Peqlab) and eluted with deionized water.
Electroporation of targeting constructs
The gene targeting DNA (300 ng) was mixed with 50 μl of cell suspension and put into a precooled 0.1-cm electroporation cuvette and electroporated with a Gene Pulser (Bio-Rad) at 1.8 kV, 200 Ohm, 25 μF for ~ 4.5 ms. Cells were resuspended in 1 ml LB broth, incubated for 1 h at 37°C on a shaker, and then plated on selective LB-agar plates.
Screening for recombinant clones
Recombinant clones were screened by a junction PCR for probing the insertion of the CreERT 2 -Neo cassette, using the targeting cassette specific primer 5'-CTCCTGTCATCTCACCTTGC-3' and the BAC specific primer Math5_90718rev 5'-CCGGACGCATTCAACATCAC-3' (expected product size: 1.6 kb). For further validation, additional junction PCRs were performed. Primers were as follows: Math5_90718rev and 5'-GGCCCGCGCTGGAGTTTC-3' (3.9 kb), Math5_90718rev and 5'-CCACTGCGGGCTCTACTTC-3' (2.9 kb), as well as with the primers 5'-CCTGACACCCTTTTAGTTAAG-3'and 5'- CCATGAGTGAACGAACCTGGTCG-3' (2.6 kb). For gap repair mediated subcloning, DNA from positive clones was purified by alkaline lysis and analyzed by restriction with Mss I (MBI Fermentas).
Results and discussion
To establish an easy, cheap, and fast protocol for recombination-based modification of BACs, we assessed the non-commercial plasmids pKM208 and pTP223. Both plasmids have been successfully used for engineering of bacterial chromosomes [10, 12, 16], but have not yet been employed for the manipulation of BACs. We tested both plasmids, as their different resistance genes provide flexibility with respect to the selection markers on the targeting constructs and substrates.
Replacement of the Math5 ORF using pKM208
Subcloning of DNA from the BAC using pTP223
It is often advisable to use only part of a BAC to generate transgenic organisms. This will prevent expression of additional genes residing in the BAC, which would result in increased gene dose, often entailing abnormal phenotypes [22, 23]. This can be achieved by two different approaches. The desired transcriptional unit can be released using appropriate restriction enzymes. Alternatively, it can be subcloned by gap repair using pBR322 , avoiding tedious gel-purification. We therefore tested next, whether the plasmids could mediate gap repair-based subcloning. For this purpose, primers were designed with a 50 nucleotide long sequence homologous to the target sequence in the BAC, an Mss I site, and 20 nucleotides, that match to pBR322 . The Mss I sites were added for easy release of the entire insert for transgenic approaches. This time we tested pTP223 (Figure 1B).
Since spontaneous loss of pTP223 has been reported , we tested positive clones on Tc containing LB-agar. Absence of growth revealed that pTP223 was lost after recombination. This beneficial feature allows stable propagation of the constructs.
We report a protocol for the modification and subcloning of large BAC-DNA by the use of the non-commercial plasmids pKM208 and pTP223. The protocol sidesteps restriction and gel-purification of large DNA fragments. Furthermore, no BAC isolation and transformation into other strains like DY380, EL250 and EL350 is required . Both plasmids are available at addgene.com and are an example for the high potential of shared sources to establish and adapt low cost protocols for everybody's use.
Here, we used pKM208 for targeting the BAC with a plasmid-derived substrate and pTP223 for subcloning. This was done to demonstrate the potential of both plasmids to promote homologous recombination using BACs as template. In future studies, only one of the two plasmids is sufficient. The choice of the plasmid solely depends on the selection marker, which should be different from those present in the BAC and the DNA substrate for homologous recombination. The availability of two different selection markers renders the system very flexible and versatile. Finally, selection for these markers in the media will ensure recombineering potential in all cells of the culture.
HH was supported by a stipend from the PhD program Hearing of the State of Lower Saxony. Financial support was provided by a DFG grant to HGN (No428 8/1) and the EWE Stiftung to HGN.
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