Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System
© Nafissi and Slavcev; licensee BioMed Central Ltd. 2012
Received: 2 October 2012
Accepted: 25 November 2012
Published: 6 December 2012
While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event.
We constructed a parent eukaryotic expression vector possessing a specialized manufactured multi-target site called “Super Sequence”, and engineered E. coli cells (R-cell) that conditionally produce phage-derived recombinase Tel (PY54), TelN (N15), or Cre (P1). Passage of the parent plasmid vector through R-cells under optimized conditions, resulted in rapid, efficient, and one step in vivo generation of mini lcc—linear covalently closed (Tel/TelN-cell), or mini ccc—circular covalently closed (Cre-cell), DNA constructs, separated from the backbone plasmid DNA. Site-specific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 105 fold lower viability than that seen with the ccc counterpart.
We offer a high efficiency mini DNA vector production system that confers simple, rapid and scalable in vivo production of mini lcc DNA vectors that possess all the benefits of “minicircle” DNA vectors and virtually eliminate the potential for undesirable vector integration events.
KeywordsMini DNA vectors Linear covalently closed plasmid vector DNA vector integration Non-viral gene delivery “minicircles” Bacteriophage PY54 Tel/pal recombination system Bacteriophage N15 TelN/telRL recombination system Bacteriophage P1 Cre/loxP recombination system Bacterial engineering
The utility of any gene therapy strategy is defined by its balance between safety and effectiveness. While virus-derived vectors offer exceptional potential to target and deliver DNA cargo with high efficiency into the target cell, viral strategies often suffer in their safety profiling. Recent viral gene therapy-related patient mortalities in clinical trials highlight some of the safety issues attributed to the use of viral gene transfer systems that include, but are not limited to unwanted immune responses to viral capsid proteins, regeneration of virulent viruses, and insertional mutagenesis . In contrast, non-viral strategies based on naked, lipoplexed or polyplexed plasmid DNA (pDNA) vectors generally offer safer gene therapy, vaccine design, and drug delivery approaches. Plasmid DNA vectors are relatively easy to generate and store and offer tremendous design capacity. Several major barriers need to be considered in order to develop non-viral gene delivery systems as a therapeutic product to be safely administered in vivo. A successful transgene delivery system depends on the entrance of the DNA vector into the mammalian host nucleus and expression of the encoded transgene(s). While simple in theory, several cellular barriers must be overcome in practice. While travelling in the extracellular surroundings, vectors must be bio- and immuno-compatible and avoid degradation by serum nucleases and immune detection by phagocytes. Plasma nucleases digest the unprotected DNA within just a few minutes, so DNA vectors need to rapidly cross the plasma membrane of target cells. This is further complicated by the fact that the plasma membrane is composed of dense lipoprotein barriers that intrinsically inhibit efficient DNA translocation. Strategies to overcome this barrier include complexing DNA vectors with synthetic nanoparticles to form a structure similar to the plasma membrane  or receptor-mediated endocytosis; i.e. targeted liposomes . However, while non-viral gene delivery techniques work toward efficiency of DNA delivery, they generally prove poor in the delivery of pDNA vectors to the nuclear compartment. Many techniques are currently being investigated to enhance levels of non-viral gene transfer by targeting vectors to the nucleus. These techniques include modification of plasmids with DNA nuclear targeting sequences (DTS), covalent linkage of nuclear localization signals (NLS) to the plasmid DNA constructs, and attachment of import receptors such as karyopherins, to vectors that promote uptake through the nuclear membrane pore complex (NPC) [2, 4, 5]. Modification of DNA with NLS-conjugates seems to result in highly efficient expression of linear, but not circular DNA, in combination with liposomal delivery vectors . This difference may be attributed to charge per unit ratio of linear versus supercoiled circular DNA and provides yet another intriguing opportunity for lcc vectors .
In addition to the aforementioned challenges, conventional non-viral gene delivery approaches may lead to unwanted immunological responses and oncogenesis, imparted by the presence of bacterial genetic elements in plasmid DNA constructs. These include prokaryotic origins of replication, antibiotic resistance genes, as well as high-frequency immunostimulatory CpG motifs that activate Toll-like receptors in mammalian hosts . In order to improve the immuno-compatibily and durability of plasmid DNA vectors, a new generation of DNA vectors have been constructed that exploit the bacteriophage λ integrase (Int)-attP or P1-derived Cre-loxP site-specific recombination systems to generate mini ccc DNA vectors . These “minicircles” provide safer minimized transgene vectors by removing unwanted prokaryotic elements, thus enhancing bio- and immuno-compatibility in the mammalian host . The smaller size compared to the parental plasmid backbone also confers improved extracellular and intracellular bioavailability leading to efficient gene delivery and hence, improved gene expression .
A second group of modified vectors offering great promise are linear covalently closed (lcc) plasmid DNA vectors. Aside from the obvious topological differences, lcc double-stranded DNA molecules are torsion-free as they are not subject to gyrase-directed negative supercoiling, and as such possess the properties of linear DNA . However, lcc DNA is not subject to ExoV exonuclease activity in prokaryotes due to covalent linkage of linear ends, preventing degradation of the lcc pDNA vector. Lcc DNA vectors have been constructed by various in vitro strategies including the capping of PCR products, and the “minimalistic immunogenic defined gene expression (MIDGE)” vectors. MIDGE is generated by the digestion of both prokaryotic and eukaryotic backbones after isolation of plasmid from bacterial cells, followed by ligation of the therapeutic expression cassette into hairpin sequences for end-refilling . This technology has shown promising results in various applications including the development of a Leishmania DNA vaccine  and a colon carcinoma treatment . MIDGE vectors have also demonstrated up to 17 fold improved transgene expression profile in vivo in some tissues, compared to conventional plasmid DNA vectors . Thus, lcc DNA vectors may in fact outperform their circular counterparts with respect to expression efficiency and bioavailability. However, large-scale production of lcc DNA vectors via existing multistep in vitro processes requires considerable time and financial cost.
E. coli phage N15 was the first discovered phage to exist in its lysogenic (prophage) state as a linear covalently closed (lcc) plasmid  that is actively partitioned to daughter cells . The lcc conformation is conferred by the cleaving-joining activity of the protelomerase protein (Prokaryotic Telomerase), TelN (~72 kDa), acting upon the 56 bp telRL target sequence that is entirely sufficient to confer TelN-mediated processing and linearization both in vivo and in vitro[19, 20]. Similarly, phage PY54, isolated from Yersinia enterocolitica, maintains its prophage as a linear, circularly permuted, and covalently closed plasmid with telomere hairpin ends and a genome size of 46 kb. The paralogous minimal protelomerase target site of PY54 is a 42 bp perfect palindrome that unlike N15, only partially functions in vivo in the absence of adjacent sequences . The paralogue of the N15 TelN protelomerase, Tel, encodes a 77 kDa protein with observably identical function, able to process recombinant plasmids containing the pal, 42 bp palindromic target site . The tel gene possesses 60% sequence identity to telN and the active recombinases are similar in size (~77 kDa). In addition, there is a partial homology between the 42 bp PY54 pal site and the 56 bp N15 telRL site, where the ten central palindromic nucleotides (5'-TACGCGCGTA-3') are identical . Despite obvious similarities between the two phages they are evolutionary quite distant, where N15 is more closely related to λ than to PY54. Purified TelN was shown to process circular and supercoiled plasmid DNA containing the identified target site, telRL, to produce linear double-stranded DNA with covalently closed ends. The lcc and mini lcc DNA vectors produced in vitro by recombinant TelN have been successfully applied in gene delivery experiments, and showed higher and more durable expression of the gene of interest in targeted human cells [20, 21]. In contrast, to the best of our knowledge, there are no reported applications of the Tel-pal system. Furthermore, current TelN-telRL applications are based on recombinant TelN production for in vitro lcc DNA vector generation . In this study, we report for the first time the development and characterization of an optimized in vivo mini lcc DNA production platform, exploiting the Tel-pal and TelN-telRL recombination systems. This one-step production system combines the biocompatibility benefits of “minicircles” with the transfection efficiency and safety profile of “MIDGE”.
R-cells exhibit temperature-regulated recombinase expression
Bacteria, Phage and Plasmids
Genotype or description
F-, Δ(argF-lac)169, ΔuidA4::pir-116, recA1, rpoS396(Am), endA9(del-ins)::FRT, rph-1, hsdR514, rob-1, creC510
E. coli Genetic Stock Center (CGSC) # 7838 
F-, Δ(argF-lac)169, φ80dlacZ58(M15), ΔphoA8, glnV44(AS), λ - , deoR481, rfbC1, gyrA96(NalR), recA1, endA1, thi-1, hsdR17
CGSC # 12384
F-, Δ(argF-lac)169,endA1, pir + , recA1
Gift from Dr. T. Charles; 
F', Δ(gpt-lac)0, glnV44(AS), λ - , rfbC1, gyrA96(NalR), recA1, endA1, spoT1?, thi-1, hsdR17, pWM5, F128-x
New England Biolabs
F-, λ - , galT22, IN(rrnD-rrnE)1, rph-1
CGSC # 4467;
F-, λ - , IN(rrnD-rrnE)1, rph-1
CGSC # 4474;
F-, λ - , IN(rrnD-rrnE)1, rph-1 lacZ::cat-cI857-cre (CmR)
F-, λ - , IN(rrnD-rrnE)1, rph-1 lacZ::cat-cI857-telN (CmR)
F-, λ - , IN(rrnD-rrnE)1, rph-1 lacZ::cat-cI857-tel (CmR)
Wild type (wt) (telN + , tos + )
Gift from Dr. S. Hertwig; 
wt (cre + , loxP + )
Gift from Dr. B. Funnell; 
wt (tel + , pal + )
Gift from Dr. S. Hertwig; 
attP λ integration plasmid (KnR)
cI857-pL-int Φ80 (ApR)
attP Φ80 integration plasmid (KnR)
cI857-pL-int λ (ApR)
lacZ::cat- cI857-pR-pL-cre-tL::lacZ (CmR)
lacZ::cat- cI857-pR-pL-tel-tL::lacZ (CmR)
lacZ::cat- cI857-pR-pL-telN-tL::lacZ (CmR)
pGL2-egfp switched for luc
pNN7 + SS (upstream of SV40 promoter)
pNN8-SS (2SS) (second SS downstream of SV40 polyA sequence)
Accession # AB248919 National Bioresource Project (NBRP); 
Integration of lcc DNA vector into the chromosome results in loss of cell viability
Linear covalently closed (lcc) plasmid confers reduced integration frequency
Plasmid SS (+/-)2
1.03 X 10-5
We next sought to investigate chromosomal linearization by constructing SS+ or SS— integrants using the same λ and Φ80 Int-attP plasmid integration system. Unlike the previous experiment, this time, rather than assessing the number of integrants formed in the presence of the linearizing recombinase, we first stably integrated the plasmid into the chromosome before inducing recombinase expression. Cells carrying the integrated SS+ or SS— vector were maintained under repressed tel and telN conditions (30°C) and several isolates were assessed for integration of single versus multiple copies of plasmid by PCR. In all cases, single integration events represented the majority of recombinants for both SS+ and SS— λ or Φ80 attP integrating plasmids (56.5-100%). Multiple copy integrants were not studied further and discarded.
Recombinase-mediated linearization of the chromosome results in cell killing
Integrated plasmid SS (+/−)2
Cell viability following induction3
5.7 X 10-4
5 X 10-3
1.3 X 10-4
1.1 X 10-3
Visualization of Cells upon induction
To date, mini DNA vector production has been limited to in vitro strategies, adding expense and complexity to the developmental process, particularly in scalability. The mini DNA vector production system, described here, is an in vivo platform to generate high quality bacterial sequence-free mini DNA vectors in both lcc and ccc topology. Modified mini vectors can be purified directly from our engineered E. coli cells (R-cells) using standard plasmid isolation methods, and without the need for digestion, ligation, and gel purification. To generate R-cells, we flanked the cI[Ts]857-protelomerase expression cassettes by homology to lacZ gene and recombined the cassette into recA + W3110 E. coli cells screening for Lac- colonies. R-cells were designed and optimized to chromosomally encode specific recombinases under control of a strong λ pL promoter. However, since strong promoters impart metabolic burden on the host cell, where protein production may occur at the expense of cell growth , we employed a simple thermally-regulated promoter system that circumvents any potential toxicity, metabolic stress or recombinase interference that might arise from the use of chemical-mediated induction strategies. As such we were able to optimize the production of mini DNA vectors through simple control over manufacture temperature using the λ CI[Ts]857 oL pL expression system . Various modifications to our parent vector construct by simple passaging through different R-cells was made possible by the insertion of the unique multi-target sequence (Super Sequence) described for the first time here. The Super Sequence inserts the cleave-joining multi-target site for TelN, Cre, and Flp enzymes within the non-coding regions of the 142 bp minimal sequence of pal site required for in vivo processing by Tel . The retained activity of Tel in processing this site indicates that the replacement of non-coding regions within the pal sequence of PY54 does not compromise cleave-joining activity of the Tel-pal system.
Mini linear covalently closed (lcc) DNA vectors, devoid of parental prokaryotic genetic elements were successfully generated in vivo exploiting the bacteriophage PY54-derived protelomerase recombination system. The mini vector produced via this system is a stable linear DNA expression cassette with covalently closed ends. Application of pDNA vectors in naked, lipoplexed, or polyplexed form for gene transfer conventionally employs plasmid vectors designed either to administer genes coding for therapeutic proteins, antigens, or antibodies into a given organism; or introduce a correct gene into a host cell to replace the mal- or non-functional allele . Derived from conventional plasmid DNA vectors, bacterial sequence-free mini DNA derivatives provide superior alternatives to traditional plasmid vectors, with some advancing in clinical trials . Conventional plasmid vectors carry a bacterial backbone in addition to the cistron expressing the gene of interest (GOI) in addition to necessary regulatory elements such as the promoter, enhancer, intron, and polyA terminal sequence. While necessary for amplification and maintenance in the prokaryotic host, the plasmid vector backbone carries prokaryotic genetic elements, including antibiotic resistance gene(s) and an origin of replication. It was previously shown that antibiotic resistance genes are undesirable for administration to human body due to potential adverse effects and events such as their horizontal gene transfer imparting resistance to naturally occurring mammalian host flora; and their compromising effect on GOI expression . CpG motifs, 20 times more common in prokaryotic DNA, induce polygonal B-cell activation and activate Toll-like receptors that in addition to generating potentially unwanted immunostimulatory responses, may also reduce or abrogate transgene expression [30, 31].
Bacterial sequence-free mini DNA vectors provide better bioavailability compared to conventional plasmid DNA vectors due to their smaller size and higher immuno-compatibility due to reduced or eliminated unwanted immune responses. Their smaller size confers higher transfection efficiency and higher copy numbers of the vector per unit mass, resulting in lower toxicity due to the need for less transfection reagents. Smaller pDNA vectors also better resist shear forces associated with the in vivo administration and delivery to the target site , important improvements to safety and efficiency of transgene delivery in treatment of human disease .
Conventional plasmid vectors are extra-chromosomal, circular, covalently closed, double-stranded DNA molecules that often possess elements or sequences such as viral promoters or cloned coding sequences that could subject them to unwanted recombination events . A major safety concern associated with gene delivery, whether by viral or non-viral carriers, is the potential for integration of plasmid DNA into the host chromosome. Integration events that activate proto-oncogenes and/or deactivate tumor suppressor genes may result in oncogenesis or silencing of adjacent genes . We showed here that the frequency of viable integrants of lcc pDNA vector into the circular prokaryotic E. coli genome is reduced at least five orders of magnitude compared to its ccc counterpart. The lethal effect of chromosomal disruption in E. coli was investigated by site-specific insertion of the lcc integrating pDNA vector using Int-attB systems from bacteriophages λ and Φ80. Our results linking site of chromosomal disruption to degree of lethality agree with previous work by Cui et al. () who reported that the closer the linearization of the chromosome occurred to the E. coli origin of replication, the stronger was the observed growth defect . Our microscopy results indicated a dramatic reduction in cell size and surrounding cellular debris following genome disruption, which we attribute to replication inhibition and associated cellular stress, phenotypes we are presently investigating further in bacterial and mammalian cells. The generation of filamentous cells similarly may be due to SOS-independent filamentation arising from inhibited or dramatically slowed replication .
Considering the lethal effect of lcc DNA vector integration into host chromosome, lcc based gene replacement vectors will likely provide a safer option for targeted recombination events. We report elsewhere that lcc vector integration into human cells results in chromosomal disruptions and cell death, preventing propagation of the lcc vector-integrated cell and its natural elimination from the transfected cell population. As such, mini lcc vectors potentially serve as ideal choice for knock-in and gene replacement studies in stem and other pluripotent cells for the generation of transgenic plant and animal models and regenerative medicine, avoiding damage and side effects of integration.
Mini lcc DNA vector constructs were generated in vivo exploiting the bacteriophage PY54-derived Tel-pal recombination system in a conditional recombinase expression scheme. The mini lcc DNA vectors provide a safer alternative to conventional pDNA vectors without compromising utility. In addition to a unique multi-target sequence, each vector is equipped with two SV40 enhancer sequences at the two covalently closed ends of the linear plasmids to facilitate the nuclear uptake and enhance the transfection efficiency and expression of the GOI. The production system reported here and its associated safety profile may serve as a basis for simplified, scalable, and safer DNA vector production that could drive the design of virtually limitless innovations with applications to health, agriculture, and industry.
Strains and plasmids
E. coli K-12 strains were used in the generation of all recombinant cell constructs and DH5α and JM109, in particular, were employed as hosts for plasmid constructions and amplification.
A list of bacterial and phage strains used in this study are shown in Table 1.
Construction of Recombinant Cells (R-cells)
W3110 was used for chromosomal engineering studies and in vivo recombinase expression as follows. Protelomerase coding gene tel was amplified from bacteriophage PY54 lysate using the following primers: Tel-F 5′-GCGGATCC TGGGTTACTTTAATTTGTGTGTT-3′ and Tel-R 5′-CGCTCGAG TTACTCCATATTTTCAGTCCATGCTTGT-3′ (annealing Tm 64°C). Protelomerase coding gene telN was amplified from bacteriophage N15 lysate using primers: TelN-F 5′-ATCGGATCC CGATATCCAGAGACTTAGAAACGGG-3′ and TelN-R 5′- ATATAAAGCTT CTTTTAGCTGTAGTACGTTTCCCATGCG-3′ (annealing Tm 62°C). As a positive control for in vivo production of modified DNA vectors, the recombinase encoding gene cre was amplified from bacteriophage P1rev6 lysate using primers: Cre-F 5′-GGAATTC CGGTCGCTGGCGTTTCTATGAC-3′ and Cre-R 5′-CGCTCGAG TGAATATTAGTGCTTACAGACAG-3′ (annealing Tm 66°C). Italicized regions denote restriction sites for BamH I, Xho I, Hind III, and EcoR I. PCR amplifications were conducted using Phusion Flash High-Fidelity PCR Master Mix (New England Biolabs) for 30 s at 98°C for initial denaturation, 30 cycles of 5 s at 98°C, 10 s at annealing Tm, 45 s at 72°C, and 2 min at 72°C for final extension to generate cre (1.3 kb), tel (2.1 kb), and telN (2.3 kb) fragments. Constructs were tested and confirmed by colony PCR and analytical digestion. PCR products were purified from 0.8% agarose gel (Qiagen Gel extraction kit), and digested with the listed enzymes (New England Biolabs). Recombinase genes were cloned into the MCS of the inducible prokaryotic expression plasmid vector pPL451 (Accession #AB248919) to produce pNN1, pNN2, and pNN3 vectors. pPL451 (4.2 kb) imparts temperature-regulated expression of the cloned gene via CI[Ts]857-mediated repression of the λ pL strong promoter. A list of plasmids used or constructed in this study is shown in Table 1. All primers were designed using the Gene Runner 3.01 (Hastings Software, Inc) and synthesized commercially (Sigma-Aldrich, Inc). R-cells were constructed via insertion of recombinase genes into E. coli W3110 chromosome using the pBRINT-cat integrating plasmids, which facilitate the homologous recombination and chromosomal integration of cloned sequence of interest into the lacZ gene of E. coli. For each plasmid construct encoding inducible expression of a cloned recombinase in pPL451, the cI857-P L -X-t L cassette (where X = cre, tel or telN) was amplified from the pNN1 to 3 constructs by the cI857X-F 5′-TCCCCGCGG AGCTATGACCATGATTACGAATTGC-3′, cI857telN/cre-R 5′-GGACTAGT CCCCATTCAGGCTGCGCAACTGTTG-3′, and cI857tel-R 5′-GCTCTAGA GCAGGCTGCGCAACTGTTGGGAAG- 3′ primers with Sac II, Spe I, and Xba I sites respectively. The amplified cassettes were cloned into the MCS of pBRINT (CmR) integrating plasmid to produce pNN4, pNN5, and pNN6 integrating pDNA constructs. Amplification have been performed by the Phusion Flash High-Fidelity PCR Master Mix (New England Biolabs) for 10 s at 98°C for initial denaturation, 30 cycles of 1 s at 98°C, 5 s at 68°C, 120 s at 72°C, and 1 min at 72°C for final extension to generate cI857-cre (2.8 kb), cI857-tel (3.2 kb), and cI857-telN (3.5 kb) fragments. Constructs were tested and confirmed by colony PCR and analytical digestion.
E. coli cells were grown in Luria–Bertani (LB) medium and plated on LB-Agar plates composed of 1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, pH 7.0. Antibiotics (Ab) (Sigma-Aldrich, Inc) were used at the following concentrations for the growth of cells carrying multi-copy plasmids: ampicillin (Ap, 100 μg/ml in H2O), chloramphenicol (Cm, 25 μg/ml in isopropanol), gentamycin (Gm, 15 μg/ml in H2O), and kanamycin (Km, 50 μg/ml in H2O). H2O used for dilution of primers, plasmids, antibiotics, and production of competent cells is nuclease-free sterile molecular grade water (Sigma-Aldrich, Inc). To achieve chromosomal integration of the pNN4 to 6 constructs into Rec+ W3110, a 1:100 dilution of fresh overnight cells was grown on SOB media (2.0% Tryptone, 0.5% Yeast Extract, 2.0% NaCl, 1.0% KCl, 1.0% MgCl2 , and 1.0% MgSO4) at 37°C to A600 = 0.4 - 0.6 and cells were harvested by centrifugation at 5K RPM for 5 min. Cells were washed in water three times and W3110 cells were transformed by 1 μg of pNN4, 5, or 6 integrating vectors via electroporation at 800 v. Cells were recovered in SOC (SOB with 2.0% Glucose) at 30°C for 1 h, then spread onto selective media on 12.5 μg/ml of chloramphenicol, 100 μg/ml of 5-bromo-4-chloro-indolyl-β-D-galactopyranoside (X-Gal) (Promega), and 0.1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, Inc) added to LB agar plates and incubated overnight at 30°C. To make a 50 mg/ml stock solution, 500 mg X-Gal were dissolved in 10 ml dimethylformamide. Protect plates from light. White colonies were selected and further screened for sensitivity to ampicillin and chloramphenicol. White ApS colonies indicated loss the pNN integrating plasmids after disruptive insertion of the cI857-P L -X-t L cassette into the lacZ gene, generating recombinant derivatives, W1NN, W2NN, and W3NN (Table 1). W3110 [lacZ::cat- cI857(cre/tel/telN)] recombinants were confirmed for presence of the cI857-P L -X-t L cassette and temperature-regulated, conditional expression of recombinanse, by colony PCR using Taq polymerase enzyme (NEB), sequencing (Sigma), and SDS PAGE (BioRad) at various temperatures between 30°C and 42°C. For the selection of cells carrying the antibiotic selection markers integrated in the chromosome, the following concentrations were used: Cm (12.5 μg/ml), Gm (5 μg/ml), and Km (20 μg/ml).
Construction of modified/ new generation of pDNA Vectors
The multi-purpose target site, named Super Sequence (SS), was designed to carry Cre, Flp and TelN minimal targets sites (loxP-FRT-telRL) respectively, all within the Tel 142 bp target site, pal. SS also carries a 78 bp SV40 enhancer sequence that flanks each side of the pal sequence to facilitate nuclear translocation and enhancing transfection efficiency. The SS fragment was synthesized by the GeneScript and cloned into the pUC57 by Eco RI and Hin dIII. Commercial eukaryotic expression plasmid vector, pGL2-promoter (5.8 kb) (Promega), was modified by replacement of the luc gene (1.65 kb) with egfp (790 bp) from pGFP (Clontech, Inc.) to form pNN7 (Genescript, Inc.) (4.9 kb) Next, SS was cloned immediately upstream of the SV40 promoter + intron site of pNN7 to form pNN8 (5.3 kb). Then the SS fragment was cloned downstream of the poly A site of pNN8 to form pNN9 (5.6 kb). The multi-copy pDNA vector pNN9 carries 2 SS sites that flank the egfp gene cassette and can be converted to a “minicircle” DNA vector (mediated by Cre-loxP; Flp-FRT), or a mini linear covalently closed DNA vector (mediated by TelN-telRL; Tel-pal). R-cells were transformed by 1 μg of pNN7 to 9 DNA constructs on LB + Ap (50 μg/ml) to A600 = 0.6 at 30°C with aeration. To induce recombinase expression and plasmid conformational conversion, transformed R-cells were heat shocked to induce the recombinase expression at 42°C for 30 min at mid-logarithmic phase of bacterial growth, before being transferred to 30°C overnight. Cells were then harvested and plasmid extracted (Omega kit, VWR). Plasmid topology was assayed by agarose gel electrophoresis and digestion. Standard recombinant DNA techniques were performed as described by Sambrook et al. (1989).
Chromosomal Integration Assays of Linear Covalently Closed (LCC) DNA
“CRIM (conditional-replication, integration, and modular)” integrating plasmids  that possess a R6K origin of replication and a phage attachment (attP) site were modified to carry the SS fragment. SS was cloned into the pAH120 and pAH153 constructs (from NBRP; Table 1) by Cla I and Bam HI (New England Biolabs) to generate the pNN10 (3.3 kb) and pNN11 (2.6 kb) constructs, respectively. Plasmids were integrated into the host bacterial attachment (attB) site by supplying phage integrase (Int) from the helper plasmids. Plasmid pNN10 and pNN11 constructs were amplified in DH5α(λpir) or BW23474, and the successful clones were confirmed by restriction pattern digestion and colony PCR. Int helper plasmids pINT-ts (λ int) and pAH123 (Φ80 int) that express int from λ pL under CI[Ts]857 control and carry a temperature sensitive pSC101 ori were used for integration of CRIM, pNN10 and pNN11 plasmids into their corresponding chromosomal attB sites of pir — hosts that are non-permissive for plasmid replication.
R-Cells W1NN, W2NN, and W3NN (Table 1) W3110 lacZ::cat- cI857(cre/tel/telN)] were grown in 2 ml of SOB cultures at 30°C to an optical density of A600 = 0.6 and then electroporated and transformed by 50 ng helper plasmids pINT-Ts (λ int) or pAH123 (Φ80 int) and selected on LB + ampicillin agar at 30°C. R-Cells carrying helper plasmids were grown in 50 ml of SOB + ampicillin at 30°C to A600 = 0.6 then transformed by 1 μg of pAH120, pNN10 (pAH120+SS), pAH153, pNN11 (pAH153+SS) DNA, suspended in SOC at 37°C for 1 h for recovery and Int expression and at 42°C for 30 min for lose of helper plasmid, then selected on LB + antibiotic (15 μg/ml Km for pAH120, pNN10 and 5 μg/ml Gm for pAH153, pNN11) and incubated overnight at 37°C. Positive bacterial growth on selective media (15 μg/ml Km, or 5 μg/ml Gm) and negative bacterial growth on ampicillin media represents stable integration of the gene of interest and loss of the helper Int expression plasmid. Single-copy integrants W4NN to W15NN represent the SS+ pAH120 (pNN10), SS- pAH120, SS+ pAH153 (pNN11), and SS- pAH153 pDNA integrated into the W3110 or W3110 lacZ::cat-cI857(cre/tel/telN)]) were screened and selected by PCR using predesigned primers .
Viability Assays of Linear Covalently Closed (LCC) DNA integration
Single copy pAH120, pNN10 (pAH120+SS), pAH153, pNN11 (pAH153+SS) DNA integrants W4NN to W15NN were isolated on selective media.
Integrants were grown in 2 ml LB media + selective antibiotic at 30°C to an optical density of A600 = 0.4 and then divided into two groups of 1 ml each and grown to A600 = 1. The first group was grown at 30°C with repressed cre/tel/telN expression and the second group was grown at 42°C, induced for cre/tel/telN expression. Cells were spread on selective plated and grew overnight at 30°C and 42°C, respectively. Viability was assayed by colony counting and size of the colonies grew at 30°C versus 42°C.
Visualization of cells
Cells were visualized by gram staining as previously described . Briefly, integrated cells were grown in 2 ml of LB media + selective antibiotic from freshly grown cells at 30°C to early log phase A600 = 0.2 and then were divided in two tubes and grown at 30°C and 42°C to late log phase, A600 = 0.8. Bacterial smears were then prepared on the slide and heat fixed and gram stained. Pictures of bacteria were taken at 1000X magnification. From pictures, 400 random cells were chosen for measurement under all tested conditions.
RAS is currently an Assistant Professor of Pharmaceutical Science at University of Waterloo, School of Pharmacy and SDM Professor of Business and Entrepreneurship. NN is currently a PhD candidate at University of Waterloo.
Circular covalently closed
DNA nuclear targeting sequence
Gene of interest
Linear covalently closed
Multiple cloning site(s)
Minimalistic immunogenic defined gene expression
Nuclear localization signal
Nuclear membrane pore complex
- o :
Polyacrylamide gel electrophoresis
- p :
Sodium dodecyl sulfate
- [ ]:
Denotes plasmid-carrier state.
This work was supported by UW Start-up funds to RAS; and Drug Safety and Effectiveness Cross-Disciplinary Training (DSECT) Scholarship provided by Canadian Institute of Health Research (CIHR), Ontario Graduate Scholarship (OGS), and Waterloo Institute of Nanotechnology (WIN) fellowship to NN. The authors also want to thank Gary Tran for his assistance in generating the pNN1 construct.
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