E. coli strains and plasmids
E. coli strain DH5α [F- φ80lacZ ΔM15 Δ( lacZYA argF)U169 deoR recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 λ- and the plasmid pVAX1 (2999 bp) were purchased from Invitrogen (Carlsbad, CA). pVAX1-GFP (3642 bp) was constructed by cloning the superfolding green fluorescent protein (sGFP) gene [27] into the multi-cloning site of pVAX1. The sGFP gene was obtained from pTrcsGFP, a gift from the Gregory Stephanopoulos laboratory (Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA). pCP40 (5029 bp) was constructed by Remaut et al. [16] and was obtained from the Belgian Coordinated Collections of Microorganisms BCCM/LMBP plasmid collection (accession number LMBP 951).
To construct pDMB02-GFP, a 1938-bp fragment of pVAX1 containing the human cytomegalovirus (CMV) immediate-early promoter/enhancer, bovine growth hormone (BGH) polyadenylation signal, and kanamycin resistance gene was PCR-amplified using primers containing AvrII and SbfI restriction sites. A 3073-bp fragment of pCP40 containing the R1 origin of replication along with the repA copA, and copB gene sequences was also PCR-amplified with the same restriction sites. The pVAX1 and pCP40 fragments were ligated to construct the plasmid pDMB02. The sGFP gene was cloned into the NheI and XhoI restriction sites downstream of the CMV promoter/enhancer. A Kozak sequence was also inserted at the start of the sGFP gene by adding the sequence ACC before the start codon and a valine codon (GTG) following the start codon. The Kozak sequence should help facilitate sGFP gene expression in mammalian cells [28]. The resulting vector, pDMB02-GFP, was 5668 bp in size (Figure 1). Correct construction was confirmed by restriction digests and sequencing.
The start codon of repA in pDMB02-GFP was mutated from GTG to ATG using site-directed mutagenesis resulting in the plasmid pDMB-ATG. Primers containing the mutation were used to amplify pDMB02-GFP using 20 cycles of PCR with Phusion high-fidelity DNA polymerase (New England Biolabs; Ipswich, MA). The primers were purified using a reverse phase cartridge by the vendor (Sigma-Aldrich; St. Louis, MO) and their sequences are shown below:
5’-GTGAAGATCAGTCATACCATCCTGCACTTACAATGCG-3’
5’-GCAGGATGGTATGACTGATCTTCACCAAACGTATTACCG-3’
After PCR, the template plasmid was digested using DpnI, and after clean-up the reaction was used to transform ElectroMAX DH10B cells (Invitrogen). Positive transformants were selected on LB/agar plates containing 50 μg/mL kanamycin, and the presence of the start codon mutation was verified by sequencing.
Preparation of working seed banks
Frozen working seed banks of DH5α[pDMB02-GFP] and DH5α[pDMB-ATG] were prepared by transforming subcloning-efficiency, chemically-competent DH5α (Invitrogen) with purified plasmid. Positive transformants were selected on LB/agar plates containing 25 μg/mL kanamycin. After overnight incubation at 30°C, a single colony was used to inoculate 3 mL of LB medium containing 25 μg/mL kanamycin. The culture was incubated overnight at 30°C. The next day, 500 μL of overnight culture was used to inoculate 50 mL of LB medium containing 25 μg/mL kanamycin in a 250-mL shake flask. The culture was incubated at 30°C until mid-exponential phase (OD600 approximately equal to 0.5), at which time 900 μL of culture was added to 900 μL of cold 30% (v/v) glycerol in a cryogenic vial and immediately stored at −80°C. Working seed bank vials were discarded after two freeze-thaw cycles.
Culture conditions
Difco LB Broth, Miller (BD; Franklin Lakes, NJ) was used for shake flask cultures and contained 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl. Cell density was monitored using OD600 measurements on a DU800 spectrophotometer (Beckman Coulter; Indianapolis, IN). All cultures were mixed and aerated by agitation at 250 rpm unless otherwise specified.
Temperature shift experiments
Temperature-induced plasmid amplification was studied using temperature shift experiments as follows: 100 to 110 mL of LB medium containing 25 μg/mL kanamycin (or 100 µg/mL ampicillin for pCP40) was inoculated to an initial OD600 = 0.00025 using working seed bank. The very low initial OD600 was chosen to avoid glycerol carry-over from the working seed bank vial. The cultures were incubated in 500-mL baffled shake flasks at 30°C until the desired growth phase was achieved (typically OD600 = 0.5 – 1.0) at which point half of the culture volume was transferred to a 250-mL shake flask and incubated at 42°C. The remaining culture was transferred to a 250-mL shake flask and incubated at 30°C.
Seed growth phase study
5 mL LB containing 25 μg/mL kanamycin was inoculated with 20 μL of DH5α[pDMB02-GFP] working seed bank and incubated at 30°C. Aliquots of the culture were collected in late exponential (OD600 = 1.1), early stationary (OD600 = 2.3), and late stationary (OD600 = 2.8) growth phases and used to inoculate 50 mL LB containing 25 μg/mL kanamycin to an initial OD600 = 0.01. The resulting cultures were incubated at 30°C for at least 24 hours. Samples were taken periodically to measure cell growth and plasmid production.
Measurement of plasmid copy number
Plasmid copy number was determined from 100 μL of culture using the quantitative PCR (qPCR) method described by Bower et al. [29] In short, SYBR Green was used to detect amplification of plasmid-based and chromosomal gene targets from total DNA samples diluted 5-fold (if necessary) to be within the linear range of the assay. The ΔΔCT method [30] was used to calculate plasmid copy number.
Measurement of plasmid DNA specific yield
Plasmid DNA was quantified from crude lysates prepared from OD600 = 10 cell pellets using the method described previously [29]. Briefly, crude lysates were run on a Gen-Pak FAX anion-exchange column (Waters Corporation; Milford, MA) and pDNA was eluted using a NaCl gradient. Plasmid detected by absorbance at 260 nm was quantified using a standard curve of known quantities of purified plasmid DNA. Specific yield could then be calculated using the correlation that 1 OD600 unit = 0.4 g DCW/L culture.
Measurement of repA mRNA expression
repA mRNA expression was measured using a quantitative real-time PCR assay. RNA was purified from OD600 = 1 pellets using the Illustra RNAspin Mini RNA Isolation Kit (GE Healthcare; Piscataway, NJ). 1 g/L lysozyme was used for cell lysis in the first step of the protocol. Due to the high plasmid DNA content of the strains used in this work, an additional DNase digestion was required after the RNA purification step to remove contaminating DNA. 43 μL purified RNA was digested with 2 μL DNase I (New England Biolabs) in 5 μL of the supplied reaction buffer for 10 min. at 37°C. RNA was purified from the other reaction components using the RNA Cleanup protocol from the RNeasy Mini Kit (Qiagen; Valencia, CA). The RNA content of each sample was measured using a NanoPhotometer (Implen; Westlake Village, CA), and 800 ng of RNA was converted to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). Control reactions containing water instead of the reverse transcriptase enzyme were included for each sample.
Quantitative PCR was performed on a 7300 Real-Time PCR System (Applied Biosystems; Carlsbad, CA). The desired reference sample was diluted 2- to 1000-fold to prepare a standard curve. repA mRNA was detected using gene-specific primers (forward primer: 5’-CAGAGCTTAAGTCCCGTGGAAT-3’, reverse primer: 5’-TGACGTTCTCTGTTCGCATCA-3’) designed by Primer Express 3.0 software (Applied Biosystems). Each 25-μL reaction contained 1X Brilliant II SYBR Green QPCR High ROX Master Mix (Agilent Technologies; Santa Clara, CA), 200 nM each of the forward and reverse primers, and the experimental sample diluted 100-fold. The thermal cycling conditions were a 95°C hold for 10 min., followed by 40 cycles of 95°C for 30 sec. and 60°C for 1 min. Dissociation-curve analysis was also performed to check for the presence of primer dimers or non-specific products. Results were analyzed using the Applied Biosystems Sequence Detection Software (v. 1.3.1).
Immuno-detection of RepA protein
Removal of cross-reactive antibodies from RepA antiserum
Polyclonal RepA antiserum was a generous gift from Prof. Rafael Giraldo (Centro de Investigaciones Biológicas, Madrid, Spain) and was prepared as described by Giraldo-Suárez et al. [24]. To reduce background binding, the RepA antiserum was incubated with plasmid-free DH5α lysate to precipitate non-specific E. coli-reactive antibodies. A lysate of plasmid-free DH5α was prepared from 50 mL of mid-exponential phase culture grown in LB medium by freeze-thaw and sonication in buffer containing 10 mM Tris–HCl at pH 7.5, 140 mM NaCl, 1% Triton X-100, 1% BSA, 1% sodium deoxycholate, and 1 Complete Mini Protease Inhibitor Cocktail tablet (Roche; Indianapolis, IN) per 10 mL. The DH5α lysate was added to an aliquot of RepA antiserum in a 1:1 ratio and incubated at room temperature for 5.5 hr with gentle rocking. After incubation, the antiserum was centrifuged for 20 min. at 20000 x g and 4°C. The supernatant was recovered and stored in single-use aliquots at −80°C.
Cell lysis
Cell lysates were prepared by resuspending pellets prepared from 4–5 mL of culture in 1 mL 10 mM Tris–HCl at pH 8.0. The 1-mL suspension was added to approximately 500 μL of 0.1 mm glass beads (Scientific Industries; Bohemia, NY) in a 1.7-mL microcentrifuge tube. The suspension and glass beads were vortexed at maximum speed for 5 min. followed by centrifugation for 20 min. at 14000 x g and 4°C. The supernatant was recovered and stored at −30°C.
SDS-PAGE
Total protein content of the lysates was measured using the modified Bradford assay described by Zor and Selinger [31]. A 7.5-μl aliquot of each lysate containing 2 μg total protein (balance water) was prepared. An equal volume of Laemmli buffer containing 5% (v/v) β-mercaptoethanol was added to each aliquot, and the samples were incubated at 100°C for 5 min. Samples were loaded on a 10% Mini-PROTEAN TGX Gel with 15 x 15-μL wells (Bio-Rad; Hercules, CA) and run at 200 V for 30 min in tris/glycine/SDS buffer.
Western blots
Protein separated by SDS-PAGE was transferred to a nitrocellulose membrane (Pall Corporation; Pensacola, FL) for 1 hr at 100 V using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) with transfer buffer containing 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol at pH 8.3. After transfer, the membrane was blocked with a 5% (w/v) bovine serum albumin solution prepared in TBS (2.42 g/L Tris, 29.24 g/L NaCl, pH 7.5) at room temperature for 2 hr. After two 10-min. washes with TBST solution (TBS + 0.05% v/v Tween-20), the membrane was incubated with a 1:1000 dilution of RepA antiserum in TBS containing 10% glycerol for 2 hr at room temperature. The membrane was washed three times with TBST for 10 min. each, and was then incubated with a 1:5000 dilution of goat anti-rabbit IgG-HRP secondary antibody (Santa Cruz Biotechnology; Santa Cruz, CA) in TBS for 1 hr. After two 10-minute washes with TBST and one 10-minute wash with TBS, secondary antibody binding was visualized using Western Blotting Luminol Reagent (Santa Cruz Biotechnology) following the manufacturer’s instructions.
Cloning and expression of repA
To verify that the band being detected on the Western blots was indeed RepA, a positive control vector was constructed by cloning the repA gene into the BamHI/HindIII sites of pETDuet-1 (EMD Millipore; Billerica, MA), in-frame with an N-terminal 6X His tag. The resulting plasmid, pETDuet-repA, was used to express RepA-His in E. coli BL21 Star (DE3) (Invitrogen). 50-mL LB cultures of BL21 Star (DE3) containing either pETDuet-repA or pETDuet-1 and 100 μg/mL ampicillin were grown at 30°C with 250 rpm shaking and induced with 0.5 mM IPTG at an OD600 of approximately 0.5. Six hours after induction, 20-mL aliquots of culture were harvested by centrifugation. Lysates were prepared from the pellets using disruption with 0.1 mm glass beads (Scientific Industries) in buffer containing 7 M urea, 0.1 M NaH2PO4, and 0.01 M Tris–HCl at pH 8.0.
Plasmid quality assessment
Plasmid DNA purified from OD600 = 2 pellets using the Zyppy Plasmid Miniprep Kit (Zymo Research Corporation; Irvine, CA) was run on a 0.7% agarose gel at 90 V for 60 min. to separate the supercoiled, nicked (open-circle), and linear isoforms. The separated DNA was visualized by staining the gel with 0.5 μg/mL ethidium bromide.