Chapter 2 Development of a Rolling Circle Replication Expression System
2.2 Materials and methods
2.2.1 In silico design
DNA plasmids were all designed, analysed and tested in silico using CLC-Main Workbench by Qiagen (Netherlands,) before any subcloning was performed.
2.2.2 Subcloning procedure
All subcloning procedures were performed using standard methodology. Typical steps involved performing a PCR reaction (section 2.2.10) or restriction endonuclease digestion (section 2.2.6) to obtain linearized of insert and vector DNA. The DNA products were separated using agarose gel electrophoresis (section 2.2.7) followed by DNA gel extraction/purification (section 2.2.16). DNA concentration was then determined using a Nanodrop 1000C (Thermo Fisher Scientific; USA) for subsequent DNA ligation reaction calculations (section 2.2.3). In some instances, PCR reactions using oligonucleotides designed to introduce specific restriction sites (specified for each instance) were used to allow for precise isolation of a DNA insert fragment and ensure its correct orientation and easy ligation into a designated target location. For all experiments, at least one restriction endonuclease site, producing a sticky end, was incorporated to ensure correct DNA orientation during ligation. Ligation reactions always used a 3:1 insert to vector ratio. After DNA ligation (section 2.2.3), the ligation mix containing the newly created plasmid was transformed (section 2.2.4) into E. coli DH5α cells and spread plated LB agar containing 50 ng/ml Ampicillin for plasmid selection. This was then incubated overnight at 37 °C and transformants on the plates were screened via colony PCR (section 2.2.10) and confirmed using restriction endonuclease digestion (section 2.2.7). PCR-positive transformants were grown in lysogeny broth (LB) (Bertani, 2004) containing the selective antibiotic (typically ampicillin 100 µg/mL) overnight. Thereafter plasmid DNA was prepared (section 2.2.5). Post screening glycerol stocks (two- parts culture to one-part 50% glycerol) (section 2.2.18) were prepared for each confirmed plasmid and stored at -80 °C.
2.2.3 General molecular techniques DNA Ligations
DNA ligations were performed per manufacturer instructions using T4 DNA ligase (Thermofisher Scientific).
Transformation procedure
Commercial E. cloni® 10G chemically competent Escherichia coli DH5α cells (Lucigen, USA) were used for plasmid transformations as per manufacturer’s instructions. Selection of transformed cells was
44 done using selective LB agar plates using either ampicillin (100 μg/mL) or carbenicillin (100 μg/mL) with overnight incubation at 37 °C.
Plasmid DNA extractions
Plasmid DNA used for subcloning and early transfections was extracted using mini or midi Zyppy™
(Zymogen, USA) endotoxin free plasmid extraction kits, as per manufacturer’s instructions. For sensitive transfections that involved expression quantification or animal inoculations ZymoPUREII™
Maxi prep kits (Zymogen, USA) were used as per manufacturer’s instructions. DNA quality (Typical
>98% supercoiled) was assessed on agarose gels and purity, A260/A280, via Nanodrop™ 1000 (Thermofisher Scientific) readouts.
Restriction digestion of DNA
All restriction endonuclease digestions used endonucleases supplied by Thermofisher (USA) or New England Biolabs (USA) as per manufacturer’s instructions.
DNA separation and extraction using agarose gel electrophoresis
DNA products were separated by electrophoresis on tris(hydroxymethyl) aminomethane (Tris), borate, ethylene diaminetetra acetic acid (EDTA), aka (TBE) agarose gels (0.8% unless stated otherwise) run using 1x TBE buffer (89 mM Tris-borate and 2 mM EDTA, pH 8.3) with (0.5 µg/mL) ethidium bromide, (Sigma-Aldrich, USA). This was imaged under 302 nm UV light using a Gel Doc™ XR+
system (BioRad, USA).
DNA extractions were done under blue light at 470 nm using a Blook blue LED transilluminator (GeneDireX, USA) and purified using the DNA IsolateII PCR and Gel Purification Kit (Bioline, USA) as per manufacturer’s instructions.
Tissue culture DNA extractions
Total DNA extractions from transfected mammalian cells were performed using a DNeasy Blood &
Tissue kit (Qiagen, USA) as per manufacturer’s instructions for cultured cells.
2.2.4 PCR oligonucleotide design
A list of primers that were designed is shown in Table 2.2.4.
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Table 2.2.4 List of oligonucleotides used. The restriction endonuclease site is underlined in the sequence.
Name Sequence (5′ → 3′) Length
BFDV LIR1 For-EcoRI GGTGGTGAATTCAGAGGTGCCCCACAGGC 29
BFDV LIR1 Rev-BamHI GGTGGTGGATCCTGTTCCCGGGCGACTGTG 30
BFDV LIR2 For-NotI GGTGGTGCGGCCGCAGAGGTGCCCCACAGGC 31
BFDV LIR2 Rev-AscI GGTGGTGGCGCGCCTGTTCCCGGGCGACTGTG 32
BFDV Rep For-HindIII GGTGGTAAGCTTATGCCGTCCAAGGAG 27
BFDV Rep Rev-BamHI GGTGGTGGATCCTCAAAAATTGATGGGGTG 30
BFDV Rep3 Rev-SphI GGTGGTGCATGCTCAAAAATTGATGGGGTG 30
BFDV Rep3 Rev-XhoI GGTGGTCTCGAGTCAAAAATTGATGGGGTG 30
LIR-Rep Forward AGGCCTAGGCGCGCCCTG 18
SIR-Rep Reverse TCCCGCCCTGCGCCATCG 18
LIR2-CP Forward GGGGCACCTCTAACTGCG 18
SIR-CP Reverse GAAGGCCAGGCCGTAGTG 18
eGFP-For-HindIII GGTGGTAAGCTTATGGTGAGCAAGGGCGAG 30
eGFP-Rev-BamHI GGTGGTGGATCCTTACTTGTACAGCTCGTCCATGC 35
2.2.5 PCR Protocols
A high fidelity Pfu Phusion (Thermo Fisher) DNA polymerase was used for cloning purposes. The thermocycling conditions were 98 °C for 30 s followed by 30-45 cycles of 98 °C for 10-15 s, Ta
(dependent on the oligonucleotide pair) for 10-15 s and 72 °C for 15 s/‘kilo base pair’ (kbp) (dependent on the expected PCR product size). A final extension of 5 minute at 72 °C was included. The reaction mix was prepared as per manufacturer’s instructions. A touch down PCR method similar in methodology to the protocol developed for difficult AT rich genes was used for oligonucleotides with a Ta difference of greater than 5 °C (Su et al., 1996).
OneTaq DNA polymerase (New England Biolabs) was used for screening of transformants instructions.
The thermocycling parameters were as follows: 94 °C for 30 s, followed by 30 cycles of 94 °C for 15 s, Ta (dependent on the oligonucleotide pair) for 15 s and 68 °C for 60 s/kbp (dependent on the expected PCR product size). A last step of 5 minutes at 68 °C was included. The PCR reaction mix was prepared as per manufacturer’s instructions. Each reaction contained cells from an identified transformed colony.
Screened colonies were also transferred to a numbered square on an agar plate with selection, for temporary storage.
2.2.6 Rolling Circle Amplification (RCA) screening
Amplification of circular DNA fragments for detection and screening was performed using a TempliPhi™ 100 Amplification Kit (GE Healthcare, USA) as per the manufacturer’s instructions.
46 2.2.7 DNA sequencing
The RCA DNA for sequencing was linearised using EcoRV – which cuts once in the viral sequence - and resolved by electrophoresis on a 0.8% TBE agarose gel and gel purified. The linearised DNA was then subcloned into the CloneJET PCR cloning kit (Thermofisher Scientific) as per manufacturer’s instructions. The ligation DNA was then transformed into competent E. coli DH5-α and the plasmid was isolated from overnight cultures. Isolated plasmid DNA was then sequenced at Stellenbosch University (Figure 2.7). Sequence alignments were done using CLCBio (Qiagen).
2.2.8 Source of eukaryotic cells and cell culture
All cell lines were obtained from the American Type Culture Collection (ATCC, USA), these included HEK-293 (ATCC® CRL-1573™), HEK-293T (ATCC® CRL-3216™), and HeLa S3 (ATCC® CCL- 2.2™). All mammalian cells were cultured at 37 °C, 5% CO2 with 80%-95% humidity in 75 cm2 tissue culture flasks. Growth media consisted of Dulbecco’s modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS) and penicillin with streptomycin (Pen/Strep) at (100 U/ml). Cells were passaged every 2-3 days at which point they were washed twice with PBS and treated with 3 mL Gibco 0.25% Trypsin containing 0.02% EDTA (Sigma-Aldrich) to free the cells. Growth media was added to neutralise the trypsin reaction and a cell count performed using a haemocytometer. Flasks were then seeded at approximately 12.5-25% confluency, depending on cell type. All handling was done in biosafety Level 2 cabinets using good laboratory practice.
2.2.9 Transfections
Tissue cultured cells were transfected with DNA plasmids by seeding 6, 12, or 24 well plates at a confluency of 30-50%. The cells were then cultured to ~60-80% confluency which took ~24-48 h before transfections were performed. Roche X-tremeGENE™ HP DNA (manufacturer?) transfection reagent (TR) was used to transfect DNA into cells and optimised per cell line used as per manufacturer instructions. In most cases this was 1 µg DNA to 1 µL transfection reagent per 100 µL DMEM with a 30-40 minute equilibration step. The transfection mix was then added directly to the growth media in TC plates and left for the duration of the experiment. Time-limited transfections were also performed whereby a transfection step of 4-6 h was used before aspirating the growth media, washing 3 × with PBS, and adding fresh growth media.
Transfections based on plasmid copy number used the same ratio of DNA to transfection reagent to DMEM. The plasmid concentration (pmols) was calculated and converted to DNA concentration with the use of Equation 2.2.9 below.
Equation 2.2.9
μgDNA = pmolDNA×(660pg/pmol) × (1μg/106 pg) x N (# bp)
47 2.2.10 Luciferase assays
The luciferase assays were performed using the Luciferase Assay System (Promega, USA), using the kit pGL4.13 expression vector as a positive control, and our customised luciferase-expressing vectors, designed in house, from this vector. The assay was optimized for transfection efficiency in BHK, HEK- 293, HEK-293T and HeLa S3 cells. Varying transfection concentrations were calculated by determining the pmol concentrations of plasmid DNA to normalize plasmid and therefore gene copy number when using plasmids of varied sizes. Transfections were performed as described above. High purity plasmid DNA was used in transfections. This DNA was >95% supercoiled, endotoxin free and supplied from Aldevron (USA) or purified using the ZymoPURE II DNA Maxiprep kits (Zymogen). Characterization assays were repeated multiple times using a high number of replicates (typically n=6) and performed as per manufacturer’s instructions. Luminescence was recorded in relative luminescence units (RLU) using a Promega GloMax® luminometer.
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