Vector and insert DNA preparation
All PCR primers were synthesized by Microsynth (Balgach, Switzerland) and used without further purification. Oligonucleotide sequences and uses are given in Figures 1 and 2, respectively. Template DNA for PCR was prepared by extraction from transformed Mach1 cells or ccdB survival cells (Invitrogen, Rotkreuz, Switzerland) using the GeneJet DNA minipreparation kit (Fermentas, Vilnius, Lithuania). About 2 ng of DNA were used as template for PCR. We used Phusion® polymerase (Finnzymes Oy, Espoo, Finland) as described [6]. Most proofreading DNA polymerases may be used, however, it is vital to avoid polymerases with terminal transferase activity like Taq that lead to A-tailing of the 3' ends of the PCR product. Before co-transformation both vector and insert were linearized by PCR. The excess of primers was removed by reaction cleanup on Minelute columns (Qiagen, Hilden, Germany). For vector preparations requiring more DNA, several PCR reactions were pooled and purified on larger spin columns (25 μg capacity, Fermentas, Vilnius, Lithuania). In general, cloning success depends on quality of the PCR product which should show a single band on agarose gel after electrophoresis. Gel purification of the PCR product and digestion of the templates with DpnI are not necessary (unless a template plasmid carries both resistances).
Co-transformation cloning using E.coli Mach1 cells
All plasmids were constructed by in vivo joining of PCR products with overlapping ends (about 15 bp) by a technique which we call co-transformation cloning. The E.coli strain Mach1 yields most colonies, but a few other strains like DH5alpha and Top10 work also. Co-transformation employs chemically competent cells [7] yielding 107 or more colonies per μg plasmid. Per co-transformation 200 ng of vector plus 50-500 ng of insert were mixed and the competent cells added to the DNA mixture which was less than 10% of the cell volume (50 μL cells). The protocol is standard: 30 minutes on ice, 45 s at 42°C, 1 min on ice and then addition of 4 volumes SOC medium [8]. In contrast to other protocols, a longer recovery time of 2 h was necessary to complete the end joining reaction before antibiotic selection was applied (Figure 2). Co-transformation works well with inserts up to 1.5 kb length. For longer inserts or cloning without positive or negative selection we use the ClonEZ kit (Genscript, Piscataway, NJ, USA).
Automation and general molecular biology techniques
All experimental procedures were carried out using a TECAN Freedom Evo II liquid handling workstation. The only procedures performed by hand were colony picking and insert DNA purification with the Qiagen Minelute reaction cleanup kit which are, however, both automatable (Reference [9], Qiacube from Qiagen).
The basic techniques applied for construction of the vectors (not described in detail) are from the Molecular Cloning Handbook [8]. DNA fragments were analyzed by fast agarose gel electrophoresis [10, 11]. DNA restriction or modification enzymes were from Fermentas (Vilnius, Lithuania) or New England Biolabs (Ipswich, MA, USA). For colony-PCR the Go-Taq Mastermix from Promega (Madison, WI, USA) was used. Cells were spread on agar plates by shaking on the TECAN workstation. Two colonies per target were picked and inoculated into 3 mL of 2xTY media with antibiotics. The cells were grown overnight at 37°C and spun down at 1900 rpm for 10 min. The plasmids were prepared on our TECAN workstation using the NucleoSpin Robot 96 Plasmid Kit. The plasmid was eluted with 200 μL of elution buffer and the yield was quantified by UV absorption.
Protein expression
All methods were carried out based on standard protocols [8] and are briefly described: The expression plasmids were transformed into E. coli expression strains (BL21(DE3) for EB1, Clip170-CapGly, TTL, CLIP170-full, PKNG and Acella for AAV2-VP3, respectively) and selected on LB-agar plates with antibiotics. Pre-cultures were grown over night at 30°C in deep 24-well blocks inoculating 4 mL LB. Expression cultures were started the next day by adding 200 μL pre-culture to 4 mL LB media. The cultures were grown at 37°C until the OD600 reached 0.4 and then moved to a 20°C incubator. The expression was induced 30 min later with 1 mM IPTG for Clip170-CapGly, EB1, CLIP170, VP3 and TTL or 0.1 mM IPTG for PKNG. The cells were harvested after overnight growth at 20°C.
The 4 mL cultures were pelleted and resuspended into 1 mL lysis buffer (50 mM HEPES pH8, 500 mM NaCl, 10% glycerol, 10 mM imidazole). The cells were lysed by sonication. The cell extracts were centrifuged for 10 min at 15,000 × g at 4°C. The soluble fractions were loaded onto 400 μL NiNTA IMAC resin (Ni SepharoseTM High Performance, GE Healthcare) in a 96 well filter plate (Novagen) pre-equilibrated with HEPES pH8, 500 mM NaCl, 10% glycerol, 10 mM imidazole. The beads were washed 3 times with 1 mL of the above buffer and the proteins eluted with 200 μL 50 mM HEPES pH8, 500 mM NaCl, 10% glycerol, 500 mM imidazole. The purified proteins were analysed by SDS-PAGE and Western blotting with anti-penta His antibodies (Qiagen).