Molecular biology
Enzymes, buffers and mass weight marker were from Fermentas, Thermo Scientific. All PCRs were performed with Phusion DNA-polymerase with HF buffer. Primers sequences are provided at Table 1.
Preparation of Bt toxins and cadherin fragment CR7-12
Crystals of Cry1Ac, Cry11Aa, Cry3Aa and Cyt1Aa were produced from Bacillus thuringiensis, purified, solubilized and activated as previously reported (Gómez et al. 2001; Pérez et al. 2005). Cadherin fragment CR7-12 was expressed in E. coli ER2566 and purified with nickel affinity column as reported (Pacheco et al. 2009).
Phagemids construct and cloning
The signal sequence PelB of the pCANTAB 5E phagemid was substituted by ssDsbA to construct pCAD phagemid. The ssDsbA was synthetized by overlapping-PCR of four primers as previously reported (Steiner et al. 2006). Briefly, 10 pmol of SP1 and SP2 primers were mixed and assembled at 72 °C for 10 min with Phusion DNA-polymerase, then 1 μl of the assembly reaction was used as template and amplified with SPIII and SP4 primers. The PCR product was separated by agarose-gel electrophoresis and ligated into pCANTAB 5E after restriction with HindIII and SfiI enzymes. Phagemid pCADS was constructed by mutagenizing the TAG amber stop codon of pCAD phagemid by CAG codon with the primer SupASC using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies) following the manual instructions. The cry1Ac toxic fragment coding-sequence (G26-A614) (GenBank No. AM949588) was amplified by PCR from Bacillus thuringiensis strain HD73 (Bacillus Genetic Stock Center-BGSC- No. 4D4) using the primers FFTc-M13/RFTc-M13. The cyt1Aa protoxin fragment coding-sequence (GenBank No. X03182) was amplified by PCR from pWF45 vector (Wu and Federici, 1993) using the primers Pro-SfiI/Pro-NotI. The cry3Aa toxic fragment coding-sequence (K63-N644) (GenBank No. AJ237900) was amplified by PCR from Bacillus thuringiensis
subsp. tenebrionis (BGSC No. 4AA1) using the primers 3Aup/3Alow. All the PCR-amplified toxin fragments were ligated after digestion with SfiI and NotI restriction enzymes on previously digested pCANTAB 5E, pCAD or pCADS phagemids.
Phage production and purification
The pCANTAB 5E and pCADS phagemids with the cry1Ac toxin gene inserted were transformed into E. coli XL1-Blue MRF’, while pCADS with cry1Ac and cyt1Aa toxin genes were transformed into E. coli HB2151. A single colony was grown overnight at 37 °C in 3 ml of 2xYT medium containing 100 μg/ml ampicillin. The overnight culture was used to inoculate 35 ml of 2xYT containing 100 μg/ml ampicillin and were incubated at 37 °C until reaching an OD600 of 0.6. The E. coli XL1-Blue MRF’ or HB2151 cultures were infected with 1011 particles of helper phages VCSM13 or Phaberge (which was kindly donated by Dr. Erik J. Wiersma), respectively. After 30 min of incubation at room temperature, kanamycin (30 μg/ml) was added and the cultures were incubated at 30 °C overnight. The supernatants were separated by centrifugation and 6 ml of PEG-NaCl solution (20 % PEG 8000/2.5 M NaCl) was added and incubated at 4 °C for 1 h. The phages were precipitated by centrifugation at 8500g for 10 min at 4 °C and the pellet was suspended in 1 ml of PBS pH 7.4. Finally the phage titer was estimated counting colonies resistant to ampicillin after E. coli XL1-Blue MRF’ infection.
Phage blot
1011 phage particles were mixed in Laemmli buffer and boiled for 5 min. The mixture was separated by SDS-PAGE and then electrotranferred to PVDF Immobilon-P membrane (Millipore). After blocking with PBS-M (PBS, 5 % skim milk) for 1 h, the membrane was incubated with polyclonal rabbit antibodies anti-Cry1Ac or anti-Cyt1Aa (1:20,000 in PBS, 0.1 % Tween-20). The membrane was incubated for 1 h with the secondary antibody anti-rabbit IgG-HRP (Santa Cruz Biotechnology) diluted to 1:30,000 in PBS-T. Finally, blots were visualized with Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and densitometry was performed with ImageJ software (http://imagej.nih.gov/ij/).
Phage binding
ELISA 96-well microplates were coated overnight at 4 °C with 100 µl of either cadherin protein fragment CR7-12 (200 nM), anti-Cry1Ac antibody (1:1000) or anti-Cyt1Aa antibody (1:1000) diluted in PBS. The plates were blocked with 200 µl/well of PBS-M for 1 h at 37 °C. Phages particles (1010) per well diluted in PBS-T were added and incubated for 1 h, then the unbound phages were removed by washing with PBS-T. The anti-pVIII-HRP secondary antibody (1:5000) was added and incubated for 1 h. The HRP enzymatic activity was revealed with 100 µl of substrate solution (1 mg/ml ortho-phenylenediamine, 0.05 % H2O2, 100 mM NaH2PO4 pH 5.0). Reaction was stopped adding 50 µl of 1 M H2SO4 and measured at 490 nm using the Emax Precision Microplate Reader, Molecular Devices.
For competition binding assay, M13-Cry1Ac phages were incubated in presence of increasing concentrations of soluble Cry1Ac toxin and the phage binding was performed as described above.
Insect bioassay
Manduca sexta neonate larvae were reared on an artificial diet in 24-well plates. Cry1Ac activated toxins (10 ng/cm2), M13-Cry1Ac or M13 wild-type phages (1011 pfu/cm2) were applied on the surface of diet and one neonate larvae was placed per well. Mortality was recorded after 7 days at 28 °C, 65 ± 5 % relative humidity and a photoperiod of 16/8 h light/dark. Mortality was scored after 7 days.
Phage selection
ELISA 96-wells plates were coated with the bait antigen (CR7-12, anti-Cry1Ac or anti-Cyt1Aa) as described for phage binding. A mixture 1:1 of M13-Cry1Ac or M13-Cyt1Aa (1010 pfu/100 μl) in PBS-T was added per well and incubated for 1 h at room temperature with gentle shaking. The wells were washed ten times with PBS-T and 100 μl of E. coli XL1-Blue MRF’ grown in 2xYT medium until OD600 0.6 were added. The plates were incubated for 30 min at room temperature to allow the infection and the cells were collected and diluted in 1.4 ml of a 2xYT medium. An aliquot of 50 μl of cells diluted were plated on agar LB plates containing ampicillin 100 μg/ml and were incubated at 37 °C for 16 h. To determine which phages were selected depending on bait was used as antigen, colony-PCR was performed to identify the phagemid by amplifying either the cry1Ac or cyt1Aa genes with the primers FFTc-M13/RFTc-M13 or Pro-SfiI/Pro-NotI, respectively. The PCR-products were evaluated by agarose gel electrophoresis.
In vitro transcription and translation
To prepare the mRNA of Cry1Ac (G26-P607), domain II of Cry1Ac (Q262-S459) or Cyt1Aa (P34-P229), the gene fragments were PCR amplified with primers FFTc/RFTc, FDIIc/RDIIc or CytF-RD/CytR-RD, respectively. After restriction with BamHI and HindIII enzymes, the PCR fragments were inserted into pFPRDV plasmid. To obtain the DNA template for in vitro transcription, the outer primers T7B and TolAkurtz were used to introduce, by PCR amplification, T7 promoter, ribosome binding site and TolA spacer sequences. The PCR products were used as DNA template for in vitro transcription using TranscriptAid T7 High Yield Transcription Kit (Fermentas, Thermo Scientific).
For in vitro translation, S30 extract were prepared as described previously (Dreier and Pluckthun 2011). Briefly, E. coli MRE600 were grown in rich medium (40 mM KH2PO4, 165 mM K2HPO4, 10 g/L yeast extract, 15 μg/ml thiamine, 2 % glucose, 1 mM MgOAc) until DO600 of 1.0. Cells were washed three times with buffer S30 (14 mM MgOAc, 60 mM KOAc, 10 mM Tris-OAc pH 7.5) and lysed with French press. Afterward, the lysate was clarified by centrifugation at 30,000 g and then it was dialyzed overnight with buffer S30. The ternary complex was prepared in a translation reaction of 27.5 μl containing 2.5 μg of mRNA, 12.5 μl of S30 extract and 11 μl of PremixZ (Dreier and Pluckthun 2011). The reaction was incubated at 37 °C for 15 min and then was placed on ice. To maintain the ternary complex, 75 μl of WB (50 mM Tris–Acetate pH 7.5, 150 mM NaCl, 50 mM Magnesium Acetate) were added; to dissociate the ternary complex, 75 μl of TBS were added.
ELISA test of the in vitro translated toxins
ELISA microplates coated with the bait antigen were blocked with TBS-BSA 0.5 %. An in vitro translation reaction prepared with cry1Ac or cyt1Aa mRNA and stopped with TBS, was applied and incubated for 1 h with gentle shaking at room temperature. Binding of Cry1Ac against cadherin fragment CR7-12 was detected with the polyclonal antibody anti-Cry1Ac followed of secondary antibody anti-rabbit IgG-HRP. In the case of immobilized antibodies anti-Cry1Ac or anti-Cyt1Aa the binding was detected with an antibody anti-RGS-His-HRP. HRP enzymatic activity was revealed as was described above.
Recovery of RNA after binding selection of ternary complexes
ELISA microplates coated with the bait antigen (CR7-12, Cry11Aa or anti-Cyt1Aa) were blocked with TBS-BSA 0.5 % and washed with WB. An in vitro translation reaction prepared with an equimolar mixture of Cry1Ac and Cyt1Aa mRNA and stopped with WB, was applied and the plate was incubated 1 h with gentle shaking at 4 °C followed by ten washes steps with WB-T. The mRNA was eluted with 100 µl of elution buffer (50 mM Tris-HOAc pH 7.5, 150 mM NaCl, 10 mM EDTA, 5 µg Saccharomyces cerevisiae RNA) during 10 min at 4 °C with gentle shaking and purified with RNeasy kit (Qiagen). Reverse transcription was performed with RevertAid RT Kit (Fermentas, Thermo Scientific) according to manual instructions. The cDNA obtained from reverse transcription with the reverse primers RFTc or CytR-RD was PCR-amplified with the primers SD-MRGS/RFTc or SD-MRGS/CytR-RD, respectively. Finally, the PCR products were analyzed by agarose gel electrophoresis.