Neb gibson assembly kit

pT7T-EcadTSMod, pT7T-EcadTL, pT7T-EcadTI, pT7T-CcadTSMod were obtained by PCR amplification of EcadTSMod, EcadTL and EcadTI plasmids (a Kind gift of Dr. N. Borghi) and C-cadherin cDNA (kindly provided by Dr. BM Gumbiner), respectively. PCR products were cloned between BglII and SpeI sites into the pT7T vector using the NEB Gibson assembly kit (NEB). pT7-CcadTSMod was constructed with the NEB Gibson assembly kit following the same scheme as for EcadTSMod: the sequence coding for the 734 first amino acid of C-cadherin were inserted after the BglII site of pT7T, the TS module containing the spider silk protein flanked by mTFP1, EYFP and glycine linker at each side, is inserted just after the first fragment. The last 146 amino acid of C-cadherin were then inserted between TS module and SpeI site of pT7T. Constructs were verified by sequencing. In-vitro transcription was performed with mMessage mMachine transcription kit according to manufacturer’s instructions (Ambion) using the EcoRI site for linearization.

A fragment containing 5′- and 3′-flanking regions of csr-1 was amplified with pLC-5-F/pLC-3-R from pCSR1 [92 (link)]. The promoter of gpd-1 was amplified from genomic DNA using primers Pgpd-NC-F/Pgpd-NC-R. The open reading frame of col-26 was amplified using Col26-ORF-F/Col26-ORF-R from cDNA which was synthesized from total RNA using a ReverTra Ace qPCR RT Kit (Toyobo, Japan). The coding sequence of gfp was amplified using primers GFP-F/GFP-R from pMF272. These four fragments were assembled using the NEB Gibson Assembly Kit (New England Biolabs, USA) to give pCSR-COL-26-GFP. The variants col-26 (S79A, S83A), col-26 (S674A, S676A), and col-26 (S4A) were generated by site-directed mutagenesis using PCR with high-fidelity polymerase. Transformation by electroporation was performed as described previously [92 (link)]. Transformants resistant to cyclosporin A were further confirmed by PCR and green fluorescent protein (GFP) fluorescence.

The ptpB derivatives plasmids were constructed using NEB Gibson Assembly kit (New England Biolabs). The ptpB gene was amplified by PCR using S. aureus Newman chromosomal DNA as a template with the primers listed in Table S1. The ptpB fragment was fused to Histidine Affinity Tag (HAT) [32 (link)] at the C-terminus and Gibson cloned into the NcoI/BamHI digested pET19b vector (Novagen, Madison, WI, USA), thus generating pET19b_PtpB. PtpB_T11I and PtpB_D111A derivatives harboring threonine to isoleucine or aspartic acid to alanine substitutions, respectively, were generated by Gibson assembly (Table S1). Transformed E. coli BL21 Star cells were grown at 16 °C in LB medium containing 1 mg/mL of glucose and 100 µg/mL of ampicillin, and protein synthesis was induced with 0.5 mM IPTG overnight. Bacteria were disrupted in a French pressure cell and centrifuged at 14,000 rpm for 25 min. Purifications of the HAT-tagged recombinants were performed using TALON^® metal affinity resins (Clontech, Mountain View, CA, USA) accordingly to the manufacturer’s instructions and eluted in 200 mM imidazole, 50 mM Tris-HCl [pH 7.4], 100 mM NaCl, and 10% [vol/vol] glycerol, to be stored at −20 °C.