Fast link dna ligation kit

Deletion of target genes (tlp4:cjj_0289; tlp6:cjj_0473; tlp8:cjj_1128; tlp9:cjj_1205; and tlp10: cjj_0046) was achieved by double crossover homologous recombination using a suicide vector containing homologous sequences on either side of the respective tlp gene as described previously (Gangaiah et al., 2009 (link); Rajashekara et al., 2009 (link)). Briefly, a 1 kb flanking region on either side of the target gene along with coding regions of the gene were amplified by PCR using the specific tlp F and R primers and C. jejuni 81–176 genomic DNA (Table S2). The PCR product was ligated into pZErO-1 to generate the plasmid pZErO-1-tlp using Fastlink™ DNA ligation kit (Epicentre, Madison). Inverse PCR was performed on pZErO1-tlp using the specific tlp INV F and INV R primers (Table S2) to amplify the plasmid without the majority of the tlp coding sequence; except for approximately 50 bp at the ends of the original tlp gene sequence. The kanamycin cassette from pUC4K was then cloned into the inverse PCR product to generate the suicide vectors pZErO-1-inv-tlp. The resulting suicide vectors were electroporated into C. jejuni 81–176 (Wilson et al., 2003 (link)). Recombinants were selected on MH agar plates containing the appropriate antibiotics. The deletion of the specific tlp gene was confirmed by PCR.

To test the effects of the parental environment on seed transcripts, we constructed Illumina Solexa sequencing libraries of 24 self-fertilized dry, ungerminated seeds derived from the same lot of seeds used for the phenotyping. The seeds experienced 1 year of after-ripening before they were used for this experiment. For each genotype and parental environment, we had three biological replicates that consist of a single seed per replicate. For each library, mRNA was isolated from a single seed using Dynabeads mRNA DIRECT Kit from Invitrogen (Product # 610.2, Grand Island, NY) and fragmented using Ambion mRNA Fragmentation Kit (Product #AM8740, Grand Island, NY), followed by cDNA synthesis using random hexamer primers. Double stranded cDNA fragments were blunt-end repaired using Epicentre End Repair (Product #ER81050, Madison, WI) and added a single A to the blunt end using Klenow Fragment 3′-5′ exo-nuclease (NEB Product #M0212L, Ipswich, MA). Ilumina adaptors were ligated to the cDNA fragments using the Epicentre Fast-Link DNA Ligation Kit (Product #LK6201H, Madison, WI). Fragments between 200-400 bp were size selected by agarose gel and samples were indexed and enriched. The libraries were indexed using 12 indices and sequenced on two lanes using the Illumina GAIIx, which generated 76 bp single-end reads.

Vectors expressing cell-surface CD28, CD28 fused C-terminally to GFP, cell-surface B7-2 and B7-2 or B7-2C fused C-terminally to Cherry have been described (4 (link), 5 (link)). Vector expressing B7-1 was generated by cDNA synthesis of human CD80 (NM_005191.3) from total human PBMC RNA using Verso RT-PCR kit (ABgene). CD80 cDNA was generated using KOD polymerase (Novagen) with phosphorylated PCR primers 5′-GAC GTC GAC ATG GGC CAC ACA CGG AGG and 5′-CAC GCG GCC GCT TAT ACA GGG CGT ACA CTT TC CC. The PCR product was inserted into pEGFP-N3 DNA (Clontech) that had been digested with SalII and NotI and lacked the GFP region, using Fast-Link DNA Ligation Kit (Epicenter). Vector expressing B7-1 fused C-terminally to Cherry was generated from B7-1 cDNA vector template with phosphorylated PCR primers 5′-TAC TCG AGA TGG GCC ACA CAC GGA GG and 5′-GTC CGC GGT ACA GGG CGT ACA CTT TCC CT TC, deleting the B7-1 termination codon. Upon digestion with XhoI and SacII, the PCR product was inserted into pmCherry-N1 DNA (Clontech).

All chemicals were from standard sources and of highest purity. Solvents used for chromatographic analysis were of gradient grade. Steroids for analysis and whole-cell biotransformation were from Sanofi, Frankfurt-Höchst (DE) and of highest purity. Restriction enzymes were obtained from New England Biolabs (Frankfurt, DE), kits for plasmid preparation and DNA purification from Machery-Nagel (Düren, DE) and the FastLink™ DNA Ligation Kit from Epicentre Biotechnologies (Madison, US). The primary antibody against arh1 was obtained from Charles River Laboratories (Sulzfeld, Germany), against etp1^fd from BioGenes (Berlin, Germany) and against bovine CYP21A2 from antikoerper-online.de (Aachen, Germany).

To construct a complementation strain, mfrA along with the potential promoter sequence was amplified from the genomic DNA of C. jejuni NCTC-11168 using specific primers (Table 1). The primers were designed to include restriction sites that facilitate directional cloning. The PCR products were digested, purified and ligated into a similarly digested pRY108 plasmid using the Fast-Link DNA ligation kit (Epicentre, Madison, WI). The ligated product was then transformed into E. coli DH5α (Invitrogen, Carlsbad, CA). The resulting plasmid (pIK01) was then purified and introduced into the ΔmfrA strain by electroporation as described previously (Wilson et al. 2003 (link)). Electroporated cells were spread on MH agar plates supplemented with kanamycin and chloramphenicol and incubated at 42°C for 3 days under microaerobic conditions. The resulting colonies were harvested and streak purified, and one colony (C-ΔmfrA), which was PCR-positive for the presence of mfrA, was selected for further studies.

The PCR amplified VP1 gene was blunt-end cloned into EcoRV-linearized pSK plasmid DNA with the Fast-Link™ DNA ligation kit (Epicentre) and transformed into E. coli DH10B to generate pSK-VP1. The pSK-VP1 plasmid was then digested with BamHI and NotI to recover the VP1 gene, which was ligated into pET28a digested with the identical restriction enzymes to yield pVP1. The recombinant plasmid was electroporated into E. coli BL21 (DE3) to generate the bacterial strain EC-VP1 that was used in all subsequent experiments. The EC-VP1 bacterial strain was stored as 0.5 mL glycerol stocks at − 80 °C until use.