The products resulting from site-specific recombination were transformed into chemically competent E. coli (DH5-α) and plated onto solid LB medium containing Zeocin. Two isolated colonies were selected for each reaction and the clones were verified by colony-PCR with pDONR™/Zeo-specific primers. The clones that had an insert of the expected size were picked for plasmid isolation
and the plasmid AP24534 supplier preparations were sequenced with a pDONR™/Zeo-specific forward and reverse primers to verify the insert from both N-terminal and C-terminal ends of the ORFs. All the sequencing reads were analyzed using NCBI standalone BLAST against the phage lambda genome to confirm the identity of each ORF. We obtained 68 entry clones out of 73 targeted lambda ORFs (see Additional file 1: Table S1). Yeast two-hybrid clones All the lambda
phage ORFs in the entry vectors are sub-cloned into yeast two-hybrid expression vectors (Table 3), by using the LR Clonase™ II Enzyme Mix (Invitrogen). The destination vectors used were pDEST22, pDEST32 (Invitrogen), pGADT7g, pGBKT7g and pGADCg, pGBKCg vectors . Yeast two-hybrid screening We carried out comprehensive Y2H interaction screening with the following Y2H vector pairs: pDEST32-pDEST22, pGBKT7g-pGADT7g, pGBKT7g-pGADCg, pGBKCg-pGADCg and pGBKCg-pGADT7g (listed as bait-prey vector pair). In the array screening we tested each protein both as activation (prey) and DNA-binding domain fusion selleck screening library (bait), including C-terminal fusions in pGBKCg and pGADCg. This way, we tested each protein pair in ten different configurations (Figure 2). The yeast two-hybrid assays were conducted as described in detail by Rajagopala et al. [10, 30]. Data availability The protein interactions from this publication have been submitted to the IMEx http://www.imexconsortium.org consortium through IntAct
http://www.ebi.ac.uk/intact/ and assigned the identifier IM-15871. Acknowledgements Svetlana Shtivelband and Kenny Huang helped in an early phase of this project with cloning lambda ORFs. We thank Johannes Goll for the PPIs statistical analysis. PU was funded by NIH grant R01GM79710 and the European Union (grant HEALTH-F3-2009-223101). SC acknowledges supported by the NIH (grant AI074825). Electronic supplementary material Additional Ketotifen file 1: Tables S1-S7(Excel spreadsheet with tables in individual sheets). S1. Lambda pDONR clones. S2. Lambda protein-protein interactions from Y2H screening. S3. Lambda protein-protein interactions with high prey count (unspecific interactions). S4. Phage Lambda Genome Anotation (Uniprot). S5. Protein interaction with different functional groups. S6. Protein interaction confidence buy ON-01910 assessment. S7. Layout of Y2H preys pGADT7g and pGADC on screening plates. (XLS 156 KB) References 1. Lederberg E: Lysogenicity in E. coli K-12. Genetics 1951, 36:560. 2. Wommack KE, Colwell RR: Virioplankton: viruses in aquatic ecosystems.