sbfi  (New England Biolabs)

Journal: Journal of Virology

Article Title: Conserved CxnC Motifs in Kaposi’s Sarcoma-Associated Herpesvirus ORF66 Are Required for Viral Late Gene Expression and Are Essential for Its Interaction with ORF34

doi: 10.1128/JVI.01299-19

Figure Lengend Snippet: ORF66 is essential in KSHV and required for late gene transcription. (A) (Left) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. (Right) The mutations were confirmed by Sanger sequencing. (B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that the introduction of mutations did not introduce large-scale changes. (C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. ****, P

Article Snippet: BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs).

Techniques: Sequencing, Recombinant, Introduce, Flow Cytometry

Journal: PLoS ONE

Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

doi: 10.1371/journal.pone.0096217

Figure Lengend Snippet: Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).

Article Snippet: Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON).

Techniques: Clone Assay, Expressing, Purification, Recombinant, Amplification, Plasmid Preparation, Transformation Assay, Sequencing, SDS Page, Affinity Chromatography, Incubation, Fast Protein Liquid Chromatography, Western Blot

Journal: bioRxiv

Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

doi: 10.1101/728139

Figure Lengend Snippet: ORF66 is essential in KSHV and required for late gene transcription A) Diagram showing the genomic locus of ORF66 with surrounding genes ORF67 (which partially overlaps ORF66) and ORF65, depicting the location of introduced mutations. Mutations were confirmed by Sanger sequencing (right). B) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that introduction of mutations did not introduce large-scale changes. C) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

Article Snippet: BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs).

Techniques: Sequencing, Recombinant, Introduce, Flow Cytometry

Journal: bioRxiv

Article Title: Conserved CxnC motifs in Kaposi’s sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and mediate its interaction with ORF34

doi: 10.1101/728139

Figure Lengend Snippet: ORF24 does not bind to late gene promoters in the absence of ORF30 or ORF66 iSLK cell lines were created using the recombinant BAC16 system. HA tags were added to the endogenous copies of the N-terminus of ORF24 (HA24) or the C-terminus of ORF66 (66HA). In select BACs, ORF24, ORF30, or ORF66 were deleted by the introduction of a stop codon early in the ORF (24S, 30S, and 66S respectively). A) Digestion of the recombinant BACs with SbfI or RsrII demonstrates that recombination did not introduce large-scale changes. B) Infectious virion production was measured by supernatant transfer from reactivated iSLK cell lines followed by flow cytometry. Data are from three independent biological replicates with statistics calculated using an unpaired t-test, where (****) p

Article Snippet: BAC quality was assessed by digestion with RsrII and SbfI (New England Biolabs).

Techniques: Recombinant, Introduce, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

doi: 10.1074/jbc.RA118.003302

Figure Lengend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

Article Snippet: Each fosmid was digested with SbfI (New England Biolabs), a unique restriction site in the pSMART FOS vector.

Techniques: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation