r0535  (New England Biolabs)

Journal: PLoS ONE

Article Title: Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha

doi: 10.1371/journal.pone.0205117

Figure Lengend Snippet: All-in-one vector systems for genome editing in M . polymorpha . (A) Designs of Gateway-based all-in-one binary vectors and entry plasmids for gRNA cloning. pMpGE010 and pMpGE011 contain a cassette for the expression of Atco-Cas9 fused with an NLS under the control of Mp EF pro , a Gateway cassette, and a cassette for the expression of hygromycin phosphotransferase ( HPT ) in pMpGE010 and mutated acetolactate synthase ( mALS ) in pMpGE011. pMpGE_En01 contains recognition sites for two restriction enzymes, SacI and PstI, upstream of a gRNA backbone for the insertion of a guide sequence by In-Fusion/Gibson cloning, which automatically places a G nucleotide for transcription initiation by RNA polymerase III (extra initial G). Expression of single guide RNAs is controlled by a 2 kbp fragment of Mp U6-1 pro . pMpGE_En02 and pMpGE_En03 contain two BsaI recognition sites upstream of the gRNA backbone for the insertion of a guide sequence by ligation without or with an “extra initial G,” whose expression is under the control of a 500 bp Mp U6-1 pro fragment. For all the entry vectors, the gRNA cassette is flanked by the attL1 and attL2 sequences and is thus transferrable to the Gateway cassette in pMpGE010 or pMpGWB011 by the LR reaction. (B) Designs of all-in-one binary vectors for direct gRNA cloning. pMpGE013 ( HPT marker) and pMpGE014 ( mALS marker) contain the Atco-Cas9-NLS expression cassette, a unique AarI site in the upstream of the gRNA backbone for insertion of a guide sequence by ligation with an “extra initial G,” whose expression is under the control of a 500 bp Mp U6-1 pro fragment.

Article Snippet: Conversely, annealed oligos of NOP1_2 gRNAs were subjected to ligation reactions with pMpGE_En03 linearized with BsaI (NEB).

Techniques: Plasmid Preparation, Clone Assay, Expressing, Sequencing, Ligation, Marker

Journal: Microbial Cell Factories

Article Title: A novel one-step approach for the construction of yeast surface display Fab antibody libraries

doi: 10.1186/s12934-017-0853-z

Figure Lengend Snippet: One step generation of YSD plasmids for the construction of large combinatorial Fab immune libraries using Golden Gate Cloning. Destination plasmids (pDest), entry plasmids (pE) and PCR amplicons contain or are flanked by Bsa I recognition sites in different orientations (B: ggtctcn, B : ngagacc). A linear and distinct assembly of those DNA fragments is ensured by the design of complementary signature sequences in defined order within the three modules after Bsa I cleavage. a The two-directional (2dir) display system enables the expression of the VH-CH1-Aga2p (Aga2p-signal-sequence; SP) gene product under control of the GAL1 -promoter whereas the cLC-CLkappa (app8-signal-sequence; App8 SP) gene product is generated under control of the Gal10 -promoter. b The bicistronic display system (bicis) allows for the expression of Fab-fragment heavy and light chains under control of the GAL1 -promoter. The generation of distinct VH-CH1-Aga2p (Aga2p-signal-sequence; SP) and cLC-CLkappa (app8-signal-sequence; App8 SP) proteins is mediated by ribosomal skipping due to the T2A (2A) peptide. c Schematic illustration of Fab-fragments displayed on the surface of yeast cells. Genes are encoded by a single plasmid and expression is either conducted by two-directional promotors or by ribosomal skipping

Article Snippet: 160 ng of VH PCR product and 160 ng VL PCR product as well as 200 U Bsa I (New England Biolabs), 800 U T4 DNA ligase (New England Biolabs) and 10 µL 10× T4 Ligase buffer (New England Biolabs).

Techniques: Clone Assay, Polymerase Chain Reaction, Expressing, Sequencing, Generated, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

doi: 10.1093/nar/gky596

Figure Lengend Snippet: Identification of suitable methylases for methylation-switching. ( A ) Initial screening of functional methylases for selective blocking of overlapping methylation/restriction sites. The diagrams show the design of overlapping sites for screening of methylase activity. The table shows the screening result using methylases expressed in vivo from an F-ori based low copy number vector. Restriction enzyme recognition sites are boxed in solid lines. The adhesive ends generated by the restriction enzyme are shown by solid lines. Methylase recognition sites are boxed in dashed lines, and methylated bases are in bold font. All the listed methylases modify N6-adenine, except M.SacI and M.AspJHL3I, which modify C5-cytosine and N4-cytosine respectively. ( B ) Experimental designs to test blocking of methylation-switchable type IIS restriction enzyme sites by in vivo methylation. For each methylase/restriction enzyme combination tested, the test plasmid contains a head-to-head potentially methylation-switchable restriction site and a non-methylatable restriction site. Restriction digestion of test plasmid prepared from a normal E. coli strain would result in cutting at both sites and the release of a 600 bp fragment from the 4.3 kb vector backbone. Restriction digestion of test plasmid prepared from a strain expressing the switch methylase would result in a single 4.9 kb band, due to blocking of the methylation-switchable restriction sites by in vivo methylation. The restriction sites of the test plasmids for each restriction enzyme are shown, with the restriction site boxed in solid line, the methylase recognition site boxed in dashed line, and the methylated bases in bold. The head-to-head arrangement of overlapping methylation/restriction site allows the same assay to be used to detect any residual single strand nicking activity of the restriction enzyme towards the methylated restriction site. ( C ) Agarose gel electrophoresis analysis of the test plasmids for each methylation-switchable restriction site after preparation of the plasmids in a normal strain (–) or in a strain expressing the appropriate DNA methylase (+) and digested with the corresponding type IIS restriction enzymes. The combinations tested were BsaI with M.Osp807II methylase using test plasmid pMOP_BsaINC, BpiI with M2.NmeMC58II methylase using test plasmid pMOP_BpiINC, and LguI with M.XmnI methylase using test plasmid pMOP_LguINC. Test conditions were 60 fmol test plasmid digested using 5 U BsaI or BpiI, or 2.5 U LguI in 10 μl reactions at 37°C for 1 h. The results show that in vivo methylation by each of the methylases successfully blocked the restriction site for the corresponding type IIS restriction enzyme when the methylase recognition site overlapped the restriction enzyme site. The data shown represents results from three independent experiments.

Article Snippet: In stage one, the fragments were assembled, seven per group, using 15 fmol of each donor plasmid prepared from DH10B cells, 15 fmol assembly vector prepared from DH10B-M.Osp807II cells, 1000 U T4 ligase (NEB), and 5 U BsaI (NEB) in 20 μl 1× T4 ligase buffer (NEB).

Techniques: Methylation, Functional Assay, Blocking Assay, Activity Assay, In Vivo, Low Copy Number, Plasmid Preparation, Generated, Expressing, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

doi: 10.1093/nar/gky596

Figure Lengend Snippet: BsaI-M.Osp807II based MetClo system. The donor plasmids contain DNA fragments to be assembled (Fragments A–D) flanked by BsaI sites that generate compatible adhesive ends (schematically labelled aaaa-eeee). The BsaI sites overlap with the M.Osp807II methylase recognition sequence. Donor plasmids were prepared from a normal strain that does not express the M.Osp807II switch methylase. As a result, the BsaI sites are not methylated and so the insert DNA fragments can be released by BsaI digestion. The recipient assembly vector contains a LacZα selection marker flanked by head-to-head BsaI sites. The outer pair of BsaI sites closer to the vector backbone overlap with an M.Osp807II methylation sequence and so are methylation-switchable, whereas the inner pair of BsaI sites are not. Preparation of the assembly vector in the M.Osp807II switch methylase-expressing DH10B strain results in selective blocking of the outer pair of BsaI sites. The LacZα fragment can be released by BsaI through cutting at inner pair of BsaI sites. Following a one-pot reaction using BsaI and T4 DNA ligase, ligation among compatible adhesive ends results in ordered assembly of DNA fragments into the assembly vector backbone. The assembled fragment in the assembled plasmid is flanked by methylated BsaI sites, which are not cut by BsaI. Following transformation into a normal strain that does not express the M.Osp807II switch methylase, methylation of the flanking restriction sites is lost, and the assembled fragment can be released by BsaI for the next stage assembly.

Article Snippet: In stage one, the fragments were assembled, seven per group, using 15 fmol of each donor plasmid prepared from DH10B cells, 15 fmol assembly vector prepared from DH10B-M.Osp807II cells, 1000 U T4 ligase (NEB), and 5 U BsaI (NEB) in 20 μl 1× T4 ligase buffer (NEB).

Techniques: Sequencing, Methylation, Plasmid Preparation, Selection, Marker, Expressing, Blocking Assay, Ligation, Transformation Assay

Journal: Nucleic Acids Research

Article Title: MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

doi: 10.1093/nar/gky596

Figure Lengend Snippet: Design of a standard MetClo vector set. Three types of adaptor sequences were designed for the standard MetClo vector set. The vectors contain two head-to-head BsaI sites (boxed) flanking the negative selection marker LacZα. The pair of unmethylated BsaI sites closest to the negative selection marker can be used to release LacZα and generates adhesive ends ‘p’ and ‘q’. This allows any fragments that start with adhesive end ‘p’ and end with adhesive end ‘q’ to be cloned into any of the three types of adaptor sequences (types ‘Start’, ‘Middle’, ‘End’). The outer pair of BsaI sites closest to the vector backbone overlap with the M.Osp807II recognition sequence and are both methylated at the adenine bases highlighted in bold when the vector is prepared in M.Osp807II-expressing E. coli strain. A DNA fragment assembled into these vectors, following transformation into a normal E. coli strain which lacks this switch methylase activity, can be released from the assembled plasmid using BsaI, which can now recognize this unmethylated BsaI site. The released fragment will carry different adhesive ends depending on the type of the vector used. Assembly into a type ‘Start’ vector will generate a fragment flanked by adaptors ‘p-a’; assembly into a type ‘Middle’ vector will generate a fragment flanked by ‘a-b’, ‘b-c’, ‘c-d’ or ‘d-e’; assembly into a type ‘End’ vector will generate a fragment flanked by ‘a-q’, ‘b-q’, ‘c-q’, ‘d-q’ or ‘e-q’. The design of these LacZα selection cassettes flanked by unique adaptors are represented in the figure by the letter codes for the outside adaptor sequences.

Article Snippet: In stage one, the fragments were assembled, seven per group, using 15 fmol of each donor plasmid prepared from DH10B cells, 15 fmol assembly vector prepared from DH10B-M.Osp807II cells, 1000 U T4 ligase (NEB), and 5 U BsaI (NEB) in 20 μl 1× T4 ligase buffer (NEB).

Techniques: Plasmid Preparation, Selection, Marker, Clone Assay, Sequencing, Methylation, Expressing, Transformation Assay, Activity Assay