Rapid dna ligation kit

Synthetic DNA encoding LldR, CanlonicSF, Green Lindoblum, GEM-IL ver.3, and FiLa were purchased from Integrated DNA Technologies. A gene encoding Laconic was obtained from Addgene (Addgene plasmid #44238; http://n2t.net/addgene:44238). Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific) was used for routine polymerase chain reaction (PCR) amplifications, and Taq DNA polymerase (New England Biolabs) was used for error-prone PCR. Restriction endonucleases, rapid DNA ligation kits, and GeneJET miniprep kits were purchased from Thermo Fisher Scientific. PCR products and products of restriction digests were purified using agarose gel electrophoresis and the GeneJET gel extraction kit (Thermo Fisher Scientific). DNA sequences were analyzed by DNA sequence service of Fasmac Co., Ltd. Fluorescence excitation and emission spectra were recorded on a Spark plate reader (Tecan).

The strains and plasmids used in this study are described in S1 Table. E. coli DH5α and E. coli DH1 were used for plasmid construction and chromosomal modification, respectively. Plasmids pKOBEG and pKOBEGA were acquired from Christophe d'Enfert [5 (link)]. Gene I-CreI and I-SceI were artificially synthesized by GENEWIZ Company and cloned into the KpnI/BamHI site of pUC19 to generate pUCIS and pUCIC. The sequences of I-SceI and I-CreI are described in S2 Table. The commercial plasmid pET28a was purchased from EMD Biosciences (Novagen). Plasmids pKOBEGK, pSNA, pSNK, and pCNA were derived from pKOBEG or pKOBEGA, and plasmid maps of all plasmids containing the λ-Red system or endonuclease gene used in this study are shown in S1 Fig. The construction process for pKOBEGK, pSNA, pSNK, and pCNA is shown in S2 Fig. The Rapid DNA Ligation Kit, DNA polymerase, Gel Extraction Kit, and Plasmid Miniprep Kit were purchased from Thermo Fisher Scientific Inc. (MA, USA), TaKaRa Biotechnology Co. (Dalian, China), or Corning Inc. (Wujiang, China). All assays were performed according to the manufacturer’s instructions.

All restriction enzymes used were fast digest enzymes (ThermoFisher Scientific, United Kingdom), the ligase used throughout this work was the Rapid DNA Ligation Kit (ThermoFisher Scientific, United Kingdom). Plasmids were transformed into chemically competent E. coli HB101 according to supplier’s instructions (New England Biolabs, United Kingdom). Plasmids are listed in Supplementary Table 1 and primers and synthetic DNA fragments are listed in Supplementary Table 2.

The sseJ-HA fusion or ompR DNA sequences were amplified using primers XhoI-PsseJ #F and sseJ-HA-BamHI #R or KpnI-PompR #F and envZ-HindIII #R from Salmonella 14028s genomic DNA. The PCR products and low copy promoter-less pWSK29 vector were double-digested with restriction enzymes XhoI and BamHI or KpnI and HindIII (Thermo Fisher Scientific), and the digested samples were ligated with the Rapid DNA Ligation Kit (Thermo Fisher Scientific). The cadA, cadB, cadC, ompC, ompF, mgtC, atpB, and sseB genes were deleted from the Salmonella chromosome using λ-Red recombination techniques as described previously [62 (link),63 (link)]. A cadBA over-expressed strain was generated by cloning cadBA into plasmid pBR322, replacing the bla gene with cadBA. SsaC-GFP and SsaJ-mCherry were constructed as follows: plasmid pWSK29 containing ssaC with the spiC promoter fused before GFP and ssaJ with the sseA promoter fused before mCherry were inserted in ssaC and ssaJ null strains, respectively. Primers are listed in S1 Table. A list of strains and plasmids are provided in S2 Table.

Unless otherwise stated, oligonucleotides were from Eurofins Operon. KOD polymerase and Escherichia coli DE3 pLysS Rosetta cells were from Novogen. RNase inhibitor murine and T7 RNA polymerase were from New England Biolabs. Glycogen was from Roche, Proteinase K from Invitrogen, and Heparin Sepharose from Sigma.
The rapid DNA ligation kit was from Thermo Scientific. QIAquick PCR purification, QIAquick Gel elution, and QIAquick nucleotide removal kits were from Qiagen. The QuikChange site-directed mutagenesis kit was from Agilent. A 2 ml HiTrap heparin column and Superdex™ 200 gel filtration column were from GE Healthcare Life Sciences.

To generate plasmid DNAs encoding 3XFLAG epitope-tagged HEXIM1, LARP7, CDK9 and RBM7, the cDNAs were amplified using Phusion High-Fidelity DNA polymerase (NEB) with primers carrying the appropriate restriction enzymes sites and cloned using Rapid DNA Ligation Kit (Thermo Fisher Scientific) into pcDNA5/FRT/TO vector, which was modified to encode 3XFLAG peptide upstream of the multiple cloning site (a gift from Dr. Ule). Q5® Site-Directed Mutagenesis Kit (NEB) was used for generating pcDNA5/FRT/TO/3XFLAG-RBM7 mRNP1 plasmid encoding the mutant mRNP1 F-RBM7 protein. RBM7 mutagenesis primer sequences and plasmids used in this study are listed in the Key Resources Table. To generate plasmid DNAs encoding the MBP-tagged RBM7 and mRNP1 RBM7 the cDNAs were cloned into a modified pET28a vector containing an N-terminal MBP sequence followed by a TEV protease cleavage site. To generate baculovirus transfer vector encoding His-tagged CDK9, its cDNA was cloned into pACEBac1 with a sequence encoding OctaHis-tag. All other cDNAs encoding full-length HEXIM1 and LARP7 as well as MePCE (aa 400-689) and CycT1 (aa 1-272) were cloned into a pGEX4T1 vector modified to contain a TEV protease cleavage site between the GST tag and the protein.