Journal: Methods in enzymology

Article Title: Quantitative Measurement of PROTAC Intracellular Accumulation

doi: 10.1016/bs.mie.2022.11.001

Figure Lengend Snippet: 2.2. KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Cell Lines E. coli derived from the BLT5403 strain T7 Select 10–3 strain Novagen 70548–3 Chemicals, Peptides, and Recombinant Proteins SEA BLOCK Blocking Buffer Thermo Scientific 37527 nLuc-BTK construct (full length) Eurofins Customized EcoRI-HF NEB R3101S HindIII NEB R0104S T4 DNA Ligase NEB M0202T T4 DNA Ligase Reaction Buffer NEB B0202S Streptavidin-coated magnetic beads Thermo Scientific 88816 biotinylated small molecule ligands Critical Commercial Assays MinElute PCR Purification Kit Qiagen 28004 T7Select ® Packaging Kit Novagen 70014–3 Open in a separate window 2.2.

Techniques: Derivative Assay, Recombinant, Blocking Assay, Construct, Magnetic Beads, Purification

Journal: mBio

Article Title: Stabilizing Genetically Unstable Simple Sequence Repeats in the Campylobacter jejuni Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes

doi: 10.1128/mBio.01401-21

Figure Lengend Snippet: Overview of the procedures of natural transformation and cotransformation in C. jejuni in this study. First, donor DNA fragments used for natural transformation were amplified by PCR. For the C. jejuni transformation, each of the resulting PCR fragments had not only a positive selection marker (e.g., antibiotic resistance gene and gain-of-function mutation) flanking regions homologous to the target gene but also an EcoRI methyltransferase-recognition sequence, GAATTC (RI) (termed “selected” fragment). Methylated RI is thought to act as a Campylobacter -specific DNA uptake signal . Therefore. the next steps were to methylate the selected fragment by EcoRI methyltransferase and to use it as the substrate for DNA uptake during transformation. Finally, resulting transformants were positively selected under appropriate conditions. On the other hand, natural “cotransformation” involved the “unselected” fragment(s) that had a negative selection marker (e.g., loss-of-function mutation) or unselectable mutation (e.g., silent mutation), in addition to the selected fragment. In this case, the selected fragment was used to replace a neutral gene with a positive selection marker, while the unselected fragments were used to introduce unmarked mutations of interest at single or multiple loci. These different DNA fragments were methylated and then mixed with a recipient strain to simultaneously introduce different mutations into the genome. The cotransformation reactions were subjected to positive selection for integration of the selected fragment and subsequently screened to assess the integration of the unselected fragments by negative selection or PCR using allele-specific primers. Cotransformation has been recently adapted to multiplex genome editing by natural transformation (MuGENT), a method for introducing multiple mutations into the genomes of Vibrio cholerae and Streptococcus pneumoniae . In this study, we optimized natural transformation and cotransformation and demonstrated its utility as a method for multiplex genome editing in C. jejuni .

Article Snippet: DNA methylation was performed using a 25-μl mixture containing 0.5 or 1.5 pmol DNA, EcoRI methyltransferase reaction buffer (New England BioLabs; 50 mM Tris-HCl, 50 mM NaCl, and 10 mM ethylenediaminetetraacetic acid [pH 8.0]), 80 μM S -adenosylmethionine (New England Biolabs), and 40 units of EcoRI methyltransferase (New England BioLabs).

Techniques: Transformation Assay, Amplification, Selection, Marker, Mutagenesis, Sequencing, Methylation, Introduce, Multiplex Assay

Journal: mBio

Article Title: Stabilizing Genetically Unstable Simple Sequence Repeats in the Campylobacter jejuni Genome by Multiplex Genome Editing: a Reliable Approach for Delineating Multiple Phase-Variable Genes

doi: 10.1128/mBio.01401-21

Figure Lengend Snippet: Schematic representation of donor DNA fragments used for the optimization of natural transformation and cotransformation in the recipient C. jejuni strains NCTC11168 and 81-176. (A) rpsL fragments with different lengths of homologous regions compared with the recombination target sequence (50 to 2,000 bp, indicated in the right) and different numbers of EcoRI methyltransferase-recognition sequences (indicated by RIs). An asterisk indicates the rpsL K88R mutation that confers streptomycin resistance. (B) flaA fragments with 1,000-bp homologous regions and two RIs. The Δ flaA :: kan was generated by replacing flaA with a kanamycin resistance gene ( kan ), whereas the flaA + contained an intact flaA sequence. In both panels, each of the fragments with underlines (indicated in the left, rpsL K88R -3 to -9, rpsL + -8, rpsL + -9, Δ flaA :: kan , and flaA + ) consisted of two sequence variants to be used for their respective recipient strains (see Table S2 , fragments labeled “A” for NCTC11168 and labeled “B” for 81-176, e.g., rpsL K88R -3 consisted of rpsL K88R -3-A and rpsL K88R -3-B for NCTC11168 and 81-176, respectively) because of genomic variations at the target sequences in these strains. Sequences of the fragments not underlined ( rpsL K88R -1 and rpsL K88R -2) were identical between NCTC11168 and 81-176 and thus were shared with these strains.

Article Snippet: DNA methylation was performed using a 25-μl mixture containing 0.5 or 1.5 pmol DNA, EcoRI methyltransferase reaction buffer (New England BioLabs; 50 mM Tris-HCl, 50 mM NaCl, and 10 mM ethylenediaminetetraacetic acid [pH 8.0]), 80 μM S -adenosylmethionine (New England Biolabs), and 40 units of EcoRI methyltransferase (New England BioLabs).

Techniques: Transformation Assay, Sequencing, Mutagenesis, Generated, Labeling