Journal: Acta Crystallographica Section F: Structural Biology Communications

Article Title: Automated gradient equilibration of macromolecular crystals to new solution conditions

doi: 10.1107/S2053230X25008398

Figure Lengend Snippet: Left: schematic of equilibration setup. The crystal (green) sits in a 40 µl pot with two tubes used for the introduction of new solution and simultaneous withdrawal of the existing solution. Right: detail of the actual system. The crystal sits in the PCR tube cap, which sits on a glass cover slip. The blue housing holding the cover slip and tubing sits on a platform (red), which includes a port for the microscope.

Article Snippet: Next, the crystal(s) are prepared as follows. (i) A crystal pot is prepared by using a razor blade to separate a single transparent PCR tube cap from a strip of caps (for example catalog No. 27-110, Genesee Scientific, Morrisville, North Carolina, USA) and inverting the cap.

Techniques: Microscopy

Journal: Acta Crystallographica Section F: Structural Biology Communications

Article Title: Automated gradient equilibration of macromolecular crystals to new solution conditions

doi: 10.1107/S2053230X25008398

Figure Lengend Snippet: Overview of the complete system, showing the Raspberry Pi touch screen, red housing, the three syringe pumps and the blue housing which holds the PCR tube cap. The first two syringe pumps are loaded with syringes. Not visible in the image are the microscope, Arduino microcontroller with motor control shield, microscope (mounted below the red platform) and the Raspberry Pi (mounted on the back side of the touch screen). Each syringe pump is comprised of a black stepper motor coupled to a lead screw that moves the syringe plunger up and down via the white housing, which is threaded on the lead screw.

Article Snippet: Next, the crystal(s) are prepared as follows. (i) A crystal pot is prepared by using a razor blade to separate a single transparent PCR tube cap from a strip of caps (for example catalog No. 27-110, Genesee Scientific, Morrisville, North Carolina, USA) and inverting the cap.

Techniques: Microscopy, Control

Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet: Anticipated results, (a-f) Appearance of mixtures in the process of emulsification/de-emulsification, (a) A 2 ml-tube containing cells suspended in the ePCR buffer, oil mixture, and the rubber stopper from a 1-ml syringe before agitation on TissueLyser. The mixture appears clear and is separated into two phases, (b) Same mixture as in a after agitation on TissueLyser. The emulsion is viscous, and appears homogeneous and of milky white color, (c) The appearance of the mixture after being aliquoted in 12 × 100-μl PCR samples, subjected to thermal cycling, and pooled together in a 1.5 ml tube, (d) The appearance of the mixture in c after 10-min centrifugation. The mixture separated into two visible layers, with a top cloudy oil phase and a bottom remaining emulsion layer. The top oil phase is to be discarded. The remaining bottom emulsion layer appears as an amorphous white solid, (e) The appearance of a broken emulsion after phenol/chloroform/isoamyl alcohol addition and vortexing. The mixture is still cloudy but exhibits a greatly reduced viscosity. The bottom amorphous solid-like layer is no longer present, (f) The same mixture as in e after 2-min centrifugation. The mixture separated into two clear phases: the top aqueous phase (to be transferred to a new tube) and the bottom organic phase (to be discarded), (g) Example of a gradient of emulsion stability that can be generated under different emulsification conditions. After 30 rounds of PCR thermal cycles, the emulsions were visually analyzed for stability. A gradient of emulsion stabilities is observed, in which unstable emulsions separated into two phases (left), while stable emulsions remained opaque, with minimal phase separation (right). Green squares, intact emulsion; red squares, oil phase separated from disrupted emulsion droplets, (h) Phase-contrast microscopy image of 50x-diluted emulsion. Scale bar, 10 μlη. (i) Superimposed GFP fluorescence/phase-contrast microscopy image of emulsified GFP-expressing DH10B(DE3) E. coli cells under 40× magnification. Scale bar, 10 μlη. (j,k) Example of mock selection data. E. coli expressing either wild-type tyrosil-tRNA synthetase from Methanocatdcococcus jannaschii (MjYRS) or its nonfunctional variant (containing a stop codon and a Notl restriction site) were mixed at the indicated ratios and subjected to a single round of ePCR. (j) After the mock selection samples were amplified by re-amp PCR and equal amounts of DNA were restriction-digested by Notl, the DNA fragments were analyzed by gel electrophoresis to distinguish active (uncut) from inactive (cut) variants of MjYRS. Several thousandfold enrichment of active enzyme variant is observed. Star, active variant fragment size; arrows, inactive variant fragment sizes. Adapted with permission from ref. 29, American Chemical Society, (k) Gel-electrophoresis image of recovery PCR. 1: pure active MjYRS amplicon; 0: pure inactive MjYRS amplicon; 10_1-10−4: amplicons of activeiinactive MjYRS dilutions. (1) Monitoring enrichment progress by GFP assay. BL21 E. coli cells carrying pACYC-GFPmut2 plasmid (in which PT7 drives GFP expression) and plasmid ligations from the initial T7 RNAP selection rounds were assayed in a microplate reader for GFP fluorescence. XX, negative control T7 RNAP with two premature stop codons; WT, parental T7-RSS plasmid reported; R0, naive library; R1–R12, the output for each subsequent round during the selections for use of PT7; CGG-R7–8, a single clone from round 7 (this mutant was subject to error-prone PCR, yielding CGG-R7 epPCR); CGG-R12-KI, a single clone from R12; other CGG-R12 variants are selected combinations of mutations seen in the round 12 population. Data represent averages of three independently grown samples. Error bars represent 1 s.d. Adapted with permission from ref. 28, Nature Publishing Group.

Article Snippet: General equipment Water bath (Fisher, cat. no. FSGPD02) Incubator/shaker (New Brunswick, model no. Innova 44) Microcentrifuge (Eppendorf, model no. 5418) Vortex mixer (Scientific Industries, model no. SI-0236) Thermocycler (Bio-Rad, model no. T100) Microwave (LG, model no. LCS1112ST) 2-ml Microtubes (Eppendorf, cat. no. 022431048) 1.5-ml Microtubes (Eppendorf, cat. no. 022431021) PCR strip tubes (Genesee Scientific, cat. no. 27–125) GFP assay 96-Well black microplates (Corning, cat. no. CLS3915) 96-Well clear microplates (Corning, cat. no. 3370) Microplate reader (Tecan, M200 PRO) Library Transformation and Expression Electroporation cuvettes (0.2 cm gap; Bio-Rad, cat. no. 1652086) Electroporation apparatus (Bio-Rad, cat. no. 1652662) Petri dishes (Thermo Scientific, cat. no. 249964) Rattler plating beads (Zymo Research, cat. no. SI001) Spectrophotometer (Biochrom WPA, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) Falcon 50-rnl conical centrifuge tubes (Corning, cat. no. 352070) AirPore Tape Sheets (Qiagen, cat. no. 19571) Round-bottom polystyrene tubes (Corning, cat. no. 14–959–IB) Emulsion PCR Spectrophotometer (Biochrom WPA, model no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) 1 ml Syringes (Covidien-Medtronic, cat. no. 1180100555) TissueLyser LT (Qiagen, model no. 69980) Microman pipette for viscous liquids (Gilson, cat. no. F148504) Capillary pistons (Gilson, cat. no. CP 100) Inverted Epi-Fluorescence Phase Contrast Microscope (Olympus, model no. 1X51) FITC/GFP filter cube (Chroma, cat. no. 41001) Cellometer cell counting chambers (Nexcelom, cat. no. SD100) High-performance near-infrared charge-coupled device (CCD) camera, IEEE 1394 FireWire (Qlmaging, model RoleraXR) Microscope camera calibration slide (0.01-mm stage micrometer; OMAX, cat. no. CS-A36CALM1) ImageJ ( https://imagej.nih.gov ) Recovery PCR Gel electrophoresis equipment (Bio-Rad, cat. nos.

Techniques: Emulsification, Emulsion, Centrifugation, Viscosity, Generated, Microscopy, Fluorescence, Expressing, Selection, Variant Assay, Amplification, Nucleic Acid Electrophoresis, Plasmid Preparation, Negative Control, Mutagenesis

Journal: Nature protocols

Article Title: Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

doi: 10.1038/nprot.2017.119

Figure Lengend Snippet: Troubleshooting table.

Article Snippet: General equipment Water bath (Fisher, cat. no. FSGPD02) Incubator/shaker (New Brunswick, model no. Innova 44) Microcentrifuge (Eppendorf, model no. 5418) Vortex mixer (Scientific Industries, model no. SI-0236) Thermocycler (Bio-Rad, model no. T100) Microwave (LG, model no. LCS1112ST) 2-ml Microtubes (Eppendorf, cat. no. 022431048) 1.5-ml Microtubes (Eppendorf, cat. no. 022431021) PCR strip tubes (Genesee Scientific, cat. no. 27–125) GFP assay 96-Well black microplates (Corning, cat. no. CLS3915) 96-Well clear microplates (Corning, cat. no. 3370) Microplate reader (Tecan, M200 PRO) Library Transformation and Expression Electroporation cuvettes (0.2 cm gap; Bio-Rad, cat. no. 1652086) Electroporation apparatus (Bio-Rad, cat. no. 1652662) Petri dishes (Thermo Scientific, cat. no. 249964) Rattler plating beads (Zymo Research, cat. no. SI001) Spectrophotometer (Biochrom WPA, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) Falcon 50-rnl conical centrifuge tubes (Corning, cat. no. 352070) AirPore Tape Sheets (Qiagen, cat. no. 19571) Round-bottom polystyrene tubes (Corning, cat. no. 14–959–IB) Emulsion PCR Spectrophotometer (Biochrom WPA, model no. {"type":"entrez-nucleotide","attrs":{"text":"C08000","term_id":"1533071","term_text":"C08000"}} C08000 ) 1 ml Syringes (Covidien-Medtronic, cat. no. 1180100555) TissueLyser LT (Qiagen, model no. 69980) Microman pipette for viscous liquids (Gilson, cat. no. F148504) Capillary pistons (Gilson, cat. no. CP 100) Inverted Epi-Fluorescence Phase Contrast Microscope (Olympus, model no. 1X51) FITC/GFP filter cube (Chroma, cat. no. 41001) Cellometer cell counting chambers (Nexcelom, cat. no. SD100) High-performance near-infrared charge-coupled device (CCD) camera, IEEE 1394 FireWire (Qlmaging, model RoleraXR) Microscope camera calibration slide (0.01-mm stage micrometer; OMAX, cat. no. CS-A36CALM1) ImageJ ( https://imagej.nih.gov ) Recovery PCR Gel electrophoresis equipment (Bio-Rad, cat. nos.

Techniques: Growth Assay, Concentration Assay, Positive Control, Western Blot, Sequencing, Expressing, Amplification, Emulsion, Plasmid Preparation, Selection, Variant Assay, Emulsification, Functional Assay

Journal: Cell reports. Medicine

Article Title: Early activation of inflammatory pathways in UBA1-mutated hematopoietic stem and progenitor cells in VEXAS.

doi: 10.1016/j.xcrm.2023.101160

Figure Lengend Snippet: Figure 1. Myeloid dominance and activa- tion of the inflammatory pathways in VEXAS BMMNCs (A) Experimental workflow. BMMNC samples from patients and healthy donors were subjected to multi-color flow cytometry to profile hematopoi- etic stem and progenitor cell (HSPC) sub- populations, and to ELISpot assay to quantify BMMNCs secreting TNF-a or IFN-g. BMMNCs and FACS-sorted LineageCD34+ cells were subjected to colony forming assay and single-cell RNA sequencing (scRNA-seq) using the 10x Ge- nomics platform. scRNA-seq libraries were sequenced on the Illumina NovaSeq system before data analysis, including single-cell tran- scriptome profiling (gene expression, gene muta- tion, and cell-cell interaction) and single-cell T cell receptor/B cell receptor (scTCR/BCR) profiling. (B) A Uniform Manifold Approximation and Pro- jection (UMAP) plot of single-cell gene expression in BMMNCs of all patients and healthy donors. Cells are colored by types (HSPC, erythroblast, neutrophil, monocyte, T cell, NK cell, B cell, plasma cell, eosinophil, and dendritic cell). A bar chart shows percentages of these cell populations in individual patients and healthy donors. The co- lor legend is the same as that in the UMAP plot. A dot plot showing a myeloid (erythroblast, neutro- phil, monocyte, and dendritic cell) vs. lymphoid (T cell, B cell, NK cell, and plasma cell) ratio in pa- tients and healthy donors. Data are presented as mean values ± standard error of the mean (SEM). p values with the two-sided unpaired Mann-Whitney test are shown. (C) Heatmap showing expression of representa- tive differentially expressed genes grouped by their functional pathways in IFN-g and IFN-a signaling, TNF-a via NF-kB signaling, inflamma- tory response, E2F targets, and apoptosis, be- tween BMMNCs from VEXAS patients (n = 9) and healthy controls (n = 4). Values are presented as log2 fold-changes (log2FC). (D) Gene set enrichment analysis (GSEA) of ex- pressed genes in BMMNC subpopulations of VEXAS patients, including neutrophils, mono- cytes, erythroblasts, T cells, B cells, and NK cells. Normalized enrichment scores for the GSEA pathways are plotted, showing higher enrichment of the inflammatory pathways in neutrophils and monocytes than those in lymphoid cells. (E) Representative ELISpot wells showing TNF-a secretion by BMMNCs from two VEXAS patients and two healthy donors in a second batch of the validation cohort, in triplicate. Bottom, quantifi- cation of TNF-a-, IFN-g-, and TNF-a/IFN-g-posi- tive spots in BMMNCs plated (VEXAS patients n = 5 and healthy donors n = 2, in triplicate). Data are presented as mean values ± standard error of the mean (SEM). p values with the two-sided unpaired Mann-Whitney test are shown.

Article Snippet: ELISpot assay to check IFN-g and TNF-a secreted by human BMMNCs IFN-g and TNF-a secretion from BMMNCs of VEXAS patients and healthy donors were measured using the Human IFN-g/TNF-a Double-Color Enzymatic ELISPOT Assay kit (Cat# SKU:hIFNgTNFa-2M, ImmunoSpot) in two separate experiments in triplicate (4 patients versus 3 healthy donors for a 1st batch, and 5 patients versus 2 healthy donors for a 2nd batch), according to the manufacturer’s protocol.

Techniques: Cytometry, Enzyme-linked Immunospot, RNA Sequencing, Gene Expression, Clinical Proteomics, MANN-WHITNEY, Expressing, Functional Assay, Biomarker Discovery