Journal: Communications Biology

Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

doi: 10.1038/s42003-021-01649-6

Figure Lengend Snippet: a Schematic illustration of spike protein expression on the cell surface and soluble ACE2-Fc staining followed by an anti-Fc antibody staining. b 293 cells transfected with spike protein with or without endoplasmic reticulum retention signal (ERRS) or with VSV-G as a negative control. The cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, flow cytometry data overlays are shown. c Schematic representation of spike protein pseudovirus generation and subsequent infection of ACE2-expressing host cells. A lentivector plasmid and a spike protein over-expressing envelope plasmid are used to co-transfect 293 cells to generate spike pseudovirus that in turn infect engineered cells over-expressing wild-type ACE2 or ACE2-mKO2 fusion. d Infection of wild-type 293 cells with either bald lentiviruses generated without envelope plasmid or spike protein pseudovirus. e Infection of 293-ACE2 cells with bald and spike lentiviruses. GFP and mKO2 markers are used to determine ACE2 over-expressing cells in ACE2-IRES-GFP and ACE2-mKO2, respectively. f The titrations of SARS-CoV-2 and SARS-CoV spike protein pseudoviruses encoding RFP. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Brown, red, salmon and orange-colored lines show direct infection, first, second and third freeze/thaw cycles, respectively. ACE2-IRES-GFP expressing 293 cells were incubated with threefold serial dilutions of virus supernatant, stored for several hours at 4 °C or serially frozen and thawed for 1, 2 and 3 cycles, and analyzed for RFP expression by flow cytometry on day 3 post-infection. Percent infection is % RFP+ cells after gating on GFP+ cells (i.e., ACE2+). Titration experiments were replicated twice except for the ‘1 freeze/thaw cycle’ for which titrations were replicated four times. Error bars represent 1 standard deviation of mean values.

Article Snippet: The sequence of mKO2 (monomeric Kusabira-Orange-2 ) obtained from Addgene (#54625) , and was added onto the C terminal of ACE2 proximal to the stop codon with a small linker peptide (ccggtcgccacc) encoding the amino acids ‘PVAT’.

Techniques: Expressing, Staining, Transfection, Negative Control, Flow Cytometry, Infection, Plasmid Preparation, Generated, Incubation, Virus, Titration, Standard Deviation

Journal: Communications Biology

Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

doi: 10.1038/s42003-021-01649-6

Figure Lengend Snippet: a Illustration of spike-protein pseudovirus blocked by soluble ACE2 or neutralizing antibodies. b SARS-CoV-2 and SARS-CoV pseudovirus neutralization with soluble ACE2. SARS-CoV-2 RFP and SARS-CoV GFP pseudoviruses were pre-incubated with soluble ACE2 for 1 h and added to 293 cells expressing ACE2-IRES-GFP or ACE2-mKO2 fusion, respectively. c Neutralization of SARS-CoV-2 and SARS-CoV with S-RBD-specific antibodies and soluble ACE2 (sACE2). Viruses were pre-incubated with antibodies (NAb#1 and SARS-CoV-2 S-RBD non-NAb) or soluble ACE2 (sACE2) proteins for 1 h at the concentrations shown and subsequently added to target cells. Expression of RFP was determined at day 3 post-infection. Infection percentages were normalized to negative controls which are the infection conditions with no blocking agent. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Red, green, blue and turquoise colored lines show SARS-CoV-2 S-RBD NAb#1, SARS-CoV-2 S-RBD non-NAb, soluble ACE2 #1 and #2, respectively. d Neutralization of SARS-CoV-2 pseudoviruses using 4 different S-RBD NAbs and two different soluble ACE2 proteins. NAb #1 and #4 were human antibodies whereas NAb #2 and #3 were mouse. Red lines represent antibodies while blue lines show soluble ACE2 molecules. Dot, triangle, square, asterisk, circle and star symbols indicate SARS-CoV-2 S-RBD NAb #1, #2, #3, #4, soluble ACE2 #1 and #2, respectively. Graphs in ( c ) and ( d ) represent three replicates of the experiments. Error bars indicate one standard deviation of mean values.

Article Snippet: The sequence of mKO2 (monomeric Kusabira-Orange-2 ) obtained from Addgene (#54625) , and was added onto the C terminal of ACE2 proximal to the stop codon with a small linker peptide (ccggtcgccacc) encoding the amino acids ‘PVAT’.

Techniques: Neutralization, Incubation, Expressing, Infection, Blocking Assay, Standard Deviation

Journal: Scientific Reports

Article Title: A Modular Assembly Platform for Rapid Generation of DNA Constructs

doi: 10.1038/srep16836

Figure Lengend Snippet: ( A ) Promoters, genes, and backbones from established GMAP-compatible collections are used in a one step isothermal assembly reaction to produce DNA constructs on demand. ( B ) Schematic of possible orderings of genes and promoters. Promoter A (pA) is flanked by sites 1 and 2, promoter B (pB) is flanked by sites 3 and 4, promoter C (pC) is flanked by sites 1 and 3, gene A (gA) is flanked by sites 2 and 3, gene B (gB) is flanked by sites 4 and 5, and gene C (gC) is flanked by sites 2 and 5. ( C ) Schematic diagram of experiment using GMAP retroviral backbone. Retroviruses expressing GFP driven by different promoters were assembled using GMAP and used to transduce murine 3T6 fibroblasts or murine lung cancer (KP) cells. PuroR, puromycin resistance; pGK, human phosphoglycerate kinase 1 promoter; CMV, cytomegalovirus immediate-early promoter; EFS, elongation factor 1α promoter; SV40, simian virus early 40 promoter; CCSP, clara cell secretory protein promoter; UBC, human Ubiquitin C promoter. ( D ) Bar graph shows flow cytometry measurements of median fluorescence intensity (MFI) of GFP from 3T6 and KP cells transduced with GMAP-generated retrovirus. Data are representative of at least three independent experiments. ( E ) GMAP-generated lentiviruses expressing mTagBFP2-A UTR (i), mKate2-B UTR (ii), or mKO2-C UTR (iii) sensor cassettes were assembled and used to transduce a Cre reporter cell line (3TB). 3TB cells were selected with hygromycin and visualized by confocal microscopy. Insets show higher magnification. Scale bar, 100 μm. ( F-H ) Histograms from 3TB cells expressing mTagBFP2-A UTR ( F ), mKate2-B UTR ( G ), or mKO2-C UTR ( H ) transfected with three inducible hairpin constructs targeting the A, B, or C 3’UTRs assembled using GMAP. After transfection 3TB cells were selected with blasticidin, treated with doxycycline, and knockdown was assessed by flow cytometry analysis on GFP + cells. Grey histograms represent cells lines transfected with an inducible shRNA targeting luciferase. Data are representative of at least three independent experiments.

Article Snippet: Accession Codes : The following plasmids were deposited into Addgene: TOPO 2-5, TOPO 1-4, RV 2-5, LV 1-5, R26TV CAG LSL 2-5, TOPO mTagBFP2, TOPO mKate2, TOPO mKO2, TOPO iRFP670, TOPO CloverCP, TOPO HygroR, TOPO ZeoR, TOPO PuroR, TOPO rtTA3-2A-Cre, TOPO tTRKRAB- rtTA3-Luc, TOPO Cre-2A-GFP, TOPO EFS, TOPO pGK, TOPO CMV, TOPO CCSP, TOPO sv40, TOPO UBC, TOPO pTRE, and TOPO HRE.

Techniques: Construct, Retroviral, Expressing, Transduction, Virus, Ubiquitin Proteomics, Flow Cytometry, Fluorescence, Generated, Confocal Microscopy, Transfection, Knockdown, shRNA, Luciferase

Journal: Heliyon

Article Title: Markerless bacterial artificial chromosome manipulation method by red proteins of phage λ mediated homologous recombination utilizing fluorescent proteins for both positive and counter selection

doi: 10.1016/j.heliyon.2023.e18983

Figure Lengend Snippet: Characteristics of fluorescent proteins used in this study.

Article Snippet: The mKO2 gene was obtained from mKO2-pBAD gifted by Michael Davidson and Atsushi Miyawaki (#54555, Addgene) [ ].

Techniques:

Journal: Heliyon

Article Title: Markerless bacterial artificial chromosome manipulation method by red proteins of phage λ mediated homologous recombination utilizing fluorescent proteins for both positive and counter selection

doi: 10.1016/j.heliyon.2023.e18983

Figure Lengend Snippet: Optimization of template plasmids. pAmp-9mScarlet without the terminator rrnB T1 was constructed (A). mScarlet protein expression in the pelleted HTS16CR cells was observed on an LED transilluminator after the cells were transformed with pAmp-9mScarlet (+) or pAmp-9mScarlet without a terminator (−) following overnight cultivation in 2 mL of LB medium at 37 °C (B) . Additionally, the plasmid yields of 2 mL of HST16CR cultures transformed with pAmp-9mScarlet (+) (with terminator) and pAmp-9mScarlet (−) (without terminator) were compared (C). The fluorescent protein gene (FP), EGFP, mClover3, YPet, mOrange, mKO2, or mScarlet were ligated into a plasmid (D), which has the same backbone as pAmp-9mScarlet, except for the fluorescent gene, as shown in . E. coli , HST16CR, or HST08 were transformed with these plasmids or pAmp-S as the negative control (control) and then cultivated on LB agar plates at either 30 °C or 37 °C overnight under ampicillin selective pressure (E) (Supplementary fig. 3E-1, -2 and -3). Note that HST08 cells transformed with mKO2 did not grow on the plates either at 30 °C or 37 °C. An unpaired t -test was used to determine the statistical significance. The calculated p values are shown above the compared groups.

Article Snippet: The mKO2 gene was obtained from mKO2-pBAD gifted by Michael Davidson and Atsushi Miyawaki (#54555, Addgene) [ ].

Techniques: Construct, Expressing, Transformation Assay, Plasmid Preparation, Negative Control, Control

Journal: bioRxiv

Article Title: In vivo characterisation of fluorescent proteins in budding yeast

doi: 10.1101/431874

Figure Lengend Snippet: Example of practical brightness quantification using the T2A peptide linker. Cells expressing either yoeCFP-T2A-mCherry or mTq2-T2A-mCherry were grown to midlog and visualized using a widefield microscope. yoeCFP shows a low brightness compared to mCherry. In contrast, mTq2 shows a higher brightness than mCherry. Calibration bar indicates the ratio value when dividing the CFP by the RFP channel (i.e. the relative brightness to mCherry).

Article Snippet: sYFP2-T2A-mTq2, tagRFPT-T2A-mTq2, tdTomato-T2A-mTq2, tagRFP-T2A-mTq2, mCherry-T2A-mTq2, mCherry-T2A-eGFP, mCherry-T2A-sGFP2, YPET-T2A-mTq2, mCitrine-T2A-mTq2, mNeongreen-T2A-mTq2, mKO k -T2A-mTq2, mClover-T2A-mTq2, mRuby2-T2A-mTq2, mScarlet-T2A-mTq2, mScarletI-T2A-mTq2, mCherry-T2A-mTq2 and mKate2-T2A-mTq2 were based on mKO2-T2A-mTq2 (addgene plasmid #98838).

Techniques: Expressing, Microscopy

Journal: bioRxiv

Article Title: In vivo characterisation of fluorescent proteins in budding yeast

doi: 10.1101/431874

Figure Lengend Snippet: In vivo brightness and photostability of FPs. A) In vivo brightness of FPs measured by normalizing the fluorescence intensity of single-cells expressing FP-T2A-FP and dividing the fluorescence of the FP of interest to the control FP (either mTq2 or mCherry). B) Photostability of FPs. Per FP, a time-lapse movie was recorded and the photostability was measured as the fluorescent fraction of the last time frame compared to the first frame. Dots represent relative brightness or photostability of an individual cell, boxes indicate median with quartiles, whiskers indicate the 0.05–0.95 fraction of the datapoints. C) Overview of the brightness and photostability of all characterised FPs. D) Coefficient of variation (CV) of the mean brightness of 3 days as an indication of day-to-day variation.

Article Snippet: sYFP2-T2A-mTq2, tagRFPT-T2A-mTq2, tdTomato-T2A-mTq2, tagRFP-T2A-mTq2, mCherry-T2A-mTq2, mCherry-T2A-eGFP, mCherry-T2A-sGFP2, YPET-T2A-mTq2, mCitrine-T2A-mTq2, mNeongreen-T2A-mTq2, mKO k -T2A-mTq2, mClover-T2A-mTq2, mRuby2-T2A-mTq2, mScarlet-T2A-mTq2, mScarletI-T2A-mTq2, mCherry-T2A-mTq2 and mKate2-T2A-mTq2 were based on mKO2-T2A-mTq2 (addgene plasmid #98838).

Techniques: In Vivo, Fluorescence, Expressing

Journal: bioRxiv

Article Title: In vivo characterisation of fluorescent proteins in budding yeast

doi: 10.1101/431874

Figure Lengend Snippet: pH sensitivity of FPs. A) Yeast cells were incubated for 2 hours in citric-acid/Na 2 HPO 4 buffers set at pH 3-8 with 2 mM 2,4-DNP and fluorescence was measured using a fluorescent plate reader. Per FP, at least 3 technical replicates were measured. Afterwards, fluorescence was normalized to the pH giving the highest fluorescence and a Hillfit was performed to determine the hill coefficient and pKa value, plotted at the y- and x-axis, respectively. B) mTq2 is an example of an FP that shows different pH sensitivity. pH calibration in vitro was performed using purified proteins in a Citric Acid – Sodium Citrate buffer (pH 3 – 5.4) and a NaH 2 PO 4 /Na 2 HPO 4 0.1 M buffer (pH 5.9-8). Dots represent mean of at least 3 replicates, error bars indicate SD. C) pH curve of sYFP2 which shows an offset (fluorescence plateau) at low pH. This offset gives different values of the pKa (red point, which is the pH that gives a 50% decrease between 1 and the offset) and the pH 50% which gives an absolute 50% decrease in fluorescence (blue point). Dots represent mean of at least 3 replicates, error bars indicate SD.

Article Snippet: sYFP2-T2A-mTq2, tagRFPT-T2A-mTq2, tdTomato-T2A-mTq2, tagRFP-T2A-mTq2, mCherry-T2A-mTq2, mCherry-T2A-eGFP, mCherry-T2A-sGFP2, YPET-T2A-mTq2, mCitrine-T2A-mTq2, mNeongreen-T2A-mTq2, mKO k -T2A-mTq2, mClover-T2A-mTq2, mRuby2-T2A-mTq2, mScarlet-T2A-mTq2, mScarletI-T2A-mTq2, mCherry-T2A-mTq2 and mKate2-T2A-mTq2 were based on mKO2-T2A-mTq2 (addgene plasmid #98838).

Techniques: Incubation, Fluorescence, In Vitro, Purification