Journal: Nature Communications

Article Title: PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

doi: 10.1038/s41467-022-30355-y

Figure Lengend Snippet: Design, Build, Test cycle for construction of plasmids from linear DNA parts. In the first step, a frontend is used to design and choose DNA fragments for assembly. After all the selected sequences passed the quality check, picklists (input for “Build” part) are generated for large-scale synthesis of primers and guides. The received oligos in either 96-well format or 384-well format are transferred into corresponding destination wells for PCR reactions to make specific DNA fragments via liquid handlers. Next, one-pot digestion, ligation, and transformation is performed inside iBioFAB to assemble DNA fragments. Pf Ago-based AREs are used for DNA assembly. In the first step, linear DNA molecules ends are digested with WT and engineered Pf Ago/AREs in a one-pot reaction. The AREs generate 5′ sticky ends of 12 nt length. After purification, the digested DNA molecules are assembled in vitro using a high-fidelity DNA ligase and assembly products are transformed into E. coli cells for screening. Finally, constructed plasmids (input for “Test” part) are checked and the correct plasmids are stocked using the robotic system.

Article Snippet: 5 μL of the dialyzed mixture was then added to 25 μL of electrocompetent NEB10β E. coli cells (New England Biolabs), mixed gently and transferred to a 2 mm electroporation cuvette.

Techniques: Generated, Ligation, Transformation Assay, Purification, In Vitro, Construct

Journal: Nature Communications

Article Title: PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction

doi: 10.1038/s41467-022-30355-y

Figure Lengend Snippet: a 10 randomly generated recognition sequences for both 9 and 12 nt AREs with different GC-contents and GC-distributions were placed on the ends of three sets of linear DNA with different overall GC content to create 60 sets of linear fragments. Each set was then digested by either WT Pf Ago or Pf Ago*/AREs creating 9 or 12 nt sticky ends and assembled by E. coli DNA ligase. v1 and v2 represent different GC-distributions. b Average cleavage/DNA assembly efficiency of both Pf Ago and Pf Ago*/AREs creating 9 or 12 nt sticky ends. Based on these results, Pf Ago/AREs can be programmed to cleave linear DNA ends with a wide range of GC-contents (0-75%). This data was used to help our guide design program to ensure efficient cleavage of DNA ends by Pf Ago/AREs (see ). Source data are provided as a Source Data file. The assembly efficiency analysis for each set was performed only once.

Article Snippet: 5 μL of the dialyzed mixture was then added to 25 μL of electrocompetent NEB10β E. coli cells (New England Biolabs), mixed gently and transferred to a 2 mm electroporation cuvette.

Techniques: Generated

Journal: bioRxiv

Article Title: Multiplex, multimodal mapping of variant effects in secreted proteins

doi: 10.1101/2024.04.01.587474

Figure Lengend Snippet: A. Secreted proteins (purple) make up approximately 10% of the human proteome . B. Missense variants in secreted proteins found in ClinVar from 2016 to 2023 colored by clinical significance. C. MultiSTEP retains secreted proteins on the cell surface, establishing a physical link between genotype and phenotype (left panel). Cells expressing a library of variants of the target protein are sorted into bins based upon intensity of fluorescent antibody binding, followed by deep sequencing to derive a functional score for each individual variant (middle panels). The result is a variant effect map (right panel). D. MultiSTEP design. Secreted protein coding sequences (pink) are cloned into an attB-containing landing pad donor plasmid. Secreted proteins are engineered to have C-terminally fused (GGGGS) 2 flexible linkers (L1 and L2, teal) attached to a single pass transmembrane domain (TMD, blue). In between the linkers is a strep II epitope tag for surface detection (green). The construct contains an IRES (purple) driving co-transcription of an mCherry fluorophore (red) that serves as a transcriptional control. E-G. Flow cytometry of known well-secreted (p.A37T, p.S220T, WT) and poorly-secreted (p.C28Y) FIX variants displayed using MultiSTEP (n ∼30,000 cells per variant). Unrecombined cells do not display FIX and serve as a negative control. Fluorescent signal was generated by staining the library with either a mouse monoclonal anti-FIX heavy chain antibody ( E ), a mouse monoclonal anti-FIX light chain antibody ( F ), or a mouse monoclonal anti-strep II tag antibody ( G ), followed by staining with an Alexa Fluor-647-labeled goat anti-mouse secondary antibody.

Article Snippet: Routine cloning was performed in homemade chemically competent Top10F’ E. coli , whereas library cloning was performed in commercially available electrocompetent NEB-10β E. coli (New England Biolabs).

Techniques: Expressing, Binding Assay, Sequencing, Functional Assay, Variant Assay, Clone Assay, Plasmid Preparation, Construct, Control, Flow Cytometry, Negative Control, Generated, Staining, Labeling

Journal: bioRxiv

Article Title: Multiplex, multimodal mapping of variant effects in secreted proteins

doi: 10.1101/2024.04.01.587474

Figure Lengend Snippet: A. Various MultiSTEP donor plasmid constructs. Secreted protein coding sequences (pink) are cloned into an attB-containing landing pad donor plasmid. Secreted proteins are engineered to have C-terminally fused (GGGGS) 2 flexible linkers (L1 and/or L2, teal) attached to a single pass transmembrane domain (TMD, blue). In between the linkers is a strep II epitope tag for surface detection (green). The construct contains an IRES (purple) driving co-transcription of an mCherry fluorophore (red) that serves as a transcriptional control. MultiSTEP: L1-Strep does not contain an L2 linker, whereas MultiSTEP: L1-Strep-L2 does. B. Experimental flow cytometry of known well-secreted (A37T, S220T, WT) and poorly-secreted (C28Y) FIX variants displayed using various MultiSTEP constructs depicted in A (n ∼30,000 cells per variant). Unrecombined cells do not display FIX and serve as a negative control. Fluorescent signal was generated by staining the library with a mouse monoclonal anti-strep II tag antibody followed by staining with an Alexa Fluor-647-labeled goat anti-mouse secondary antibody. C. Heatmaps showing strep tag secretion scores for nearly all missense FIX variants. Heatmap color indicates antibody score from 0 (blue, lowest 5% of scores) to white (1, WT) to red (increased antibody scores). Black dots indicate the WT amino acid. Missing data scores are colored gray. D. Density distributions of strep tag secretion scores for FIX missense variants (orange) and synonymous variants (blue). Dashed line denotes the 5th percentile of the synonymous variant distribution. E. Scatterplot comparing MultiSTEP-derived strep tag secretion scores for seven different variants to the geometric mean of Alexa Fluor-647 fluorescence measured using flow cytometry on cells expressing each variant individually. Error bars show standard error of the mean (n = 3; >10,000 cells per replicate). Black dashed line indicates the line of best fit. Pearson’s correlation coefficient is shown. F-G. Scatterplots of median MultiSTEP-derived strep tag secretion scores vs. heavy chain ( F ) or light chain ( G ) at each position in FIX. Points are colored by chain architecture, using the same color scheme as . Black dashed line indicates the line of perfect correlation between secretion scores. Gray background indicates <30% deviation from perfect correlation.

Article Snippet: Routine cloning was performed in homemade chemically competent Top10F’ E. coli , whereas library cloning was performed in commercially available electrocompetent NEB-10β E. coli (New England Biolabs).

Techniques: Plasmid Preparation, Construct, Clone Assay, Control, Flow Cytometry, Variant Assay, Negative Control, Generated, Staining, Labeling, Strep-tag, Derivative Assay, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Multiplex, multimodal mapping of variant effects in secreted proteins

doi: 10.1101/2024.04.01.587474

Figure Lengend Snippet: A. Factor IX domain and chain architecture. Signal: Signal peptide. Pro: Propeptide. Gla: Gla domain. EGF1: Epidermal growth-like factor 1 domain. EGF2: Epidermal growth-like factor 2 domain. Activation: Activation peptide. Protease: Serine protease domain. B-C. Heatmaps showing FIX heavy chain secretion scores ( B ) or FIX light chain secretion scores ( C ) for nearly all missense FIX variants. Heatmap color indicates antibody score from 0 (blue, lowest 5% of scores) to white (1, WT) to red (increased scores). Black dots indicate the WT amino acid. Missing data scores are colored gray. D-E. Density distributions of heavy chain ( D ) or light chain ( E ) secretion scores for FIX missense variants (orange) and synonymous variants (blue). Dashed line denotes the 5th percentile of the synonymous variant distribution. F-G. Scatterplots comparing MultiSTEP-derived heavy chain ( F ) or light chain ( G ) secretion scores for seven different variants to the geometric mean of Alexa Fluor-647 fluorescence measured using flow cytometry on cells expressing each variant individually. Error bars show standard error of the mean (n = 3; >10,000 cells per replicate). Black dashed line indicates the line of best fit. Pearson’s correlation coefficients are shown. H. Scatterplot of median MultiSTEP-derived heavy chain and light chain secretion scores at each position in FIX. Points are colored by chain architecture, using the same color scheme as A . Black dashed line indicates the line of perfect correlation between secretion scores. Pearson’s correlation coefficients are shown. Gray background indicates <30% deviation from perfect correlation. Points with median positional scores outside gray background are labeled with their corresponding FIX position. I. AlphaFold2 model of mature, two-chain FIX (positions 47-191 and 227-461). Positions labeled in ( H ) are shown as colored surfaces where color corresponds to the FIX heavy chain (magenta) or light chain (green). J. Magnified view of FIX EGF1 domain in the light chain (orange). Putative epitope positions with discordant light and heavy chain antibody scores ( H-I ) are shown as a green colored surface with visible amino acids labeled. K. Magnified view of FIX serine protease domain in the heavy chain (yellow). Putative epitope positions ( H-I ) are shown as a purple colored surface with visible amino acids labeled.

Article Snippet: Routine cloning was performed in homemade chemically competent Top10F’ E. coli , whereas library cloning was performed in commercially available electrocompetent NEB-10β E. coli (New England Biolabs).

Techniques: Activation Assay, Variant Assay, Derivative Assay, Fluorescence, Flow Cytometry, Expressing, Labeling