pb ta erp2 piggybac transposon destination vector  (Addgene inc)

Journal: bioRxiv

Article Title: A scalable transposon mutagenesis system for non-model bacteria

doi: 10.64898/2025.12.22.696024

Figure Lengend Snippet: ( A ) Schematic of the transposon variant library components. LE, left mosaic end; RE, right mosaic end; oriT , origin of transfer; ABR, antibiotic resistance. ( B ) Mean transcription strength and GC content (%) of 16 promoters selected for inclusion in the transposon variant library (green). Transcription strength for 421 promoter sequences was measured across 10 bacterial strains by Yim et al. 2019 . ( C ) Himar1-only library variant performance in E. coli DH10B, with either the catP (CAM) or ( D ) aadA(9) (SPEC) ABR gene. Relative transposition efficiency (enrichment) was measured by Tn-seq after pooled conjugative delivery. ( E ) Correlation between previously published transcriptional strength of promoters and their transposition efficiencies in E. coli when driving the Himar1 transposase. ( F ) Correlation between previously published transcriptional strength of promoters and their transposition efficiencies in E. coli when driving the catP ABR.

Article Snippet: All transposon/ABR promoter part plasmids and transposon plasmid destination vectors used in this study have been made available via Addgene (see Supplementary Data Table 1 and Supplementary Data Table 2 for Addgene IDs).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: A scalable transposon mutagenesis system for non-model bacteria

doi: 10.64898/2025.12.22.696024

Figure Lengend Snippet: ( A ) Bacterial strains ranked by the number of unique genomic insertion events measured by Tn-seq analysis. ‘High confidence’ strains received over 100 insertions per sample, while ‘low confidence’ strains received between 10-100. Each data point represents the total number of unique genomic insertions sites obtained from delivery of a 324-member transposon variant library. Note the data for Cupriavidus necator is a combination of two different strains (see Supplementary Table 2) ( B ) Himar1 transposase promoter efficiencies in strains with at least 100 observed insertion events in multiple samples. Each row represents a single replicate, and rows are clustered by relative transposase promoter efficiencies. Strains are colored by their class: Gammaproteobacteria (red), Alphaproteobacteria (green), Betaproteobacteria (purple), or Verruomicrobia (yellow). ( C ) The replicability of promoter efficiencies as measured by Tn-seq across all microbes screened consistently increases with transformation efficiency for both transposase and ABR promoters. ( D ) Validation of promoter efficiencies. Promoters with high (H), medium (M), and low (L) abundances from the screen results were individually cloned and then tested for their conjugation efficiencies in three strains. Conjugation efficiency is reported as the number of transconjugants per recipient.

Article Snippet: All transposon/ABR promoter part plasmids and transposon plasmid destination vectors used in this study have been made available via Addgene (see Supplementary Data Table 1 and Supplementary Data Table 2 for Addgene IDs).

Techniques: Variant Assay, Transformation Assay, Biomarker Discovery, Clone Assay, Conjugation Assay

Journal: bioRxiv

Article Title: A scalable transposon mutagenesis system for non-model bacteria

doi: 10.64898/2025.12.22.696024

Figure Lengend Snippet: ( A ) Number of genomic insertions observed for different recipient strains undergoing dual-transposon promoter screen, as determined by sequencing. Conjugations were performed either under standard petri dish-based format (‘high input’) or 96-well plate-based format (‘low input’) and transconjugants sequenced after growth in selective media. ( B ) Comparison of Tn5 (orange) or Himar1 (blue) promoter abundance between the Himar1-only or Tn5-only transposase library screens (y-axis) and Himar-Tn5 dual-transposase library screen (x-axis). ( C ) Comparing the efficiencies of promoters associated with Himar1 (y-axis) versus Tn5 (x-axis) transposons for three strains.

Article Snippet: All transposon/ABR promoter part plasmids and transposon plasmid destination vectors used in this study have been made available via Addgene (see Supplementary Data Table 1 and Supplementary Data Table 2 for Addgene IDs).

Techniques: Sequencing, Comparison

Journal: bioRxiv

Article Title: CRISPR loss of function screening to identify genes involved in human primordial germ cell-like cells development

doi: 10.1101/2022.05.23.493064

Figure Lengend Snippet: (A) Diagram of piggyBac -based Cas9 expression under a tet-On inducible system with a PBase (transposase) helper vector. (B) A phase-contrast image of 17C-2 (iCas9) hiPSCs. Bar, 200 µm. (C) Phase-contrast images of iCas9 hiPSC-derived iMeLCs (top) and day 5 floating aggregates containing hPGCLCs (bottom). – Dox, without Dox; + Dox, treated with 1 µg/ml doxycycline. Bars, 200 µm. (D) Fluorescence-activated cell sorting (FACS) analysis of day 5 hPGCLCs derived from iCas9 hiPSCs. AG; TFAP2C-2A-EGFP, VT; DDX4-2A-tdTomato. (E) rtTA (top) and Cas9 (bottom) expression during hPGCLC induction, as measured by qPCR. For each gene examined, the ΔCt from the average Ct values of the two independent housekeeping genes ARBP and PPIA (set as 0) were calculated and plotted. Error bars indicate the standard deviation (SD) of biological replicates.

Article Snippet: For the construction of an inducible Cas9 expression piggyBac vector (PB-iCas9-Neo), a Gateway entry vector containing Cas9, pENTR-hSpCas9, was cloned into a PB-TA-ERN plasmid (all-in-one piggyBac transposon destination vector for doxycycline (Dox)-inducible expression with constitutive rtTA and neomycin resistance) ( Kagawa et al , 2019 ; Kim et al , 2016 ) with Gateway LR Clonase II enzyme mix (Invitrogen).

Techniques: Expressing, Plasmid Preparation, Derivative Assay, Fluorescence, FACS, Standard Deviation

Journal: Cell systems

Article Title: Gene Regulatory Network Analysis and Engineering Directs Development and Vascularization of Multilineage Human Liver Organoids

doi: 10.1016/j.cels.2020.11.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For constructing the dox-inducible expressing GATA6 vector, pENTR_L1_hGATA6–2A-EGFP_L2 previously published ( ) was cloned into an All-in-One PiggyBac transposon destination vector from Addgene (Addgene plasmid ID: 80479) via gateway reaction.

Techniques: Recombinant, Transfection, Expressing, Plasmid Preparation, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Titration, RNA Sequencing Assay, Isolation, Sequencing, Variant Assay, Software