input plasmid dna  (New England Biolabs)

Journal: bioRxiv

Article Title: A novel functional gene delivery platform based on a commensal human anellovirus demonstrates transduction in multiple tissue types

doi: 10.1101/2024.03.27.586964

Figure Lengend Snippet: (A) (Top) Schematic representation of the Anellovector comprising the nrVL4619 NCR, CMV promoter, eGFP, WPRE, and bGH polyA flanked by the mutant lox sites, Lox71 and Lox66. (Bottom) Comparison of DNase-protected vector DNA (using eGFP (4) primer-probe set) when viral proteins are supplied by plasmids of nrVL4619 WT single or tandem or hEF1α-nrVL4619-CDS; data reported as vector genomes per liter of production culture (vgs/L) and presented as mean (n=3) ± SEM. (B) Immunoblots probing for GAPDH and the viral proteins (ORF1 and ORF1/1 left; ORF2, ORF2/2, and ORF2/3 right) from nrVL4619 WT and hEF1α-driven contexts in MOLT-4 cells. The blue arrows denote the expected sizes of the viral proteins. Red arrow denotes the GAPDH loading control. (C) Southern blot probing against nrVL4619 CDS sequences for conditions outlined in (Top). The plus and minus signs indicate whether the sample was digested with DpnI to remove input plasmid DNA. See (top) for lane identities and restriction enzyme digests. Sizes of expected digestion products: nrVL4619 tandem 5479bp + 2876bp (single unit genome); nrVL4619 single copy 5479; hEF1a-nrVL4619-CDS 6636. D) Schematic diagram of the nrVL4619 SRR (Top) and the SATURN system (bottom). The SRR contains a hEF1α promoter driving the co-expression of the nrVL4619 and SV40 large T antigen. The downward arrow and multiple smaller SRRs denote the self-amplification of the plasmid by large T antigen and the SV40 origin of replication. The SATURN system comprises a three-plasmid transfection which circularizes the vector genome out of a plasmid (“Vector”) through the expression of Cre (“Accessory plasmid”) and the SRR plasmid supplying anellovirus proteins for replication and packaging. (E) Southern blot probing against nrVL4619 CDS and eGFP transgene sequences (multiplex) for conditions outlined in (bottom). The plus and minus signs indicate whether the sample was digested with DpnI to remove input plasmid DNA. See (bottom) for lane identities and restriction enzyme digests. Sizes of expected digestion products: ANV.eGFP plasmid 5452bp; ANV.eGFP single unit replication intermediate 2839bp; nrVL4619 SRR single digestion 9391bp; ORF1-KO SRR single digestion 9391bp. (F) Quantitation of ANV.eGFP DNase-protected vector DNA recued with either the nrVL4619 SRR or ORF1-KO SRR using eGFP (4) primer-probes. Data presented as mean (n=3) ± SEM.

Article Snippet: To digest input plasmid DNA, each sample was also subjected to treatment with DpnI (NEB catalog # R0176) which selectively cleaves methylated GATC sites.

Techniques: Mutagenesis, Comparison, Plasmid Preparation, Western Blot, Control, Southern Blot, Expressing, Amplification, Transfection, Multiplex Assay, Quantitation Assay

Journal: bioRxiv

Article Title: A novel functional gene delivery platform based on a commensal human anellovirus demonstrates transduction in multiple tissue types

doi: 10.1101/2024.03.27.586964

Figure Lengend Snippet: (A) Diagram representing the discovery of anellovirus species from various tissues of origin and their utilization to encapsidate the vector of ANV.eGFP. (B) Comparison of productivity as measured by DNase-protected qPCR detecting ANV.eGFP DNA packaged into its own capsid versus being packaged into ANV.LY1 or ANV.LY2. Data presented as mean (n=3) ± SEM; *: p< 0.05 (one-way ANOVA); ns: p > 0.05 (one-way ANOVA with Brown-Forsythe test). (C) Southern blot probing against eGFP transgene. All samples were digested with the AgeI restriction enzyme to linearize DNA. The plus and minus signs indicate whether the sample was digested with DpnI to remove input plasmid DNA. Expected band sizes: ANV.eGFP plasmid 5452bp; ANV.eGFP replication intermediate 2839bp.

Article Snippet: To digest input plasmid DNA, each sample was also subjected to treatment with DpnI (NEB catalog # R0176) which selectively cleaves methylated GATC sites.

Techniques: Plasmid Preparation, Comparison, Southern Blot

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

doi: 10.3389/fbioe.2021.727584

Figure Lengend Snippet: Optimized in-house cell-free reactions compared to commercial alternatives. (A) Left: Schematic representation of testing the performance of home-made or commercial cell-free systems using the sfGFP constitutive reporter. Right: Example of the endpoint sfGFP reaction and negative control (without input DNA) in a home-made cell-free system supplemented with maltodextrin energy source. Tubes were photographed under white light or blue light plus an amber filter that allows visualizing the sfGFP fluorescence. (B) Endpoint sfGFP fluorescence (plasmid DNA at 9 nM final concentration) was measured in four different cell extracts (Batch A, B, C, D) supplemented with either maltodextrin and polyphosphates (light blue) or 3-PGA (green) as energy source. Grey dots represent the arithmetic mean of three measurements performed on each batch, and error bars represent standard deviations of the means of the four batches tested (N = 4). t -test for paired measurements was performed and statistically significance was found between the two groups ( p -value = 0.03, shown by *). Assumptions of the paired t -test were verified using the Shapiro-Wilk test for normality of the differences between energy sources for a given batch ( p -value = 0.97), and Levene test for homoscedasticity of the 3-PGA and maltodextrin data sets ( p -value = 0.25). (C) sfGFP production dynamics from plasmid DNA (9 nM final concentration) in reactions performed at 29°C using NEB PURExpress and Promega S30 T7 High Yield commercial kits along with four optimized in-house cell-free reactions (Batch A, B, C, D) using maltodextrin and polyphosphates as the energy source. Error bars represent the standard deviations of three independent replicates, dots are centered at the arithmetic mean for each time point.

Article Snippet: Plasmid DNA input was produced by midi prepping an overnight culture of 200 ml LB with the appropriate strain (Promega, A2492) and cleaned again using PCR cleanup (Promega, A6754).

Techniques: Negative Control, Fluorescence, Plasmid Preparation, Concentration Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

doi: 10.3389/fbioe.2021.727584

Figure Lengend Snippet: Performance of ZIKV toehold sensors in low-cost cell-free lysate reactions. (A) Schematic representation of toehold-mediated RNA sensing. (B) Dynamics of the RNA sensing reactions performed with ZIKV toehold sensor 8 (0.7 nM plasmid DNA) and 27 (2 nM plasmid DNA), regulating the expression of the full-length LacZ in home-made cell extracts and PURExpress cell-free reactions. Error bars represent the standard deviations of three independent experiments, dots are centered at the arithmetic mean for each time point. (C) Example of the endpoint visualization of the experiments after 4 hours of incubation at 29°C. (D) Endpoint measurement of RNA sensing reactions performed with ZIKV sensor 27 and trigger 27 in a range of concentrations with and without NASBA isothermal amplification. Gray dots represent data from six independent measurements performed from two independent NASBA amplifications performed on different days. Black error bars correspond to standard deviations of these six measurements.

Article Snippet: Plasmid DNA input was produced by midi prepping an overnight culture of 200 ml LB with the appropriate strain (Promega, A2492) and cleaned again using PCR cleanup (Promega, A6754).

Techniques: Plasmid Preparation, Expressing, Incubation, Amplification

Journal: PLoS ONE

Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

doi: 10.1371/journal.pone.0199563

Figure Lengend Snippet: A. The mutant constructs were first amplified with two partially overlapping primers designed to incorporate the desired alteration (shown in green), using the corresponding wild type block as a template. The input template DNA was then eliminated by digestion with the restriction enzyme DpnI. In the second step, the PCR-derived plasmid was circularized in an assembly reaction, which merged and sealed the overlapping ends. B. Oligonucleotide pairs that were used to generate the mutants are illustrated. The mutant block 1-derived constructs were E1B19K-null, E1B55K-null or modified to express an endogenous E1B55K protein with a c-terminal FLAG epitope. Similarly, the E4ORF3 open reading frame was disrupted by a mutation at the initiation codon or the introduction of a c-terminal FLAG tag. The targeted codons are boxed, single base mutations are shown in red. At positions where the initiation codon of the target protein overlapped with a codon for a different protein, base substitutions were selected so that the mutation would be silent with respect to the second open reading frame. C. hTERT-RPE1 cells were infected with the synthetic Ad5 virus and the E1B mutant viruses generated in this study. Cells were lysed 16 h post-infection, and assessed by immunoblot with antibodies against p53 and the FLAG epitope, as indicated. D. hTERT-RPE1 cells were uninfected (no virus) or infected with wild type Ad5 and the E4 mutants. For all viruses, the MOI was 100. GAPDH was probed as a loading control.

Article Snippet: Input plasmid DNA templates were digested with 20 U of the restriction enzyme DpnI (New England Biolabs) for 30 min at 37°C.

Techniques: Mutagenesis, Construct, Amplification, Blocking Assay, Derivative Assay, Plasmid Preparation, Modification, FLAG-tag, Infection, Generated, Western Blot