Journal:

Article Title: Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

doi: 10.1002/jgm.1499

Figure Lengend Snippet: Vector retrofit. (A) gWIZ EGFP kanR vector with annotation of functional sequences CMV enhancer, promoter, intron, EGFP transgene, bovine growth hormone (BGH) terminator, and pUC origin. The kanR selection marker and nonessential spacer DNA (TN903 inverted repeat, polyC, polyG, ampR promoter) that accounts for the 2kb increased size compared to NTC8685 are shown. The kanR promoter and kanR gene (black annotation) were replaced with the 140 bp RNA-OUT antibiotic free marker in both orientations. (B) pVAX1 EGFP kanR vector with locations of kanR gene, and other functional sequences (CMV enhancer, promoter, EGFP transgene, BGH terminator, pUC origin) indicated. The kanR promoter and kanR gene (black annotation) were replaced with the 140 bp RNA-OUT antibiotic free marker in both orientations.

Article Snippet: The gWIZ-EGFP vector ( ) is a 5.746 kb EGFP transgene version of the kanR pUC origin gWIZ DNA vaccine plasmid vector (Gene Therapy Systems, San Diego, CA).

Techniques: Plasmid Preparation, Functional Assay, Selection, Marker

Journal:

Article Title: Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

doi: 10.1002/jgm.1499

Figure Lengend Snippet: Vectors <3 kb have reduced copy number. The relative fermentation copy number for monomer and dimer plasmid in DH5α (Table 3) versus monomer vector size for: (A) kanR NTC7485 derivatives; and (B) AF NTC8485 derivatives. Monomer kanR or AF plasmids <3 kb had a much lower relative copy number than larger plasmids. The dimer plasmids of the 2.8 and 3.5kb NTC7485 kanR and the 2.1 and 2.7 kb NTC8485 vectors (plotted at the monomer kb size) had up to two fold improved copy number. Since a dimer plasmid also contains 2 fold more DNA, this resulted in dramatically higher plasmid yields than the corresponding monomer plasmids. (C) The inducible fermentation process maintained AF monomer and dimer plasmid stability with all vector sizes and produced high quality supercoiled plasmid. Plasmid DNA was prestained with SYBR Green I (Sigma, St. Louis, MO) and resolved on a TAE agarose gel. Fermentation harvests (see Table 3 for yields) of 2.1 kb NTC8485-U6-shRNA monomer (RF230; lane 1) and 4.2 kb dimer; (RF240; lane 2) and 2.7 kb NTC8485E-U6-shRNA monomer (RF232; lane 3) and 5.4 kb dimer; (RF241; lane 4) are shown. M is marker lanes (1 kb DNA ladder; Invitrogen). (D) Supercoiling linking number analysis of NTC7485-U6-shRNA fermentation samples. Samples were treated with T5 exonuclease to remove nicked plasmid prior to AGE in the presence of chloroquine. Plasmid from 42°C induction (lanes 1 and 4 = 8 h induction; lanes 2, 5 and 7 = 10 h induction) and post-induction 25°C hold stages (lanes 3, 6 and 8) of DH5α fermentations RF226 (lanes 1-3), RF279 (lanes 4-6), and XL1-Blue fermentation RF273 (lanes 7 and 8) are shown. Lanes C1 and C2 are 8 h 42°C induction sample from DH5α fermentation RF226 (without T5 treatment to remove nicked species) with (C2) or without (C1) E. coli DNA gyrase (New England Biolabs, Beverly, MA) treatment [10] to increase negative supercoiling linking number. In this assay negatively supercoiled plasmid topoisomers with higher linking numbers migrated faster. M is marker lanes (1 kb DNA ladder; Invitrogen).

Article Snippet: The gWIZ-EGFP vector ( ) is a 5.746 kb EGFP transgene version of the kanR pUC origin gWIZ DNA vaccine plasmid vector (Gene Therapy Systems, San Diego, CA).

Techniques: Plasmid Preparation, Produced, SYBR Green Assay, Agarose Gel Electrophoresis, shRNA, Marker

Journal:

Article Title: Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields

doi: 10.1002/jgm.1499

Figure Lengend Snippet: Small plasmids have impaired induction. (A) Typical plasmid yield profiles and biomass growth for the inducible fed-batch fermentation (30 to 42°C shift) in DH5α. Induction of this 6.5 kb kanR plasmid was at 29hrs, plasmid yield reached 2130 mg/L after 12 hrs induction. (B) Plasmid yield profiles and biomass for 2.8 kb kanR gWIZ derived plasmid (Table 3; RF213). Induction was at 36 hrs; plasmid yield reached a peak of 120 mg/L and dropped to 70 mg/L after 10.5 h induction.

Article Snippet: The gWIZ-EGFP vector ( ) is a 5.746 kb EGFP transgene version of the kanR pUC origin gWIZ DNA vaccine plasmid vector (Gene Therapy Systems, San Diego, CA).

Techniques: Plasmid Preparation, Derivative Assay