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Method for genetic selection of high-plasmid producing e. coli clones

A plasmid, high-yield technology, applied in biochemical equipment and methods, microbial assay/inspection, introduction of foreign genetic material using vectors, etc., can solve problems such as time-consuming and laborious

Inactive Publication Date: 2009-06-10
MERCK & CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although high-producer screening has been successfully implemented to isolate high-producing clones of several DNA vaccine candidates, the process is laborious and time-consuming

Method used

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  • Method for genetic selection of high-plasmid producing e. coli clones
  • Method for genetic selection of high-plasmid producing e. coli clones
  • Method for genetic selection of high-plasmid producing e. coli clones

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Identification of IS1 Transposon in DNA Vaccine Plasmids

[0084] Strains, DNA vaccine plasmids and growth media - the host strain for all DNA vaccine constructs was E. coli DH5[F - deoR recA1 endA1 hsdR17(r k - , m k + )supE44λ - thi-1gyrA96 relA1]. This strain was originally purchased from Invitrogen (Carlsbad, CA; formerly GibcoBRL), adapted in synthetic medium DME-P5, and became electrocompetent for subsequent transformation. E.coli DH5α[F - φ80lacZ Δ M15 Δ(lacZYA-argF)U169 deoR recA1 endA1hsdR17(r k - , m k + ) gal - phoA supE44λ - thi-1 gyrA96 relA1] were purchased from Invitrogen (Carlsbad, CA) as electrocompetent cells. The construction of the HIV DNA vaccine plasmid V1Jns-nef is described in detail in International PCT Application No. PCT / US00 / 34162, filed December 15, 2000 (published June 21, 2001 as International Publication No. WO 01 / 43693). Briefly, the DNA vaccine plasmid consists of a pUC19-derived bacterial origin of replication and a neomy...

Embodiment 2

[0091] Comparison of IS1 content in high and low producer genomes using restriction fragment length polymorphism (RFLP) analysis

[0092]Strains, DNA vaccine plasmids and growth media - see, Example 1, supra. Alternatively, non-adapted, non-transformed cells were purchased from Invitrogen and maintained in LB medium. The construction of the HIV DNA vaccine plasmid V1Jns-tpa-nef (5540bp) is described in detail in International PCT Application No. PCT / US00 / 34162 (above). The construction of HIV DNA vaccine plasmid V1Jns-tpa-pol (7516bp) is described in detail in International PCT Application No. PCT / US00 / 34724 filed on December 21, 2000 (published as International Publication No. WO01 / 45748 on June 28, 2001 public). The construction of HIV DNA vaccine plasmid V1Jns-tpa-gag (6375bp) is described in detail in International PCT Application No. PCT / US98 / 02293 filed on February 3, 1998 (published as International Publication No. WO 98 / 34640 public).

[0093] Shake Flask Cultivat...

Embodiment 3

[0101] IS1 transposon integration site in the V1Jns plasmid

[0102] Materials and Methods - Plasmid DNA from V1Jns-nef clone NLB-5 propagated in DME-P5 medium was obtained as described in Example 1 . A total of sixteen oligonucleotide primers complementary to the intact, insert-free plasmid were designed to anneal in ~700 bp increments in the forward (8) and reverse (8) directions. The second set of primers is specific for the forward and reverse ends of the IS1 insert. A series of 32 PCR reactions were performed consisting of (i) one of 16 V1Jns-nef-specific primers and one of 2 IS1-specific primers, (ii) cloning of NLB-5 plasmid DNA as template, (iii) and HotStarTaq PCR Master Mix reagents (Qiagen). PCR reactions were performed using standard protocols. Each sample was analyzed on a 0.7% agarose gel to identify amplified fragments. The presence of amplified fragments is a preliminary indication of a vector-IS1 junction, but the possibility of false initiation events (fa...

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Abstract

The present invention relates to methods of selecting for highly productive clones of E. coli for the production of plasmid DNA comprising measuring the frequency of IS1 transposon insertional mutagenesis within either the plasmid or genomic DNA of transformed clonal subtypes. An increase in IS1 insertional mutagenesis is correlated with clonal subtypes likely to exhibit a low specific productivity. The PCR-based, genetic selection assays disclosed herein are amenable to high throughput analysis, reducing the time to identify highly productive clones capable of cultivating large quantities of plasmid DNA on an industrial scale.

Description

field of invention [0001] The present invention relates to a method for selecting highly productive clonal subtypes of E. coli strains carrying plasmid DNA, comprising comparing IS1 transposition activity between clonal subtypes of the same strain, wherein those clones showing relatively low transposition activity are representative Potentially productive clonal subtypes. A PCR-based assay is disclosed to measure the frequency of IS1 transposon insertion mutations within the plasmid or genomic DNA of transformed clonal subtypes. These genetic selection assays can be amenable to high-throughput analysis, reducing the amount of time to identify highly productive clonal subtypes capable of growing large quantities of plasmid DNA on an industrial scale. Background of the invention [0002] The manufacture and purification of large quantities of pharmaceutical-grade plasmid DNA is critical for the suitability of polynucleotide vaccines and gene therapy regimens for therapeutic u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCC12N15/69C12N15/1034C12Q1/6818
Inventor M·C·埃德蒙兹J·赫罗德K·J·普拉瑟尔
Owner MERCK & CO INC
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