Host-vector system for antibiotic-free CoIE1 plasmid propagation

a coie1 plasmid and host technology, applied in the field of plasmid dna as gene transfer vehicle, can solve the problems of inconvenient use of antibiotics, additional genes on the plasmid, and potential contamination of the final product with residual antibiotics, and achieve the effect of inhibiting transcription

Inactive Publication Date: 2006-03-23
BOEHRINGER INGELHEIM RCV GMBH & CO KG
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Benefits of technology

[0084] In a preferred embodiment, the toxic marker protein is not lethal or toxic per se or due to a toxic effect upon its expression, but a repressor protein which acts by repressing the transcription of a gene that is essential for growth of said bacterial cell.
[0085] In this embodiment of the invention, RNA I-mediated down-regulation in the presence of the plasmid affects the repressor. This means that the presence of RNA I and its interaction with the repressor mRNA (the RNA II-like sequence) leads to inhibition of the repressor and thus to activation or up-regulation o

Problems solved by technology

A second drawback of plasmids that bear antibiotic resistance genes is a potential contamination of the final product with residual antibiotic.
In view of possible immune sensitization, this is an issue, especially in the case of beta-lactam antibio

Method used

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  • Host-vector system for antibiotic-free CoIE1 plasmid propagation
  • Host-vector system for antibiotic-free CoIE1 plasmid propagation
  • Host-vector system for antibiotic-free CoIE1 plasmid propagation

Examples

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example 3

[0158] Expression / Suppression of Marker Gene During Fermentation

[0159] The E. coli strains IS11 and IS5 are analyzed during a fed-batch fermentation process, with and without the presence of plasmid pBR322. Table 4 summarizes the experimental set-up of four fed-batch fermentations. Each strain is grown either in the presence or absence of pBR322.

TABLE 4Fed batch fermentationsExperimentHost strainpBR322AS1IS 11−AS2IS 11+AS3IS 5−AS4IS 5+

[0160] All four cultivations show very similar trends for online signals such as CO2, O2, base consumption or capacity and the course of total BDM varies also in a very small range of less than ±10% from the calculated mean as shown in FIGS. 7a and 7b. FIG. 7a shows bacterial dry mass (BDM) and GFP expression of IS II with or without maintenance of pBR322. While the total BDM is identical for both fermentations, the GFP concentration is drastically decreased when pBR322 is present (50%). The curve progression of GFP measurements strongly indicates i...

example 4

[0161] Use of a Repressor for Regulating the Expression of an Essential Gene

a) Generation of Constructs for Essential Genes

[0162] The first essential gene to be tested is map (Li et al., 2004), the gene for the methionine aminopeptidase, which is located at min 4 of the E. coli chromosome, 357 base pairs from the rpsB-tsf operon and 201 bp from the T44-RNA gene. The two genes are transcribed divergently and promoters do not overlap. This is an essential point, because the promoter of the essential gene is to be removed entirely and replaced by an inducible promoter that is specific for a chosen repressor. Chang et al, 1989 described a conditionally lethal mutant strain which has the map gene controlled by the lac promoter. By the map cassette, a 67 bp chromosomal section is replaced containing the map promoters (Chang et al, 1989). To circumvent possible transcripts from the genome, two strong transcriptional terminators T1 and T2 from the rrnB operon (Brosius et al, 1981) are ad...

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Abstract

A host-vector system that uses the RNA-based copy number control mechanism of ColE1-type plasmids for regulating the expression of a marker gene allows for antibiotic-free selection of plasmids and is useful for production of plasmid DNA and recombinant proteins.

Description

RELATED APPLICATIONS [0001] This application claims priority from EP 04 022201 filed Sep. 17, 2004. [0002] The present invention relates to the field of plasmid propagation, in particular for production of plasmid DNA and recombinant proteins. In addition, the sequence listing submitted herewith is incorporated herein by reference in its entirety. BACKGROUND AND DESCRIPTION OF THE INVENTION [0003] The use of plasmid DNA as gene transfer vehicle has become widespread in gene therapy. In gene therapy applications, a plasmid carrying a therapeutic gene of interest is introduced into patients; transient expression of the gene in the target cells leads to the desired therapeutic effect. [0004] Recombinant plasmids carrying the therapeutic gene of interest are obtained by cultivation of bacteria. For selecting bacterial transformants and in order to assure maintenance of the plasmids in the bacterial host cell, traditionally, an antibiotic resistance gene is included in the plasmid backbo...

Claims

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Application Information

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IPC IPC(8): C12P21/06C12N15/74C12N1/21C12N15/70
CPCC12N15/70
Inventor GRABHERR, REINGARDPFAFFENZELLER, IRENE
Owner BOEHRINGER INGELHEIM RCV GMBH & CO KG
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