# US 5,346,818 A · Method for the conjugative transfer of mobilizable vectors for E. coli to gram-positive bacteria and vectors suitable for use in such a method

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- Number: US 5,346,818 A
- Document ID: US5346818A
- Status: Granted
- Kind: A
- Application number: 07/447,139
- Filed: 1989-12-08
- Granted: 1994-09-13
- Earliest claimed: 1988-12-09
- Assignees: Degussa AG
- Inventors: Andreas Schafer; Jorn Kalinowski; Alfred Puhler
- Web page: https://patentgrep.com/patent/US5346818A

## CPC

- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N 15/77: for Corynebacterium; for Brevibacterium
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a method of effecting the conjugative transfer of a mobilizable vector from cells of an E. coli mobilizer strain to gram-positive bacterial cells. The invention also relates to vectors suitable for use in such a method.

## Claims

### Claim 1

A method for the conjugative transfer of a mobilizable vector from cells of an E. coli mobilizer strain to Coryneform bacterial cells which comprising producing restriction-defective cells of said Coryneform bacteria and crossing said defective cells with cells of said E. coli strain carrying said vector under conditions such that transfer of said vector to said defective cells is effected.

### Claim 2 (depends on 1)

The method according to claim 1, wherein said vector comprises a mob site DNA segment and said E. coli mobilizer strain contains a plasmid that codes for a Mob-site that effects mobilization of said vector.

### Claim 3 (depends on 2)

The method according to claim 2 wherein said plasmid is present as an extrachromosomal element.

### Claim 4 (depends on 2)

The method according to claim 2, wherein mob site is a 1.9 kb DNA segment of the plasmid RP4, an RP4 derivative being transferably by conjugation in the presence of a transfer function encoded by a gene that acts in trans, integrated into the chromosome of said E. coli mobilizer strain.

### Claim 5 (depends on 1)

The method according to claim 1, wherein said E. coli mobilizer strain is selected from the group consisting of E. coli S17-1, E. coli SM10, and E. coli S68-7.

### Claim 6 (depends on 1)

The method according to claim 1, wherein said vector is selected from the group consisting of pSUP101, pSUP102, pSUP201, pSUP202, pSUP203, pSUP205, pSUP301 and pSUP401.

### Claim 7 (depends on 1)

The method according to claim 1, wherein said defective cells of said Coryneform bacteria are produced by heat shocking said Coryneform bacteria prior to said crossing step.

### Claim 8 (depends on 1)

The method according to claim 1, wherein said defective cells are intact bret res cells.

### Claim 9 (depends on 1)

The method according to claim 1, wherein said defective cells are spheroplastized cells.

### Claim 10 (depends on 1)

The method according to claim 1, wherein said mobilizable vector comprises a gene, or fragment thereof, active in said Coryneform bacteria, which gene undergoes homologous recombination when transferred into said Coryneform bacteria.

### Claim 11 (depends on 1)

The method according to claim 1, wherein said mobilizable vector comprises a Procaryotic transposon.

### Claim 12 (depends on 1)

The method according to claim 1, wherein said mobilizable vector comprises a shuttle vector fused with a plasmid or vector which replicates in said Coryneform bacteria.

### Claim 13

A mobilizable, non-self-transferrable vector, comprising:
i) a first DNA segment containing a replicon functional in E. coli;
ii) a second DNA segment containing DNA sequences coding for mobilization (mob site) and transfer (oriT); and
iii) a third DNA segment that recombines homologously in Coryneform bacteria or that contains a replicon functional in Coryneform bacteria.

### Claim 14 (depends on 13)

The vector according to claim 13, wherein said third DNA segment further comprises a Procaryotic transposon.

### Claim 15 (depends on 13)

The vector according to claim 13, wherein said third DNA segment is derived from a vector that replicates in Coryneform bacteria.

### Claim 16 (depends on 15)

The vector according to claim 15, wherein said third DNA segment is derived from the vector pCV35.

### Claim 17 (depends on 13)

The vector according to claim 13, wherein said first and second DNA segments are derived from a vector selected from the group consisting of pSUP101, pSUP102, pSUP201, pSUP202, pSUP203, pSUP205, pSUP301 and pSUP401.

### Claim 18

A mobilizable, non-self-transferrable vector comprising:
i) a first DNA segment containing a replicon functional in E. coli;
ii) a second DNA segment containing DNA sequences coding for mobilization (mob site) and transfer (oriT); and
iii) a Procaryotic transposon.

### Claim 19

A vector pECM1, accession number DSM 4982.

### Claim 20

A vector pECM3 having the restriction map shown in FIG. 2.