# US 2010/0167379 A1 · Bacteria for high efficiency cloning

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- Number: US 2010/0167379 A1
- Document ID: US20100167379A1
- Status: Published application
- Kind: A1
- Application number: 12/646,828
- Filed: 2009-12-23
- Published: 2010-07-01
- Earliest claimed: 2003-01-16
- Assignees: Life Technologies Corporation
- Inventors: Fredric R. Bloom; Brian Schmidt
- Web page: https://patentgrep.com/patent/US20100167379A1

## CPC

- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed are novel bacterial hosts that are capable of high efficiency transformation with methylated and/or unmethylated nucleic acids, and that are bacteriophage resistant. Such bacteria contain: (1) an F′ episome that confers high efficiency transformability; (2) one or more mutations that allow transformation of methylated nucleic acids; (3) one or more mutations that allow transformation with unmethylated nucleic acids; and/or (4) one or more mutations that confer resistance to bacteriophage infection. Also disclosed are methods for transforming such bacteria, and kits that contain such bacteria (e.g., that have been made competent for transformation).

## Claims

### Claim 1

An isolated bacterium comprising: an F′ episome that confers high efficiency transformability; one or more mutations that allow transformation of methylated nucleic acids; one or mutations that allow transformation with unmethylated nucleic acids; and one or more mutations that confer resistance to bacteriophage infection.

### Claim 2 (depends on 1)

The bacterium of claim 1 wherein said bacterium is an Escherichia coli strain.

### Claim 3 (depends on 2)

The bacterium of claim 2 wherein said strain is K-12.

### Claim 4 (depends on 3)

The bacterium of claim 3 wherein the genotype of said F′ episome comprises proAB+ lacIq lacZΔM15 Tn10(TetR).

### Claim 5 (depends on 3)

The bacterium of claim 3 wherein said one or more mutations that allow transformation of unmethylated nucleic acids involve the hsdR, hsdM and/or hsdS genes.

### Claim 6 (depends on 3)

The bacterium of claim 3 wherein said one or more mutations that allow transformation of methylated nucleic acids involve the mcrA, mcrBC and/or mrr genes.

### Claim 7 (depends on 3)

The bacterium of claim 3 wherein said bacteriophage is phage T1.

### Claim 8 (depends on 3)

The bacterium of claim 3 wherein said one or more mutations that confer resistance to bacteriophage infection involve the tonA gene.

### Claim 9

An isolated Escherichia coli K-12 bacterium, said bacterium having a genotype comprising mcrA Δ(mrr-hsdRMS-mcrBC) tonA/F′ proAB+ lacIq lacZΔM15 Tn10(TetR).

### Claim 10 (depends on 9)

The bacterium of claim 9, wherein said genotype comprises: mcrA Δ(mrr-hsdRMS-mcrBC) φ80(lacZ)ΔM15 Δ(lacZY A-argF) U169 endA1 recA1 supE44 thi-1 gyrA96 relA1 deoR tonA panD/F′ proAB+ lacIq lacZΔM15 Tn10(TetR).

### Claim 11 (depends on 9)

The bacterium of claim 9, wherein said bacterium is Escherichia coli BRL3946 (NRRL accession No. B-30640).

### Claim 12 (depends on 9)

The bacterium of claim 9, wherein said bacterium is a derivative of Escherichia coli BRL3946 (NRRL accession No. B-30640).