# US 5,707,841 A · Process of producing highly transformable bacterial cells and cells produced thereby

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- Number: US 5,707,841 A
- Document ID: US5707841A
- Status: Granted
- Kind: A
- Application number: 08/637,003
- Filed: 1996-04-18
- Granted: 1998-01-13
- Earliest claimed: 1993-11-12
- Assignees: Stratagene
- Inventors: Alan L. Greener
- Web page: https://patentgrep.com/patent/US5707841A

## CPC

- C12N 9/2417: {from microbiological source}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- 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

The invention provided herein includes gram negative bacteria cells containing a gene encoding an enzyme with carbohydrate degrading activity that had been rendered competent to transformation. Carbohydrate degrading enzymes of interest for use in the invention include alpha-amylase. The competent cells of the subject invention may be frozen so as to provide for prolonged storage. Other aspects of the invention include methods for rendering gram negative bacterial cells, such as E. coli cells competent to transformation. These methods involve the step of transferring a gene encoding an enzyme with carbohydrate degrading activity into E. coli cells and subsequently rendering the cells competent using any of a variety of competency inducing procedures.

## Claims

### Claim 1

A method of preparing competent gram negative bacterial cells, said method comprising the steps of:
transferring a vector comprising an alpha-amylase gene into gram negative bacterial cells, and
treating the cells with a competency inducing procedure, whereby competent cells are produced.

### Claim 2 (depends on 1)

The method of claim 1, wherein the alpha-amylase gene is isolated from a thermophilic bacterium.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the competency inducing procedure is a standard high competency induction procedure employing the step of washing the cells with a buffer comprising potassium acetate, KCl, MnCl.sub.2, CaCl.sub.2, glycerol, and hexamine cobalt chloride.

### Claim 4 (depends on 1)

The method of claim 1, said method further comprising the step of freezing the competent cells.

### Claim 5 (depends on 4)

A competent bacterial cell produced by the method of claim 4.

### Claim 6 (depends on 1)

A competent bacterial cell produced by the method of claim 1.

### Claim 7 (depends on 1)

The method of claim 1, wherein the bacterial cells are E. coli.

### Claim 8 (depends on 7)

The method of claim 7 wherein the E. coli have a genotype selected from the group consisting of:
(1) ›e14.sup.- (mcrA), .DELTA. (mcrCB-hsdSMR-mrr)171, SbcC, recB, recJ, umuC:: Tn5 (Kan.sup.r), UvrC, supE44, lac, gyrA96, relA1, thi-1, endA1 ›F' proAB, lacI.sup.Q Z.DELTA.M15, Tn10, (tet.sup.r)!!,
(2) ›recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, ›F'proAB, lacI.sup.Q Z.DELTA. M15, Tn10 (tet.sup.r)!!, and
(3) ›.DELTA. (mcrA) 183, .DELTA.(mcrCB - hsdSMR - mrr) 173, endA1, supE44, thi-1, recA, gyrA96, relA1, Lac, ›F'proAB, laCI.sup.Q Z .DELTA. M15, Tn10 (tet.sup.r)!!.

### Claim 9

A competent gram negative bacterial cell comprising a genetic construction for the expression of an alpha-amylase gene.

### Claim 10 (depends on 9)

The competent bacterial cell according to claim 9, wherein the alpha-amylase gene is isolated from a thermophilic bacterium.

### Claim 11 (depends on 9)

The competent bacterial cell according to claim 9, wherein the bacterial cell is E. coli.

### Claim 12 (depends on 11)

The competent bacterial cell according to claim 11 wherein the E. coli has a genotype selected from the group consisting of:
(1) ›e14.sup.- (mcrA), .DELTA. (mcrCB-hsdSMR-mrr)171, SbcC, recB, recJ, umuC:: Tn5 (Kan.sup.r), UvrC, supE44, lac, gyrA96, relA1, thi-1, endA1 ›F' proAB, lacI.sup.Q Z.DELTA.M15, Tn10, (tet.sup.r)!!,
(2) ›recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, ›F'proAB, lacI.sup.Q Z.DELTA. M15, Tn10 (tet.sup.r)!!, and
(3) ›.DELTA. (mcrA)183, .DELTA.(mcrCB - hsdSMR - mrr)173, endA1, supE44, thi-1, recA, gyrA96, relA1, Lac, ›F'proAB, lacI.sup.Q Z .DELTA. M15, Tn10 (tet.sup.r)!!.

### Claim 13 (depends on 9)

The competent bacterial cell according to claim 9, wherein the cell is frozen.