AU611404B2 - Improvements in or relating to organic systems - Google Patents

Description

Davies Collison, Melbourne and Canberra.

-r I- i i r I COM MON w E A LT H OF 'A US T RA LI A PATENTS' ACT 1952 COMPLETE SPECIFICATION 611404 (Original) FOR OFFICE USE Application Number: Lodged: Complete Specification Lodged: Accepted: 4 Published: 0 (9 Priority: 0 Related Art: Class mnt. Class 4 4 Name of Applicant: 4 Aldress of Applicant: SANDOZ LTD.

CH-4002 Basle,

SWITZERLAND

4 4 4 0 4 A~tual Inventor(s): Address for Service: Dimitri KARAMATAI Jean-Christophe PlOT DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete specification for the invention entitled: "IMPROVEMENTS IN OR RELATING TO ORGA~IC SYSTEMS" The following statement is a full description of this invention, including the best method of performing it known: to us 1- Signature of declarant(s) (no 1 attestation required) .11 I.

Note: Initial all alterations.

DAVIES COLLISON, MELBOURNE and C BEP drzed jOfVicer -la- The present invention relates to a method of transforming Bacillus thuringiensis cells.

The terms "transforming" and "transformation", as used herein are S0o intended to relate to a mechanism of genetic transfer whereby O o exogenous DNA is introduced in a recipient bacterium, thereby inducing 0o genetic changes in said recipient bacterium.

SBacillus thuringiensis (BT) are gram-positive bacteria containing a° a crystal protein, the delta-endotoxin (DET) which is toxic to the larvae of a number of insects. Depending on the sub-species, BT is used as a selective biological pesticide against different pests. The 0 sub-species thuringiensis, alesti and dendrolimus are for example o pathogenic against Lepidoptera: the sub-species israelensis, 0 o00 darmstadiensis 73-E-10-2, kyushuensis and morrisoni PG14 against Diptera; the sub-species tenebrionis against Coleoptera; the So°° sub-species kurstaki HD-1, kenyae, aizawai and colmeri against 0 00..o* Lepidoptera and Diptera, whereas the sub-species dakota, indiana, tokokuensis and kumamotoensis are not known to be toxic to any pests.

0 o 0o° From the industrial and ecological point of view it is desirable 00 0 0. to have additional biological pesticides with different e.g. higher or broader spectrum of activity.

This aim can, for example, be achLeved by the development of new isolates from nature, by conjugation of bacteria or by transformation of bacteria.

Thus new BT strains with interesting activity have been isolated recently var. tenebrionis with activity against beetles) and recent successes with regard to the conjugation of BT strains have been reported as well.

Transformation of bacteria has the advantage that, if-successful, it allows the introduction of specific genetic information into bacteria.

L iI 2 130-4004 Thus a gene coding for a DET has been cloned in various microorganisms such as Escherichia coli, Bacillus subtilis and Pseudomonas fluorescens and even in higher plants (tobacco) by recombinant DNA techniques, more specifically by transformation.

Such genetically manipulated organisms produce however low amounts of DET compared to the amounts produced by natural BT strains.

The commercial value of such organisms is accordingly questionable, at least as long as no way has been found to improve the expression of So a the exogenous DNA encoding for DET.

SoO°° It would accordingly appear indicated to try and obtain a better 00 oa expression of exogenous genes (DNA) by using a BT bacterium as 0 0 o o0 recipient bacterium in transformation techniques.

0 0 0 o Known transformation techniques are essentially effected o 00 1 0 4 employing either cells or protoplasts.

The transformation of cells implies the presence of competent 0 cells, i.e. cells in a precise physiological stage allowing binding

S

t and uptake of exogenous DNA. There is however no evidence for the 0,0 existence of competent BT cells.

The transformation of BT protoplasts by DNA has been reported to 00 0 0o succeed only in very low yields, i.e. substantially lower than those oo000, obtained with the transformation of B. subtilis. The low yields may he partly due to the poor regeneration of the protoplasts, including the Gtransformed protoplast. Although Shall et al. (Fundamental and applied aspects of invertebrate Pathology, edited by R.A. Samson, J.M. Vlak and D. Peters, 1986, page 402) report that they optimized the protoplasting procedure and developed improved regeneration media to transform BT or B. cereus with plasmid DNA, they do not specify the nature of the optimization or improvement.

The transformation frequencies indicated by Shall et al. are accordingly difficult to interprete.

The present invention now provides an improved method of transforming BT. It is based on the finding that BT microorganisms develop a so-called competence status when they are introduced in a hypertonic aqueous medium.

3 130-4004 The term hypertonic as used herein refers to a medium which is hypertonic vis a vis the conventional BT (cell culture or growth) media.

The method of the invention involves the steps of a) growing BT cells in a hypertonic aqueous medium b) introducing in the cell culture obtained by step a) and in the presence of polyethylene glycol, exogenous DNA while maintaining the hypertonic status, and o O c) isolating and resuspending the thus treated BT cells in hypertonic S" aqueous medium to allow expression.

0o In principle any compound which does not pass the semi-permeable 0 0 0 o o0 cell membrane and is not metabolized by or toxic to BT cell may be 00 0 o oo employed to obtain the desired hypertonic status. In general, the o 0 desired hypertonic status will conveniently be achieved with the aid of saccharides, particularly mono- or disaccharides, which are not metabolized by BT. Suitable examples of such saccharides are sucrose 00 o and lactose.

0 00 o°o0 The concentration of saccharides to be employed to achieved the desired hypertonic status is conveniently of the order of 0.4 M 00 0 00 o saccharide per litre of aqueous medium or higher. In general, good 0.°o results will be obtained with concentrations which are essentially isotonic with respect to the BT cytoplasm. Such osmotic status is in a000 general obtained with a concentration of from 0.4 M to 0.5 M of o, saccharides per litre of aqueous medium. Higher saccharides concentrations may however be employed, but offer in general no advantages.

J The term "hypertonic" employed hereinafter refers to a status or medium as specified hereinbefore.

It is important that the hypertonic conditions are essentially maintained throughout the various steps a) to c) of the process.

The hypertonic aqueous media should be essentially neutral, i.e.

they should conveniently have a pH of 7 2, more preferably of 7 1.

In addition to the saccharides (to maintain the hypertonic U 4 130-4004 status) and eventually buffers (to maintain an essentially neutral status of the medium) other ingredients may and will be added e.g. to allow growth and development of the BT culture when required, etc.

Such additional ingredients are conventional and known by those skilled in the art, they comprise e.g. nutrients and salts.

Examples of suitable nutrients are e.g. beef extract, yeast extract, peptones, tryptones, amino acids tryptophan), nucleosides such as thymidine and the like.

o1o Examples of suitable salts are NaCl and MgCl 2 .6H 2 0. A suitable o hypertonic medium may contain from 0.05 to 0.1 M of salts per litre.

The salts wil comprise preferably magnesium salts, such as MgC12.6H 2 0.

The BT cell culture (starting material) will conveniently be oo0 0 0o prepared and grown under conventional conditions, i.e. with aeration °0 and at ambient temperature, in an appropriate nutrient medium, e.g. in the minimal medium disclosed by J. Spizizen in Proc. natl. Acad. Sci.

(Wash) 44, 171-175 (1958), eventually supplemented with amino acids, 00 0 ooo salts, e.g. catalytic amounts of a manganese salt such as MnSO 4 etc.

o 00 000oo It is advantageous to employ in step a) a BT cell culture which is in the exponential growth phase.

00 0 o o The freshly prepared BT cell culture is then diluted in a .0 ao hypertonic medium to a starting cell concentration of substantially less then 109 cells per ml, e.g. of 10 to 10 cells per ml and the 0 0o0 cell culture is grown, in said hypertonic medium up until a cell concentration of slightly less than 10 cells per ml, e.g. 10 to 5.108 cells per ml medium is obtained.

The hypertonic medium employed to dilute the freshly prepared BT cell culture is conveniently at 20 to 40 0 C, e.g. at 37°C. The culture is then allowed to grow at this temperature. Thorough aeration should of course be ascertained. A slight amount of silicon is conveniently added to the cell culture medium to prevent foaming.

When the desired final cell concentration (of slightly less than cells per ml) is reached, the thus prepared competent BT cells may be treated with DNA in the presence of polyethylene glycol (PEG), according to step b) of the process of the invention.

130-4004 It is however advantageous to treat the competent BT cells, obtained according to step a) of the invention, with moderate concentrations of lysozyme in hypertonic medium, and to isolate and resuspend the lysozyme treated BT cells in hypertonic medium, before subjecting them to process The amount of lysozyme to be employed should be less than that normally used for the preparation of protoplasts. Such amount (concentration) will of course depend on various factors such as the osmotic pressure of the medium, its o' temperature, the desired reaction time etc. In general a suitable Soo0 lysozyme concentration is of 20 to 300 microgram, e.g. of 200 o CO microgram per ml of hypertonic aqueous medium (which is substantially 0 0 0 S0 lower than the 2 to 15 mg per ml which vould be normally required for 0 0 o 0o protoplasting purposes). Adequate distribution of lysozyme in the cell 0 00 °o oo° culture medium should be ascertained. The reaction time will i.a.

depend on the concentration and the quality of the lysozyme 0o solution employed. The optimum reaction time may be determined by 0 0. standard assays.

0 o 0 0 0 The reaction temperature is conveniently between 20 to 40 0

C,

0o preferably above room temperature, e.g. at about 37 0

C.

0 Go During the lysozyme treatment the hypertonic status, as specified o above, should be maintained.

The treatment with lysozyme is then terminated by centrifugation 0 of the cell suspension and resuspension of the pellet in hypertonic SImedium, conveniently at room temperature.

The thus prepared BT cell culture obtained according to step optionally followed by treatment with lysozyme is then treated with DNA, e.g. plasmid DNA, in the presence of polyethylene glycol (PEG). For that purpose, the DNA as well as the PEG are employed as suspensions/solutions in a hypertonic solutions, such that the osmotic pressure of the cell suspension remains essentially unchanged after addition of DNA and PEG to said cell suspension.

The amount of PEG employed will be conveniently selected such that its concentration in the BT cell culture lies within the range of from lOOg to 400g per litre., e.g. at 3QOg per litre cell culture medium.

CJI i 6 130-4004 The transformation step b) can essentially be effected under the conditions known to be appropriate for conventional protoplast transformation processes.

Accordingly, the selection of the appropriate amount and type of PEG and of the appropriate amount of DNA to be employed can conveniently be made by those skilled in the art of protoplast transformation.

Thus, an example of PEG suitable for use in this process is PEG o.oo° 6000.

0 8 9 0o DNA amounts of from 100 nanogram to 20 microgram per 10 8 to 10 9 o°o BT cells will in general allow good results.

0o0o 0 The incubation is conveniently effected with gentle mixing at 0 00 oo room temperature. The required incubation time is short, in general of 0 oo the order of a few minutes (see the example).

The suspension comprising the transformed cells is then worked up Oo employing conventional methods but while securing the hypertonic 0 000 o 00 status of the solvent of the cells (when in solution/suspension). Thus 000 othe suspension is for example diluted with hypertonic solution, tilhe oOo suspension mixed, centrifuged and the pellet resuspended in hypertonic 0 00 o medium.

000900 The resulting suspension is then incubated at a temperature of to 40°C, e.g. at 37 0 C, to allow expression. The suspension is 00 conveniently aerated, employing e.g. a shaking water bath. An appropriate incubation time is 30 minutes to 5 hours, more preferably between 2 to 4 hours, e.g. 3 hours.

Appropriate dilutions of the thus obtained cell cultures may then be placed on culture plates for determination of colony forming units (CFU). The transformation frequency may be determined by known methods employing standard techniques such as antibiotic containing culture plates, visual observation etc.

The method of the present invention allows the transformation of BT cells in high yields. Transformation allows gene cloning of genomic libraries in BT cells, cloning and expression of DET genes in BT, cloning and expression of in vitro and in vivo modified DET genes in

C

7 130-4004 BT, the synthesis of useful polypeptides, etc.

Where the transformed BT cells are intended for use as biological pesticides they are conveniently employed in insecticidal composition form, e.g. in suspension concentrate form or powder form. Such compositions may be obtained in conventional manner.

In the following non-limitative example the starting materials (BT cells and plasmid DNA) were selected such that the results are Soo unambigous and cannot be due to plasmid interaction; the BT cells used o 0 as starting material did not contain plasmids, the plasmid DNA used as 0 00 o 0o 0o.. transforming agent encodes for resistance against tetra- 0o oo cycline.

o0o It will be appreciated that other BT cells and/or exogenous DNA, oo0 particularly plasmid DNA may be used in the method of the invention 0 00 with similar results.

Temperatures are in centigrade and parts by weight unless 0 oo specified otherwise.

O 00 0 0 0 00 00 0 0 0 0 0 00 0

I

8 130-4004

EXAMPLE

Starting Materials Strain :Bacillus thuringiernsis subsp. kurstaki HDi cry B, (obtained from Lecadet, Institut Pasteur, Paris) having no

DNA

plasmids.

zpBCl6.l (Kraft. J. et al. (1978) Molec. gen. Genet. 162 59-67) extracted from EIDi cry B (pBCl6.1), in which it was introduced by conjugation via cell mating with B. subtilis BD224 (pBCl6.l), coding for tetracycline resistance.

0 6p0 00 0 00 00 0 04000 00 0 0 a 0 00 0 04 Media SA Trp :Spizizen minimal medium (Spizizen J. (1958) Proc. natl. Acad.

Sci. (Wash.) 44 :171-175) suppleinented with 1% Casamino acids (Difco), 5x10- 6 M MnSO 4 and 20 iag/ml Tryptophan.

Hypetonic medium (HM): Beef Extractc Pep tone NaCl Sucrose Maleic Acid MgCl 2 *6H 20 pH 6.7 Luria Medium (LA) Tryp tone Yeast Extract NaCl Agar (Difco Bacto) Thymidine Antibiotics :Tetracycline, 1.50 g/l 5.00 g/l 3.50 g/l 171.15 g/l 2.32 g/l 4.07 g/l 10 g/l 5 g/l 10 g/l 15 g/l 20 mg/l 10-100 p~g/ml in LA plates 9 130-4004 Solutions SMM Sucrose 171.15 g/l Maleic Acid 2.32 g/l MgCl 2 6H20 4.07 g/l pH PEG PEG 6'000 40 g SMM ad 100 ml Lysozyme 2 mg/ml in HM, freshly prepared.

Method An overnight culture of HD1 cry B is prepared in 15 ml of SA Trp 0 o and grown with aeration at 20 0 C. The following morning, 'he culture is S0 diluted 50-100 x in prewarmed HM medium to a starting cell conceno tration of 7.5 x 105 ml. Silicon (2 pl) is added to prevent foaming.

0 00 °Qo o The culture is grown at 37°C with moderate aeration for 3h 30 min., i.e. to a cell concentration of 2.5 x 10 3 x 10 8 /ml. Lysozyme is co added to a final concentration of 200 pg/ml and 1 ml of cell suspension is incubated for 30 min. at 37 0 C in a shaking water bath 'oo" (150 rpm). The cell suspension is then centrifuged 1 min. at 10'000 g and the pellet is resuspended in 1 ml fresh HM at room temperature.

S 0.5 ml cell suspension is added to 50 pl SMM to which 100 0 ug of plasmid DNA have been added. The cells are transformed by addition of 1.5 ml of PEG solution, gentle mixing and a 2 min.

incubation at room temperature. 5 ml of HM is added to the cell 00 0 0 .o suspension, which is gently but thoroughly mixed and centrifuged for min. at 3'000 g. The pellet is resuspensed in 0.6 ml of HM and incubated 3h. at 37°C in a shaking water bath (150 rpm) to allow expression. Appropriate dilutions are plated on LA plates for CFU determination and on Tetracycline-containing LA plates for transformant selection.

1-2 x 103 transformants per pg of intact plasmid DNA, with a frequency of 5 x 10 10 4 are obtained.

i

Claims (12)

1. Process of transforming Bacillus thuringiensis cells comprising the steps a) growing Bacillus thuringiensis cells in a hypertonic aqueous medium b) introducing in the cell culture obtained by step a) and in the presence of polyethylene glycol, exogenous DNA while maintaining the hypertonic status and So c) isolating and resuspending the thus treated Bacillus thuringiensis 0 o cell in hyperonic aqueous medium to allow expression. o oo S.o

2. The process of Claim 1, whereby the Bacillus thuringiensis cell a culture of step a) is treated with moderate concentrations of 0 on lysozyme while maintaining the hypertonic conditions, and the thus o o0 treated Bacillus thuringiensis cells are then isolated and 0 resuspended in hypertonic aqueous medium prior to the treatment according to steps b) and c). S"

3. The process of Claims 1 or 2, wherein the hypirtonic status is o obtained employing saccharides which are not metabolized by Bacillus thuringiensis. C A

4. The process of Claim 3, which comprises employing at least 0.4 M of saccharides per litre aqueous medium.

Process of Claims 1 to 4, wherein the initial Bacillus sc thuringiensis cell concentration introduced in step a) is of from S104 to 106 cells per ml medium and the cells are grown up to a concentration of from 10 to slightly less than 10 cells per ml medium.

6. The process of Claims 2 to 5, wherein the lysozyme cone ntration is of 20 to 300 microgram per millilitre aqueous medium.

7. The process of Claims 1 to 6, wherein the hypertonic medium has a pH in the range of 6 to 8.

8. The process of Claims I to 7, wherein the hypertonic medium comprises a magnesium salt.

9. The process of Claims 1 to 8, effected at a temperature between and 400C. a) growing Bacillus thuringiensis cells in a hypertonic aqueous %p@y /2 11 130-4004 process of Claims 1 to 9, which comprises employing 100 nanogram to 20 microgram of DNA per 10 to

10 Bacillus thuringiensis cells.

11.The process of Claims 1 to 10, which comprises employing 100 to 400g of polyethylene glycol per litre of cell culture.

12.The process of Claims 1 to 11, which comprises maintaining the o on hypertonic status of step c) for 30 minutes to 5 hours. 0 .0 o °o -1 The-s-aeepts-,-f-atues-,-sompQ-&i-t-i-o-n-a-nd--Gmpea4 cells or organisms, referred to or i -ea-te in o o the specification and/or c -ms this application, individually or c-L-ecti-vely, and any and all O o combina j.n-s-o any two or more of said steps or ures. o oa 0 00 Dated this 21st day of December 1987 o0 SANDOZ LTD. o 0 o By its Patent Attorneys o 0- DAVIES COLLISON 0 0 o0 00 0 0 Sc e