JPS596889A - Production of modified methyl metabolizing bacteria and plasmid and host organism suitable therefor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the genetic engineering of methyl-utilizing microorganisms, particularly the preparation of obligate methyl-utilizing bacteria to express foreign DNA, cilasmids for introducing foreign DNA, and hosts. Regarding. One of the preparation methods according to the present invention includes (a) isolating a plasmid derived from an obligate methyl-assimilating bacterium; (0) introducing the hybrid plasmid into a host organism by transformation and amplifying it there; (d) following a selection process, performing appropriate conjugation. (-) conjugating the clone thus obtained with a unique methyl-assimilating bacterium as a recipient; The process consists of selecting clones of .

The plasmid used in step (c) of the preparation method is isolated from a methyl-assimilating bacterium of the genus Methylomonas, preferably from the species Methylomonas clara. Plasmid pB from Methylomonas clara strain deposited with the German National Strain Archive as DM Day No. 2!197.
F! 5 and other plasmids with the same replicon are particularly preferred.

The cilasmid carrying the selectable marker used in step (b) of method a may be the plasmid pBR332 described in Gone J, Vol. 2, pp. 95-113 (1977). The plasmid is designated below as pRMz.

The hybrid plasmid (European Patent Application No. 0ρ32425) containing genetic information for the expression of insulin is suitable as a plasmid with a selection marker for use in step (b) of the preparation process.

The hybrid plasmid obtained in step Φ) of the preparation method is then introduced into a suitable host organism, preferably E. coli, by transformation and amplified there.

In step (d) of the preparation method, a suitable conjugative plasmid (
Advantageously RP4) is introduced into this host organism containing the hybrid plasmid. Furthermore, the mobility defect is Ool
This is complemented by the introduction of appropriate plasmids such as K and 0o1v.

The host organism thus prepared is then subjected to (e) in the preparation method.
In step ), the hybrid plasmid introduced in step (C) can be transferred by conjugation to an obligate methyl-assimilating bacterium, preferably without cilasmid, as a receptor. Preferred receptors are bacteria of the genus Methylomonas, especially Methylomonas clara sp.
It is the 22nd evening strain. This strain is known, for example, to German Patent No. 2.655,451 and US Pat.
No. 4 is described in each specification.

Finally, in step (f) of the preparation method, the desired clones are selected by culturing in a medium containing methanol as carbon source and also on the basis of transfer of resistance or sensitivity to antibiotics. The methyl-assimilating bacteria thus obtained can express the foreign DNA they contain and thus produce, for example, insulin.

Details of these steps are further embodied in the examples below.

Furthermore, the present invention provides eL) and (in the above preparation method)
e) a simplified preparation method in which the steps follow, i.e. the hybrid plasmids are isolated after selection in @);
It also relates to a modified preparation method in which isolated cilasmids are introduced into spheroplasts of obligate methyl-assimilating bacteria by transformation at (θ).

The invention thus also relates to spheroplasts of methyl-assimilating bacteria, in particular of individual methyl-assimilating bacteria, in particular of bacteria of the genus Methylomonas, preferably of the species Methylomonas whales. Methylomo 9- Solanum clara ATOO strain No. 51226 is particularly preferred.

Spheroplasts according to the invention are prepared by culturing bacteria in a glycine-rich medium containing osmotic stabilizers. Preferably, this medium is slightly hypotonic. Further preferred embodiments of this preparation method are described in detail below.

Furthermore, the present invention relates to the use of novel spheroplasts to insert external Mum into these bacteria. This external DNA is basically introduced in the form of a plasmid. Suitable cilasmids are the hybrid plasmids described above.

To produce spheroplasts, methyl-assimilating bacteria are cultured and grown to a suitable number in a suitable medium (preferably a minimal medium). An appropriate number of bacteria is within the range of 0.5 to 1.8, particularly 0.9 to 1.4, at an optical density of 01)600. An aliquot of these cultures is then added to about 5 times the volume of the medium described above, which is rich in glycine and contains osmotic stabilizers. The glycine-containing powder may reach saturation, and 2
~4 inches (weight) is preferred.

Suitable osmotic pressure stabilizers include sugars such as sucrose, sugar alcohols such as sorbitol, and glycols such as polyethylene glycol. Preferably, the medium is slightly hypotonic, and this condition is provided by appropriate concentrations of osmotic stabilizers. For example 10
% sucrose or cicada, 1 molar sorbitol are suitable.

Bacteria are shaken in this medium until no rod-shaped methyl-assimilating bacteria can be detected under a phase contrast microscope. generally 6
After shaking at 100-180 times per minute for 1 hour at 7°C, the formation of spheroplasts as non-motile spherical structures is observed. This process is usually complete after about 4 hours.

For example, the generated spheroplasts were incubated at 4°C for 2,
Carefully centrifuge at 600XP for 10 minutes and resuspend in appropriate medium. This resuspension medium also contains an appropriate concentration of an osmotic stabilizer such as 15% (by weight) polyethylene glycol (molecular weight: 000) to stabilize the spheroplasts.

A solution consisting of a mixture of equal volumes of the medium used for resuspending spheroplasts and the carrier for the DNA to be introduced dissolved in a suitable buffer! ! ! ! Add to the suspension in a ratio of about 1:5.

A suitable 11JA vehicle is essentially a hybrid plasmid, as detailed above. A buffer suitable for this purpose is a buffer containing 1 mmol of Tris [2231049 moles of tris(hydroxymethyl)aminone and ethylenediaminetetraacetic acid] Licum in 1β (Tll
l buffer). Osmotic pressure stabilizer, e.g. 4t (heavy t)
Add the polyethylene glycol solution in three times the volume of the spheroplast suspension and mix carefully and thoroughly. Let the resuspension medium stand briefly at room temperature, add 10x the spheroplast suspension to the fcvk, and carefully centrifuge the mixture (3.+500X?).

The precipitate is resuspended in twice the volume of the spheroplast suspension and streaked onto agar plates to assay whether transformation has taken place.

The agar plate medium used for this has a composition corresponding to the resuspension medium and is selected in addition to Pact Agar (trade name) 1.5 H if the hybrid plasmid used contains the appropriate resistance gene. Antibiotics suitable for example ampicillin 5
Contains 0 μt/- or 10 μm/- of tetracycline. Plate 13 - Incubate the medium at 37° for 1 to 3 days,
Colonies of resistant bacteria were then cultured in the above-mentioned liquid medium containing the appropriate antibiotic and then assayed for the presence or absence of plasmid DNA [Humphreys et al.
BBAJ Vol. 583, pp. 457-463, (1975)]. By this means, plasmids used for transformation can be isolated from methyl-assimilating bacteria.

Furthermore, the present invention provides a DBM of Methylomonas clara, a hybrid flask with a replicon specific to a methyl-assimilating bacterium, in other words a bacterium of the genus Methylomonas, preferably Methylomonas whale, not specified. is prokaryotic DNA and eukaryotic DNA, especially DNA for the expression of insulin.
Regarding ATOO No. 21, No. 21, and No. 26, which are integrated.

1 4 - Furthermore, the present invention provides host organisms having the above-mentioned plasmids, and in addition host organisms having a conjugative plasmid, and furthermore host organisms having a plasmid for eliminating mobility defects. Regarding. Preferred host organisms include RP4 as a conjugative plasmid and also Oo as a plasmid to eliminate the mobility defect.
Includes IK or 001 V.

The advantage of the invention lies in the use of plasmid replicons of obligate methyl-assimilating bacteria, in other words cilasmids with a very narrow host range.

The preparation method according to the present invention has a high degree of safety and can be applied to recombinant DNA containing harmful and dangerous genetic information.

The invention will be demonstrated in more detail in the examples described below. All percentage data in the description is by weight, p!, unless otherwise indicated. - Cents.

In the examples, the following abbreviations were used.

i.e. ATP - adenosine triphosphate EIDTA - ethylenediaminetetraacetic acid or its sodium 0D6oo - optical density at 600 nm TI buffer - Tris HOIt adjusted to pH 8,0 per λ
10 mmon and the same (ID adjusted to pH 8,0
Aqueous Tris(HO2) containing TA 1 mmofi
- Tris(hydroxymethyl)aminomethane (coral acid salt) Example 1 Isolation of plasmid 1 of Methylomonas O whale DSM 2nd 26th et al. Volume 383, pages 457-463)
isolated by. For this purpose, 0D600
Bacteria are centrifuged from 1 L of medium in which the value has reached approximately 1.0,
The sedimented bacteria were resuspended in 5 ml of sucrose solution (25 sucrose dissolved in 50 mmol/λ Nodris HO°C solution at pH 8.0). Lysozyme solution while cooling with water
Lysozyme 5nll/- pH 8, 0)) Squirrel ao
A (250 mmol/n) dissolved in solution) Lmj and 0.2 molar EDTA solution (pH
8.0) 2- was added. The mixture was placed on ice for 5 minutes and the container was gently moved several times. Then Tris HOfi of 50 mmol/L, EI of 75.5 mmol/λ
Bacteriolysis was caused by adding a mixture of DTA and 0.2 t of a nonionic surfactant (Triton X-100) at pH 8.0. High viscosity mixed liquid at 48,0O
Centrifugation was performed in Oxr for 30 minutes, and the lysate was obtained as the supernatant. 1.5 molar saline solution per 10 − lysate solution.
1 glt and 1 7- and polyethylene glycol (average molecular weight 6,000) 1
, 1f was added. The mixture was kept at 4°C overnight.

The aggregated precipitate was collected by centrifugation at 1,500
0Q5 mmol/i, I) H8. O) 5.
The amount of solution was measured. After adding ○θ0L11 and 1% concentration ethidium bromide aqueous solution 1/15- to 1- of solution, 10 at 16,0OOxr
Centrifugation was performed for minutes. The resulting supernatant was centrifuged to equilibrium in a vertical rotor at 18°C.
Two bands were produced that were clearly visible even without ultraviolet irradiation, at an interval of about 1 yn (4000 revolutions per minute).

The fluorescent band became more clear by irradiation with ultraviolet light having a wavelength of 566 nm, and the lower band was punctured from the side of the gradient with a needle and then collected by suction into a syringe.

Ethidium bromide is 080! Isoproprone saturated with 1 day--was removed by repeated extraction and shaking with Panol.

Tl! After dialysis six times for 2 hours each time against 2μ of i buffer, the plasmid DNA was obtained in a purity suitable for subsequent steps.

If the presence of impurities in the DNA solution is significant, adjust the pH to 8.
,0 of a phenol-saturated 0.1 molar Tris-Hd solution twice and then three times with pure ether. Excess ether was stripped off and Δ amount of 6 molar acetic acid solution was added to the solution followed by an equal volume of isoprozonel to precipitate the Dl. After standing overnight, it was centrifuged at 12,000Xr for 60 minutes, and the precipitated DNA was washed with 90% ethanol. The DNA was then lyophilized and collected in TK buffer.

Example 2 Isolation of DNA from Agarose The following preparation method uses the method described by Langrage et al.
+n1stry J Vol. 106, pp. 264-271 (1
980)] was used.

Spread the DNA horizontally on a 0.5% agarose gel (Sigma product, low melting point agarose type ■Model number A-4018)
applied on top. Bands were visualized by ethidium bromide staining after electrophoresis and excised from the gel. DNA
The gel disc containing the was melted at 70°C in a glass tube, the volume was measured, and it was cooled to 37°C. To it were added equal amounts of the butanol and aqueous phases described below.

n-butanol 150- and water 150- in a separatory funnel
After shaking and separating into two phases, hexadecyltrimethylammonium bromide 1f'i was added to the water-saturated butanol phase. This solution was extracted with 100 ml of water from the aqueous phase. At this time, an antifoaming agent (Antiform A50 μJ2 manufactured by Sigma) may be added.

The mixture was allowed to stand overnight to separate into two phases, which were then fractionated separately.

The mixture obtained by adding the butanol phase and the aqueous phase was mixed well by carefully rotating the glass tube, and then the two phases were separated at 37°C. The upper butanol phase containing 1 person was separated, and the remaining aqueous phase was extracted twice with butanol by the method described above. 0.2 molar concentration Na0j corresponding to the amount of A in the combined butanol phase! Add the solution and mix well.

After the aqueous phase was separated, brine extraction was performed once more, and an equal amount of chloroform (previously purified using an aluminum oxide column) was added dropwise to the combined aqueous phase. After 30 minutes on ice, the lower chloroform phase was discarded and the remaining chloroform was evaporated with air. The DNA was precipitated with Isopro/JNol and after separation, TI! 1 buffer.

21 - Example 3 Polyacrylamide Gel Electrophoresis An 8-15% concentration polyacrylamide (FAA) gel was used for visualizing and characterizing DNA fragments below 0.3 megadaltons. The thickness of the gel is 1m.

The length was 30crn and the width was 14α. 1 for buffer solution
A mixture containing 89 mmol of boric acid, 289 mmol of Tris HO and 2.5 mmol of KDTA per pH 8.
, 2) was used.

To prepare 8 ml of PAA gel, add 35 m of a 24% concentrated FAA stock solution (10 (23.22 F of acrylamide and 0.789 F of N,N'-methylenebisacrylamide in 1 of water) as described above. Electrophoresis buffer (all components at 10x concentration) 10mg, 6
．． 4% concentration 3-ethylaminopropionitrile solution 4
25 mg and 50.75 mg of water were mixed. The mixture was degassed using a water pump, and the polymerization was initiated using solid ammonium peroxonisulfate. Gels were injected immediately and electrophoresis was started after at least 2 hours. Before loading the sample, remove the residual ammonium peroxonisulfate by
The battery was energized at 100V for about 1 hour.

Electrophoresis was performed at 100V and 12mA.

Example 4 Characterization of plasmid pBE'3 of Methylomonas clara DSM No. 2 No. 97 et al. Plasmid pBm3 was digested with the restriction endonucleases shown in Table 1, and the generated fragments were separated by gel electrophoresis. . The results recorded in Tables 1 and 11 were obtained by double digestion and characterization of the individual fragments cloned into pBR 522 as described below.

Plasmid pBIBj 6 is a deletion mutant of the large plasmid. Additionally, smaller plasmids also exist. These plasmids are all equivalent within the meaning of the invention if they carry Methylomonas-specific replicons.

Table 1 General structure of enzymes used to characterize pBE5
Number of fragments AcC7 1 XOr f ( 1EIQOR
1 5H1nc R5 Aval 12 Bal I At least 19 that does not cause decomposition Bam Hll Bst Ffll
Hlncl [IBgl ll:・t
1nQQ RV Hpa 1Kpn I
Small Xba IN
ru I' 13ph l
Xho IPst l 8
st l Xmn IPvu i
f Sst ■Sal Sumire
stu 12 5- Table 2 Size of fragment generated from restriction endonucleus of pBK 3 F'l 4.14 Hl 6.8
A, 1 2, OF2 5. o9
H2t42 A2 172F! l
1.27 H50,87A31.26F
4 0.91 H4Q, 51 A
4a 105F5 α63 H50,
28A4 1.0! IA5 0.85 A6 0.66 A7 0.55 A8 0.50 Mu9 0.13 Mu10 0.08 A11 Q, 07 Decomposition by aase (megadalton) Bco RI Eco R1+
+DI 1.35
K1 2.29D2 1.22
H21,83D3a 1.03 E! +
1.42D! 1 1.05 H41,
27D4 0.85 H50,91D6
0.63 to 6 0.65D7 0.55
H70,46n8 0.54 H80,
4゜H90,55]! t9 0.51 D10 0.50 )1f10 0.2BD
11 0.40 DI2 0.14 DI3 0.12 DI4 0.1 DI5 0.1 DI<S O,1 26- Example 5 Limited Decomposition Individual digestions were carried out using the manufacturer's recommended buffers. 5
Performed in 0-100 μλ. Samples were incubated overnight to ensure complete degradation of the DNA.

For dual and multiple degradations, appropriate multiple enzymes were usually added to the DNA simultaneously. In these cases, 1n
50 mmol of sodium chloride, Tris l1Cff
A buffer solution consisting of a solution containing 5 mmol (adjusted to pH 75), 6 mmol of magnesium chloride, 6 mmol of 2-merikabutoethanol, and 100 to 100 g of bovine albumin was used.

Control experiments showed that the enzyme gave the same results in this buffer as in the manufacturer's recommended buffer. The exceptions were enzymes that required above average salt. In these cases = 27 - the digestion was first carried out with one enzyme at a low salt concentration, then the salt concentration was increased before the second enzyme was added.

Example 6 Partial degradation using Boo RI 2 μV of plasmid DNA was incubated at 37° C. with 1 unit of restriction enzyme in a total volume of 50 μL.

Incubating the same mixture for different times,
At different times, the reaction was stopped by heating the mixture to 70° C. for 10 minutes. The unique nomoturn of bands determined by gel electrophoresis indicated the extent to which degradation had progressed after a certain point.

Example 7 Growth Conditions All strains of E. coli used were cultured in L broth (11 per Bactro Trisiton 10.?7 yeast extract 52 and sodium chloride 57) f28-. Methylomonas clara strains were cultured in one of the following minimal media.

M3 (S medium methanol 1.5 chia3po4
2SO4Q to 0.1%,
083% Na2SO4・10H20α018% Mg804・7H200,056 Chi OaC! 04 0.004
%Citric acid 11.015 t (N
H4)2Fe(804)2.6H200,005% trace element solution 80μjQ/QV155L
Medium methanol 1.5 cm 2SO4
0.16% MgE104・7H200.0 (S-Ch Na2804 0.025
%aaao3
0.014%Fθ2(BO2)s
O, 01 Chi H3PO40, 2nd Section NH5 (25th Section) 0
28 Chi KNO5Q, 3% NaHOO5G, 5% light solution 1-/! Trace element solution HgBOs O, 05t/
ItK engineering 0.01r/Q
MnSO4・4H200,04f/ItZnSO4・7
H200,049714(NH4)6MO70240,
02f/λ Example 8 Constituent plasmid pBF of hybrid vector! Restriction enzyme 1! so that the main part of 5 is cleaved at only one place and exists in a linear form. ico
pBR322 DNA was partially digested with Fl
ea R1 and then treated with alkaline phosphatase. The DNA thus prepared was mixed and incubated with 'r4 DNA synthetase at 14°C overnight. 50 μf of this synthesized mixture was used to transform E. coli HB101 Sono strain sensitized by OaOλ2.

The bacterial cells were spread on an L broth plate medium containing 20 μt/− of tetracycline and incubated overnight. The resulting resistant colonies were inoculated onto new plates, and well-growing clones were isolated from PBR52 by single colony lysis.
Tested for the presence of plasmids larger than 2. Clones with this type of plasmid DNA were finally cultured in L broth with or without tetracycline and plasmid DNA was obtained after bacterial growth was promoted with chloramphoenic 31-ml.

Restrictive digestion using the enzymes FiOOR1 and Ava I showed that the hybrid plasmid pRM 21 shown in FIG. 2 and the hybrid plasmid pRM 54 shown in FIG. 3 were obtained.

In addition to this, it is also possible to identify hybrid plasmids containing fractions of pBK3.

Example 9 Synthetic Enzyme Reaction The synthetic enzyme reaction is carried out at a total volume of 50 t<total concentration of DNA in A.
Performed at 0 μf/me. The buffer contained 50 mmol of Tris non (adjusted to pa 75), 4 mmol of magnesium chloride, 10 mmol of dithioerythritol and 2 mmol of ATPO, per bruise. T4
Synthetic enzyme (equivalent to 400 units) was added to these mixtures. However, the 32-1 unit mentioned here is the λ decomposed by H1n4 [1
50% of the DNA was incubated in a volume of 20 μλ at -16°C for 3
This corresponds to the amount of enzyme required for synthesis in 0 minutes, and the concentration of DNA in these mixed solutions is about 330 μV-.

The optimal amount ratio between two types of DNA in a particular synthetic enzyme experiment was calculated by the method of Dugaiczyk et al. [r, TMB, Vol. 96, p. 171 (1975)]. Incubation was at 14°C for at least 16 hours.

Example 10 A conjugative recipient bacterium and a donor bacterium were cultured overnight, and the recipient bacterium had an optical density of 0.
D600 is 1.0-1.2 and donor bacteria is 1.4-1.
6, and equal amounts of recipient and donor boxes were mixed so that the mixture had a large available surface area.

Incubate the mixture at 37° C. for 2 hours without shaking, and use 200 μμ of the mixture to form a composition that has screening and removal properties against donor bacteria and recipient bacteria having traits provided by cilasmids transferred from the donor bacteria. was applied on an agar plate medium. For the conjugation of Escherichia coli and Methylomonas whales, the plate is prepared in methanol minimal medium, and an antibiotic selected according to the type of plasmid to be transferred is added thereto.

In another experiment, the recipient and donor bacteria were incubated as described above, and 200 μl of the mixture was plated on methanol minimal plates without antibiotics. The plate medium was incubated overnight at 67°C, and the grown bacterial cells were poured into methanol minimal medium 2-. 200μ2 of this bacterial suspension was applied again to a methanol minimal plate medium supplemented with an appropriate antibiotic. Clones appeared after incubating the plates at 57°C for 48-72 hours.

Example 11 Expression of eukaryotic DHk in Methylomonas clara a) Plasmid pBE5 was transformed into a plasmid containing a eukaryotic gene, namely the monkey insulin gene as a-DHk.
Cloning and recloning were performed using the preparation method described for the derivatives of BR322.

The monkey insulin c-DNA is Pθt of pBR322.
Since this foreign genetic information was incorporated into the region divided by (1), this foreign genetic information was expressed in E. coli under the control of the β-lactamase promoter. This effect is caused by anti-insinillin antibodies (European Patent Application No. 0.052,6).
75) was produced.

35- Monkey insulin c-DNA has two internal sites that are split by Pst. Therefore, the encoded genetic information is not completely removed from the plasmid by dividing it with Pst (2).

In order to enable replication of this plasmid in Methylomonas whale, it is necessary to incorporate the D11A sequence of Methylomonas whale plasmid pBB5 into a derivative of pBR522 expressing insulin genetic information. Experimental operations were as described above. By this means, various hybrid cilasmids were obtained which structurally differed from the hybrid vector only in the nine monkey insulin c-DNA incorporated into the pBR522 portion. Since the sequences of the insulin genes in the 1) BR522 portions of these buffers are consistent, these clones can also express the insulin antibody determinant in E. coli. Of these clones, 36-encompassing the entire sequence of pBK 322 included
It was given the name S8.

b) The mobility defect found in pBR322 is due to O
complemented by plasmids such as oI K and Ool V [Young and POulis Ge
ne J Vol. 4, pp. 175-179 (1978)]o
1. Then, the sensitive Toji9 pRM series hybrid vector and the conjugative plasmid RP4 were introduced into the donor bacteria by transformation using the calcium chloride treatment described above. The colicin-susceptible strain AB 11'57 was used to detect plasmid Oo'l K [Warren et al.
1979)]. The donor bacterium Escherichia coli HB 101 strain (RP
4.001 K% pRM 54 ) or HB101
In conjugation experiments between strains (RP 4, ooIK, PRM 54) and the recipient Methylomonas clarum TOO5122+S strain, high concentrations (50 μg/-) of tetracycline were used to select clones harboring the hybrid vector. Selection was performed on methanol minimal medium (M56) in the presence of M.

In the analysis of a very large number of Methylomonas clara clones, approximately 10 clones harboring only the hybrid vector were obtained.

Donor strain of E. coli HB 101 (RF 4, coIK
.

A Methylomonas whale clone harboring only the plasmid pInMc 6B was obtained in a similar conjugation experiment between pInMc 6B ) and the recipient Methylomonas whale ATGG 51226.

C) Obtained plasmid structure of Methylomonas clara
Clones harboring Mc68 were cultured and insulin function was examined by radioimmunoassay or adipocyte assay.

Findings in E. coli (European Patent Application No. 0.032,67
5), Methylomonas clara ATOO51
In the case of strain 226, the insulin value was also measured to be 1 to 5 σ/no. In this way, the hybrid vector pBR! 122
The insulin genetic information contained in the part is Methylomonas Q
It is also expressed correctly and efficiently in whales.

Example 12 Methylomonas O clara ATOO51226 strain M3<S
Cultivate in minimal medium and adjust the optical density OD600 to 0.9-1.
Grow up to 4. These cultures were treated with glycine 4
- and into MS6 minimal medium supplemented with 10 g of sucrose. Bacteria were grown in this medium 100-180 times per minute.
Shake for 4 hours at 7°C. No oblong motile bacterial cells are observed under phase contrast microscopy.

The generated spheroplasts were incubated at 2,600XP at 4°C.
The mixture was centrifuged for 10 minutes and resuspended in M56 medium 1-1 supplemented with 10% sulphate.

89- In place of sucrose 10%, 1 mol/λ sorbitol or 15% polyethylene glycol having an average molecular weight of 6,000 may be added to the above medium.

Spheroplasts were grown at 5,600x10 at 4°C.
Separation can also be carried out by centrifugation for minutes.

Example 15 0.1- of a mixture containing eb and plasmid pRM21 from equal volumes of resuspension medium and TB buffer are added to 0.5- of the suspension obtained according to Example 12. Add 1.5 mm of a 4% strength polyethylene glycol 6000 solution to this mixture and mix carefully and thoroughly. This mixture is allowed to stand at room temperature for 2 minutes, resuspension medium 5- is added, and then centrifuged at 5,600Xf.

The sediment is collected in 1- resuspension medium and streaked onto agar plates. The composition of the plate medium was the same as that of the suspension medium, except for Bacto Agar (trade name) 1.5%.
and ampicillin 50μ2/-. The plates are incubated at 57° C. for 6 days, and the resulting colonies are cultured in M56 medium containing 50 μt/− of ampicillin. It is possible to isolate plasmid pRM21 used for transformation from the bacteria thus obtained.

Similar results are obtained when plasmid pRM54 is used instead of the above plasmid.

Example 14 The operation is carried out analogously to Example 13. However, using plasmid engineering nMc 6B, selection is carried out with 50 μf/- of tetracycline. This allows the plasmid engineering nMa 6
Methylomonas that contains B and produces insulin.
Obtains a clone of Clara.

[Brief explanation of drawings]

In the attached drawings, Figure 1 shows the DNA of pBK3 plasmid.
FIG. 2 is a schematic diagram of the whole of A and the 7 fragments generated when it is cut with restriction enzymes, and FIGS.
The figure shows pBR! on plasmids pRM21 and pRM54, respectively. It is a schematic diagram showing the location where +22 is inserted. Patent applicant Hoechst Akchengesellschaft F
IG, 1 Continued from page 1 Priority claim @ April 15, 1983 ■ West Germany (DE
) ■ P 3313643.2 0 Inventor Paul Plehue Day-6232 Partsoden am Taunus Pfarstrasse 5 @R Author Wurz Stahl Day-4630 Bochum Biermansve 2 0 Inventor Rüdiger Markwald Germany 8000 Germany Day 80 Berg Ordschüllerstrasse 1 0 Inventor Gerhard Würder Germany Day 6093 Frelsheim am Main Frelsheimerstrasse

Claims (1)

[Claims] 1) A hybrid plasmid having a replicon specific to an individual methyl-assimilating bacterium. 2) Bacteria of the genus Methylomonas, preferably Methylomonas.
Clara species, specifically the plasmid described in ATOO No. 31, No. 31, No. 26, Paragraph 1. 3) The cilasmid according to claim 1 or 2, which has integrated eukaryotic DNA, particularly DNA involved in insulin expression. 4) The plasmid according to claim 1 or 2, which has an integrated nine prokaryotic DNA. 5) The following operational steps include (a) isolating cilasmids from obligate methyl-assimilating bacteria; (b) generating hybrid plasmids having replicons specific to individual methyl-assimilating bacteria from this plasmid and a plasmid carrying a selection marker; (0) this hybrid plasmid is introduced into the host organism by transformation and amplified there; (1) after selection, the clones are treated with the appropriate conjugative plasmid and mobility defects are removed; , (,) The clone thus obtained is conjugated with an individual methyl-assimilating bacterium, preferably without a plasmid, as a recipient bacterium, and (f) a desired clone is selected. Production method of assimilating bacteria. 6) The specific methyl-assimilating bacterium is preferably a member of the genus Methylomonas, Methylomonas clara vertebrae, specifically the production method described in item 5 of ATOO No. 31, No. 31, No. 26. 7) The bacterium from which the plasmid is isolated is DSM No. 2, Section 2, 97, or the production method described in Section 6. 8) The plasmid with selection marker is pBR 322
and/or contains genetic information involved in the expression of insulin. 9) The host organism is E. coli and the conjugative plasmid is RP.
9. The process according to any one of claims 5 to 8, wherein the mobility defect is eliminated by the introduction of plasmid 001K or 001v. 10) Instead of operating steps 1fi) and <6), individual methyl-assimilating bacteria serving as recipient bacteria are converted into spheroplasts and hybrid cilasmids are introduced by transformation. The manufacturing method described in any one of paragraphs. 11) Methyl-assimilating bacteria are cultivated in a medium rich in glycine, containing osmotic stabilizers, preferably slightly hypotonic and containing 2 to 4 g glycine (by weight). Method for preparing spheroplasts of methyl-assimilating bacteria as described. 12) DSM no. A host organism harboring a cilasmid that eliminates the defect. 14) The host organism according to claim 15, which contains RP4 as a conjugative plasmid and, in the case of Tateshima, the plasmid coIK t or 001 V. 15) Methyl-assimilating, preferably obligate methyl-assimilating bacteria, especially Methylomonas spp., specifically Methylomonas clara species, specifically Methylomonas clara ATOO No. 3
1 No. 31 Yoshi 25 Eroplast.