JP2648674B2 - Transformation promoter and transformation promotion method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to the field of the so-called biotechnology industry, which has been recently developed, in which a gene is directly introduced into a microorganism, specifically a bacterium, using a vector to produce a transformant. The present invention relates to a transformation promoter and a transformation promotion method that can significantly increase the yield of a transformant when used in the transformation.

[Conventional technology]

Hitherto, it has been an important requirement in genetic recombination operations to improve the yield of transformants, and various technical devices have been devised. For example, various additives such as cell wall degrading enzyme and calcium chloride are used to transform the bacteria into which the gene is to be introduced into protoplasts or competent cells, or yeast extract, glucose, etc. are added as a medium to be used after the addition of the vector. Or a rich medium.

[Problems to be solved by the invention]

However, it is still difficult to reliably obtain a sufficient amount of transformants by the above-mentioned various measures.
In some cases, this was an important requirement that influenced the success of genetic manipulation experiments. This became a problem especially when the gene to be introduced was a rare and valuable one or was difficult to enter the host cell.

Also, when analyzing the base sequence in long-chain DNA,
You will be cloning a huge number of DNA fragments,
Since a large amount of labor and time are required for this, it has been desired that the yield of the transformant is stable and high when the gene is introduced.

An object of the present invention is to provide a transformation promoter and a transformation promotion method which can dramatically improve the yield of a transformant in order to solve the problems of the prior art.

[Means for solving the problem]

The present invention provides, as an active ingredient, at least one selected from the group consisting of an acid extract of grass seeds, an acid extract of cruciferous plants, an acid extract of brown algae plants, and an acid extract of red algae plants. The present invention relates to an agent for promoting the transformation of bacteria.

The present invention also relates to a method for promoting transformation, comprising transforming a bacterium with a vector in the presence of the transformation promoting agent.

As used herein, the term "transformation" means that a foreign gene is introduced into a target microorganism, for example, a bacterium, using a plasmid vector or a phage vector, and as a result, a transformed microorganism is obtained.

The term “transformation promoter and transformation promotion method” refers to an additive and an operation method for improving the yield of a transformant obtained in a transformation operation. The yield of this transformant, also referred to herein as transformation efficiency, is expressed by the number of transformed colonies or plaques obtained per microgram of the vector used.

Hereinafter, the present invention will be described in detail by dividing the sections into I. a transformation promoter and II.

I. Transformation promoter i) Raw material The transformation promoter of the present invention can be used as a raw material at least one selected from the group consisting of grass seeds, cruciferous seeds, brown algae plants, and red algae plants. It is. Specific examples of these are listed below.

Gramineous plant seeds Preferable ones of cereal seeds include rye, rice, barley, wheat, oats, millet, corn, barley,
Seeds such as millet, sugarcane, pigeon wheat, milo and the like can be mentioned.

Particularly preferred among these are rice, wheat, rye, and corn seeds, and among the seeds, the skin is particularly preferred. Wheat bran and corn hull are suitable as raw materials from the viewpoints of effect and economy.

Brassicaceae plant seeds Preferred examples of cruciferous plant seeds include seeds of rape, Chinese cabbage, cabbage, turnip, mustard, radish, wasabi, rape and the like.

Among them, rapeseed seeds, that is, rapeseed are particularly preferable, and rapeseed meal is preferably used from the viewpoint of effect and economy.

Brown algae plants Brown algae plants include:
Examples include plants belonging to the order of Pterolactyla, Cyperidae, Muchimo, Kerarimo, Urushigusa, Kombu, Cyropteris, Chlorophyta, Amidigthida, Hibamata and the like.

Among these, Hibamata, Ezoishige, Yabanemoku,
Plants of the order Himbamata, such as Jiromoku, Rappermoku, Hijiki, and Honwara, are preferred, and Hijiki is particularly preferred in terms of economy and effect.

In these plants, it is preferable to use leaves.

Red algae plants As red algae plants, the order of the primitive red algae
Examples include plants belonging to the order of the genus Chrysoriformes, the order of the Benidimates, the order of the genus Echinacea, the order of the genus Echinacea, the order of the genus Red algae, the order Echizonosidae, the proboscis, the caclates, the cynoliths.

Among these, Hoonoo, Lycopodium, Venetianago, Susukabeni, Okamuragusa, Mirin, Tosakanori, Yukari, Ibaranori, Isodantsuuatsubanori, Ogonori, Itanigusa, Suginori, Akabaginnansou,
Plants of the order Cygnaceae, such as hornworms, are preferred, and Ogonori are particularly preferred from the viewpoint of economy and effect.

ii) Acid extraction In the present invention, an acid extract from the above-mentioned grass seeds, cruciferous seeds, brown algae plants and red algae plants is used as an active ingredient of a transformation promoter. The processing will be described.

Type of Acid The acid that can be used in the present invention may be a mineral acid or an organic acid, but it is preferable to use an organic acid. Examples of the mineral acid include hydrochloric acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, and the like. Since it is easily hydrolyzed and nitric acid and formic acid have properties such as corrosiveness, they are generally less preferable than organic acids, and in the step of selecting and culturing transformants, treatments such as neutralization and removal are performed. Is preferred.

Organic acids include acetic, propionic, butyric, caproic, valeric, oxalic, malonic, succinic,
Maleic acid, fumaric acid, lactic acid, tartaric acid, citric acid, malic acid, benzoic acid, p-hydroxybenzoic acid, p-aminobenzoic acid, phthalic acid, cinnamic acid, salicylic acid, ascorbic acid, methanesulfonic acid, and the like. You can,
Acetic acid is preferred.

Acid concentration The concentration of the acid used for the extraction is suitably about 0.1 to 1.0 mol. When the amount is less than 0.1 mol, extraction of the active ingredient is incomplete, and when the amount is more than 1.0 mol, it takes time to remove the acid in a subsequent step.

Extraction temperature and time Extraction does not require heating, and room temperature is sufficient.

 Also, the time required for extraction is sufficient if 5 to 10 minutes.

Form of Transformation Promoter The transformation promoter of the present invention can be used in any form, such as an acid extract from a plant, a concentrated solution, a dry powder and the like. The most convenient use is to add it to the medium in advance.

II. Transformation promotion method i) Microorganism In the present invention, the target of the transformation operation is Escherichia coli and other transformable bacteria. Illustrative examples including microorganisms that can be transformed by known techniques are as follows.

Prokaryotes Prokaryotes include bacteria, myxobacteria, actinomycetes,
Although spirochetes and the like are included, Escherichia coli and Bacillus subtilis are preferably used.

Yeasts Yeasts include brewer's yeast, sake yeast, wine yeast, baker's yeast, and the like.

Molds As molds, Aspergillus niger, mustard mold, black mold, Neurospora, Aspergillus, Aspergillus,
Blue mold and the like.

Algae Examples of algae include Euglena, Chlorella, Juremo, Spirulina and Nostmo.

Bacteria are suitable for use in the present invention because they are fast growing and easy to operate. Yeasts, like bacteria, can be said to be fast growing and simple in transformation.

ii) Pretreatment step In order to carry out the transformation usually, the bacteria are subjected to some pretreatment to prepare a microorganism in a state in which the foreign gene carried by the vector can be easily introduced into the body. Examples of such pretreatment include methods such as preparation of protoplasts and competent cells, and electroporation. The present invention can be applied to any of these methods.

The effect of the transformation promoter of the present invention can be obtained by adding it in this pretreatment step, but it is preferable to add it after the addition of the vector as described later.

iii) Introduction of vector The vector is added to the suspension of the bacteria that has been subjected to the above pretreatment.

The present invention can be applied to both cases where a plasmid vector is used as a vector and where a phage vector is used.

The transformation-promoting agent of the present invention may be added in any of the previous steps.However, after adding the vector to the bacterial suspension pretreated as described above, the transformation-promoting agent of the present invention is added. It is most effective to add.

iv) Culture of transformants In general, in the method of introducing a gene by physical or chemical means, the survival rate of the bacteria is reduced because the bacteria are damaged during the treatment. Increase the yield of transformants.

It is most effective to add the transformation promoter of the present invention to the medium used for this culture. The medium used at this time was an LB medium (10 g of tryptone, 5 g of yeast extract).
g NaCl5g /) pH7.5, SOC medium (trypton 20g, yeast extract 5g, NaCl 0.584g, KCl 0.186g, 1M MgCl ₂ + 1M
It is preferable to use a rich medium such as MgSO ₄ solution 10 ml and glucose 20 ^{mM /)} pH 7.0, but any known medium according to each technique may be used. Although the yield of the transformant tends to increase as the amount of the transformation promoter added to the medium increases, it is sufficient to add 10 mg by dry matter weight per 1 ml of the medium.

v) Selection of Transformant The bacteria cultured as described above may be selected by any known method according to each purpose.

〔The invention's effect〕

According to the present invention, a transformant can be obtained at a 25- to 67-fold yield compared to the conventional method.

Although the compositional characteristics of the extract of the present invention and the cause of the effect of improving the transformation efficiency have not been sufficiently investigated, it is thought that other than mere nutritional or growth promoting effects are involved. Can be

The reason is that, in the transformation operation described in each Example, after adding the plasmid vector to the competent cells and cooling with ice-warming-ice, the medium containing the acid extract according to the present invention was added to the agar medium. Time to smear is 30 minutes to 1
This is because it is difficult to imagine that the growth of the transformant proceeds rapidly during this time.

Therefore, even if the agent for promoting transformation of the present invention has the effect of promoting the growth of microorganisms, the effect of the method of the present invention is completely different from this, and the effect of improving the transformation ability of competent cells is the greatest. It is determined to be the cause.

The causative substance relating to the effect of the transformation promoter of the present invention is:
It is presumed that it is not a single thing but a compound. Further, since the present extract is easily soluble in water and exerts a sufficient effect even when added in a small amount, the effect can be obtained without separating and purifying the active ingredient and using it.

The effect of the extract in the present invention can exhibit high transformation efficiency even with competent cells degraded by long-term storage. For example, after 3 months storage, the transformation efficiency was at a low level of 3 × 10 ⁵ ,
A ^10-7 were allowed to recover to a high level.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 (1) Sampling of cereal husk extract A 0.5M acetic acid aqueous solution 4 was added to 250 g of a by-product (wheat bran) having a high wheat husk content generated by wheat milling, and the mixture was stirred with a homogenizer type stirrer for 3 minutes, and then meshed with 40 mesh. The solution was filtered through a filter cloth, the filtrate was centrifuged at 3,000 × G for 10 minutes, the supernatant was concentrated under reduced pressure to evaporate and remove acetic acid, then dissolved in a small amount of water, and insolubles were removed by filtration and vacuum freeze-dried ( Yield 18
%).

(2) Use for transformation Escherichia coli (K12 strain) was placed in an Erlenmeyer flask in a 200 ml SOB medium.
(Difco tryptone 20g, Difco yeast extract 5g, NaCl 0.584g, KCl 0.186g, 1M MgCl ₂ + 1M MgSO ₄ 10m
1 in 1 and adjusted to pH 7.0) at 37 ° C 2.
After shaking culture for 5 hours, cool on ice for 10 minutes, centrifuge (2,000 × G,
10 minutes), precipitate FSB solution of _{68ml (KCl7.4g, MnCl 2 · 4H} 2
O 8.9 g, CaCl ₂・ 2H ₂ O 1.5 g, hexaamine cobalt trichloride 0.8 g, 1 M potassium acetate pH 7.5 10 ml, glycerin 100
g in 1 and the pH adjusted to 6.4 with HCl) suspended in ice (15 minutes), centrifuged (2,000 × G, 10 minutes), and the precipitate was suspended in 16 ml of FSB solution. DMSO (Dimethyl
Sulfoxide), ice-cooled (5 minutes), and 0.1 ml.
Add 56 ml of DMSO and cool on ice (15 minutes). Escherichia coli is transformed into competent cells by the above-mentioned treatment, and 0.2 ml of each was dispensed into a microtube and stored at -80 ° C.

Next, the process proceeds to the step of transforming the competent cells. First, during storage at -80 ℃, after thawing in the ice in the competent cells, this plasmid DNA pUC118 (Ap ^r, la
cZ), ice-cooled (20 minutes), heated (42 ° C. for 45 seconds), further ice-cooled (1 minute), and then added to the above-mentioned wheat bran extract in an LB medium (10 g of wheat bran extract). , Difco Tripton 10g, Difco East Extract 5
g, 5 g of NaCl in 1 and adjusted to pH 7.5 with NaOH), dilute 5-fold and incubate at 37 ° C for 1 hour, take a part of it, agar plate medium for selecting transformants in a Petri dish (Difco Tryptone 1%, Difco yeast extract 0.5%, NaCl0.5
%, PH 7.5 basic medium, ampicillin 50 μg / ml, X-G
al100 μg / ml, IPTG 47 μg / ml), and cultured at 37 ° C. overnight to count the number of blue colonies formed.

As a result, the number of colonies was an average of 200 per plate, and there was a great difference as compared with the average of three controls to which no wheat extract was added.

The transformation efficiency (number of transformed colonies / plasmid
DNA μg), the result was 2 × 10 ⁷ when the wheat bran extract was added, and 3 × 10 ⁵ in the control.

Example 2 (1) Harvesting method of cereal skin extract Extract of 0.4 mol of hydrochloric acid in 100 g of hull, which is a by-product of hulls, produced in the dry crushing process of corn
After stirring for 3 minutes with a homogenizer-type stirrer, suction filtration was performed with a 40-mesh filter cloth, and the filtrate was concentrated under reduced pressure. Next, the mixture was neutralized with caustic soda and centrifuged at 3000 × G for 10 minutes to obtain 250 ml of an extract. The concentration of the solid in the extract was about 100 mg / ml, and the yield of the solid was about 25 g. The solid portion contains about 13 g of NaCl generated by the neutralization.

(2) Use for transformation Escherichia coli (K12 strain) was placed in an Erlenmeyer flask in a 200 ml SOB medium.
(Difco tryptone 20g, Difco yeast extract 5g, NaCl 0.584g, KCl 0.186g, 1M MgCl ₂ + 1M MgSO ₄ 10m
After shaking culture at 37 ° C. for 2.5 hours, the mixture was cooled on ice for 10 minutes, and centrifuged (2000 × G, 10
Minutes). The precipitate FSB solution of _{68ml (KCl7.4g, MnCl 2 · 4H} 2
O 8.9 g, CaCl ₂ .2H ₂ O 1.5 g, hexaamine cobalt trichloride 0.8 g, 1 M potassium acetate 10 ml, glycerin 100 g
And adjusted to pH 6.4 with HCl), cooled on ice (15 minutes), and centrifuged (2,000 × G, 10 minutes). The precipitate is a 16 ml FSB solution containing Hull extract (Hull extract 1
_{g, KCl7.4g, MnCl 2 · 4H} 2 O 8.9g, CaCl 2 · 2H 2 O1.5g, trichloride hexamine cobalt 0.8 g, 1M potassium acetate pH7.5
The suspension was suspended in 10 ml of 100 g of glycerin in 1 and adjusted to pH 6.4 with HCl, 0.56 ml of DMSO (dimethyl sulfoxide) was added, and the mixture was cooled on ice (5 minutes). 0.56 more
ml of DMSO was added and cooled on ice (15 minutes). Escherichia coli is transformed into competent cells by the above treatment, and 0.2 ml is dispensed into a microtube and stored at -80 ° C.

Next, the process was shifted to transformation of the competent cells.

First, during storage at -80 ° C., after thawing the competent cells on ice, this plasmid ^{DNA pBR322 (Ap r, Tc r} ) added and ice (20 min), then warmed (42 ° C. 45 Seconds)
After further cooling with ice (1 minute), the LB medium to which the hull extract was added (10 g of hull extract, 10 g of Difco tryptone,
Difco yeast extract 5g, NaCl5g in 1
(Adjusted to pH 7.5 with OH), and cultured at 37 ° C for 1 hour. Thereafter, a part of the agar was taken, and agar plate medium for selecting transformants in a Petri dish (Difco tryptone 1%, Difco yeast extract 0.5%, NaCl 0.5%, pH7.5, basic culture medium, tetracycline 15 μg / ml) And ampicillin
(50 μg / ml added) and cultured at 37 ° C. overnight, and the number of colonies formed was counted.

As a result, the number of colonies was 240 on average per plate, and there was a great difference compared with the control without the hull extract having an average of 4 colonies.

The transformation efficiency (number of transformed colonies / plasmid
Compared with DNAμg), the case of hull extract addition was 2.4 ×
To 10 ^7, in the control it was 3 × 10 ^5.

Example 3 (1) Collection of cereal bark extract 0.5M acetic acid aqueous solution 4 was added to 250 g of wheat bran, and the mixture was stirred with a homogenizer-type stirrer for 3 minutes, and then filtered through a 40-mesh filter cloth. After centrifugation for 10 minutes, the supernatant was concentrated under reduced pressure to evaporate and remove acetic acid, and then dissolved in a small amount of water. Insoluble matters were removed by filtration and then freeze-dried in vacuum (18% yield).

(2) Utilization for transformation Escherichia coli (K12 strain) was placed in an Erlenmeyer flask in a 200 m SOB medium.
l (Difco tryptone 20g, Difco yeast extract 5g, NaCl 0.584g, KCl 0.186g, 1M MgCl ₂ + 1M MgSO ₄ 10m
After shaking culture at 37 ° C for 2.5 hours at 37 ° C and adjusted to pH 7.0, the mixture was cooled on ice for 10 minutes and centrifuged (2,000 x G, 1
0 min) and the precipitate is RF1 solution of _{68ml (RbCl12g, MnCl 2 · 4H} 2 O
9.9 g, potassium acetate pH 7.5 30 ml, CaCl ₂・ 2H ₂ O 1.5
g, glycerin 150g in 1, pH 5.8 with 0.2M acetic acid
The mixture was suspended on ice (15 minutes), centrifuged (2,000 × G, 10 minutes), and precipitated in 16 ml of RF2 solution (20 ml of 0.5 M 3- (N-morpholino) propanesulfonic acid, RbCl
1.2 g, CaCl ₂ .2H ₂ O, 11 g, glycerin 150 g contained in 1, pH was adjusted to 6.8 with NaOH, suspended in ice,
5 minutes). Escherichia coli becomes competent cells having a transforming ability by the above treatment, and 0.2 ml of each was dispensed into a microtube and stored at -80 ° C.

Next, the process proceeded to the step of transforming the competent cells. First, the competent cells stored at -80 ° C are thawed on ice, and then phage DNA M13mp18 (lacZ) is added thereto.
After cooling with ice (30 minutes), warming (42 ° C, 45 seconds), and ice cooling (1 minute), dilute 5-fold with the host E. coli suspension cultured with shaking in LB medium. did. Next, a part thereof was taken, that is, a soft top agar (soft top agar (10 g of wheat bran extract, 10 ml of 1M MgCl ₂ , and 0.6% of agar) was added to softtop agar (LB medium 1). Agar plate medium for selecting transformants in a Petri dish (Difco tryptone 1)
%, Difco yeast extract 0.5%, NaCl 0.5%, pH 7.
(X-Gal 100 μg / ml, IPTG 47 μg / ml added) to the basal medium of No. 5, and the number of blue plaques formed by culturing overnight at 37 ° C. was counted.

As a result, the average number of plaques was 250 per plate, and the average of the controls without the addition of wheat bran extract was 120.
Was individual.

In addition, transformation efficiency (number of transformed plaques / phage DN)
Aμg), the case of wheat bran extract addition
To 2.5 × 10 ^6, in the control it was 1.2 × 10 ^6.

Example 4 (1) Collection of rapeseed meal extract 0.5 g acetic acid aqueous solution was added to 180 g of rapeseed meal, stirred with a homogenizer-type stirrer for 3 minutes, filtered through a 40-mesh filter cloth, and the filtrate was centrifuged at 3,000 × G for 10 minutes. Separate and concentrate the supernatant under reduced pressure to evaporate and remove acetic acid, then dissolve with a small amount of water,
The insolubles were removed by filtration and then freeze-dried in vacuo (yield 14
%).

(2) Utilization for transformation Escherichia coli (K12 strain) was placed in an Erlenmeyer flask in a 200 m SOB medium.
l (Difco tryptone 20g, Difco yeast extract 5g, NaCl 0.584g, KCl 0.186g, 1M MgCl ₂ + 1M MgSO ₄ 10m
l in 1 and adjusted to pH 7.0) at 37 ° C 2.5
After shaking culture for 10 hours, cool on ice for 10 minutes and centrifuge (2000 × G,
10 minutes), precipitate FSB solution of _{68ml (KCl7.4g, MnCl 2 · 4H} 2
_{O 8.9g, CaCl 2 · 2H 2} O1.5g, trichloride hexamine cobalt 0.8 g, 1M potassium acetate pH 7.5 10 ml, glycerin 100g
Was suspended in ice-cold (15 minutes), centrifuged (2,000 × G, 10 minutes), and the precipitate was suspended in 16 ml of FSB solution. DMSO (dimethyl sulfoxide) was added, and the mixture was ice-cooled (5 minutes). Further, 0.56 ml of DMSO was added and the mixture was ice-cooled (15 minutes). Escherichia coli was transformed into competent cells by the above treatment, and 0.2 ml of each was dispensed into a microtube and stored at -80 ° C.

Next, the process proceeded to the step of transforming the competent cells. First of all, after thawing the competent cells during storage at -80 ℃ in ice, this plasmid DNA pUC18 (Ap ^r, lac
Z), cool on ice (20 minutes), and warm (42 ° C, 45 minutes).
After chilling for 1 second and then ice-cooling (1 minute), the LB medium (10 g of rapeseed meal extract, Difco
Tryptone 10g, Difco yeast extract 5g, NaCl5g
Was adjusted to pH 7.5 with NaOH), and the mixture was cultured at 37 ° C. for 1 hour. Then, a portion was taken and agar plate medium for selecting transformants in a Petri dish (Difco
Tryptone 1%, Difco yeast extract 0.5%, NaC
l0.5%, pH7.5 basic medium, ampicillin 50μg / ml,
X-Gal 100 μg / ml, IPTG 47 μg / ml added)
The number of blue colonies generated by culturing overnight at ℃ was counted.

As a result, the number of colonies was 320 on average per plate, which was much different from the average of 9 controls without the rapeseed meal extract being added.

The transformation efficiency (number of transformed colonies / plasmid
DNAμg), the rapeseed meal extract added was 3.2
The value was × 10 ⁷ , whereas the value was 9 × 10 ⁵ in the case of no addition.

Example 5 (1) Collection of brown algal plant extract To 250 g of hijiki, 4 0.5M acetic acid aqueous solution was added, and the mixture was stirred with a homogenizer-type stirrer for 3 minutes, filtered through a 40-mesh filter cloth, and centrifuged at 3000 × G for 10 minutes. The supernatant was concentrated under reduced pressure to evaporate and remove acetic acid, and then dissolved in a small amount of water. Insolubles were removed by filtration and then freeze-dried under vacuum (yield 11).
%).

(2) Utilization for transformation Escherichia coli (K12 strain) was placed in an Erlenmeyer flask in a 200 m SOB medium.
l (Difco tryptone 20g, Difco yeast extract 5g, NaCl 0.584g, KCl 0.186g, 1M MgCl ₂ + 1M MgSO ₄ 10m
After shaking culture at 37 ° C. for 2.5 hours at 37 ° C., the mixture was cooled on ice for 10 minutes, and centrifuged (2000 × G,
And 10 minutes), the precipitate 68 ml FSB solution (KCl7.4g, MnCl ₂ · 4H _{2
O 8.9g, CaCl 2 · 2H 2} O1.5g, trichloride hexamine cobalt 0.8 g, 1M potassium acetate pH 7.5 10 ml, glycerin 100g
Was suspended in ice-cold (15 minutes) and centrifuged (2,000 × G, 10 minutes), and the precipitate was suspended in 16 ml of FSB solution in 0.56 ml of DMSO (DMSO). Dimethyl sulfoxide), cool on ice (5 minutes), and add 0.56 ml
DMSO was added and the mixture was ice-cooled (15 minutes). Escherichia coli becomes a competent cell having a transforming ability by the above treatment, and 0.2 ml was dispensed into a microtube and stored at -80 ° C.

Next, the process proceeds to transformation of the competent cells. First of all, after thawing the competent cells during storage at -80 ℃ in ice, this plasmid DNA pUC118 (Ap ^r, lac
Z), cool on ice (20 minutes), and warm (42 ° C, 45 minutes).
For 2 seconds) and further ice-cooled (1 minute), and then added to the above-mentioned hijiki extract in an SOC medium (hijiki extract 10 g, difco-trypton 20 g, difco-yeast extract 5 g, NaC
0.584 g, KCl 0.186 g, 1 M MgCl ₂ +1 M MgSO ₄ solution 10 ml, Gl
ucose 3.6g, adjusted to pH 7.0) and diluted 5 times.
Incubated at ℃ for 1 hour. Then, one part was taken and agar plate medium for selecting transformants in a Petri dish (Difco tryptone 1%, Difco yeast extract 0.5%, NaCl 0.5%
Ampicillin 50 μg / ml, X-Gal100
μg / ml and IPTG 47 μg / ml) and cultured at 37 ° C. overnight, and the number of blue colonies formed was counted.

As a result, the number of colonies was 350 on average per plate, which was much different from the average of 8 controls without the addition of the hijiki extract.

The transformation efficiency (number of transformed colonies / plasmid
DNA μg), the case of the addition of the hijiki extract was 3.5
The value was × 10 ⁷ , whereas the value was 8 × 10 ⁵ in the case of no addition.

Example 6 (1) Collection of Red Algae Plant Extract To 250 g of Ogonori, 4M aqueous acetic acid solution was added, and the mixture was stirred with a homogenizer-type stirrer for 3 minutes, then filtered with a 40-mesh filter cloth, and the filtrate was 3000 × G for 10 minutes. After centrifugation, the supernatant was concentrated under reduced pressure to evaporate and remove acetic acid, then dissolved in a small amount of water, and insolubles were removed by filtration, followed by vacuum freeze-drying (yield 7%).

(2) Utilization for transformation Escherichia coli (K12 strain) was placed in an Erlenmeyer flask in a 200 m SOB medium.
l (Difco tryptone 20g, Difco yeast extract 5g, NaCl 0.584g, KCl 0.186g, 1M MgCl ₂ + 1M MgSO ₄ 10m
l in 1 and adjusted to pH 7.0) at 37 ° C, 2.5
After shaking culture for 10 hours, cool on ice for 10 minutes and centrifuge (2000 × G,
10 minutes), the precipitate, FSB solution of 68ml (KCl7.4g, MnCl ₂ · 4
_{H 2 O 8.9g, CaCl 2 ·} 2H 2 O1.5g, trichloride hexamine cobalt 0.8 g, 1M potassium acetate pH 7.5 10 ml, glycerin 100
g in 1, pH adjusted to 6.4 with HCl), ice-cooled (15 minutes), centrifuged (2000 × G, 10 minutes), and the precipitate was suspended in 16 ml of FSB solution. , 0.56 ml of DMSO (dimethylsulfoxide) was added, and ice-cooled (5 minutes).
0.56 ml of DMSO was added and the mixture was cooled on ice for 15 minutes. Escherichia coli becomes competent cells having a transforming ability by the above-mentioned treatment, and 0.2 ml of each is dispensed into a microtube and stored at -80 ° C.

Next, the process proceeds to the step of transforming the competent cells. First of all, after thawing the competent cells during storage at -80 ℃ in ice, this plasmid DNA pUC118 (Ap ^r, lac
Z), ice-cooled (20 minutes), heated (42 ° C., 45 seconds), and ice-cooled (1 minute), and then the LB medium (Ogonori extract 10 g, Difco・
Tryptone 10g, Difco yeast extract 5g, NaCl5g
And the pH is adjusted to 7.5 with NaOH).
After culturing at 7 ° C. for 30 minutes, a portion is taken and agar plate medium (Difco tryptone 1) for selecting transformants in a Petri dish is taken.
%, Difco yeast extract 0.5%, NaCl 0.5% pH7.5
In a basic medium, ampicillin 50 μg / ml, X-Gal 100 μg / ml
ml, and IPTG 47 μg / ml), and cultured at 37 ° C. overnight, and the number of blue colonies formed was counted.

As a result, the number of colonies was 290 on average per plate, and there was a great difference compared to the average of 7 controls to which no Ogonori extract was added.

The transformation efficiency (number of transformed colonies / plasmid
DNAμg), the case of Ogonori extract added
In contrast to 2.9 × 10 ⁷ , the value was 7 × 10 ⁵ when no additive was added.

Examples 7-14 An acid extract of various cereal bark parts was produced in the same manner as in Example 1, and the transformation efficiencies were compared.

Table 1 shows a comparison of the effect of water or acid extracts on the transformation efficiency of various cereal bark contents. The water extract was 1.6-2.7 times the control and 25% in the acid extract. The difference was as large as ~ 67 times.

Even when the same type of cereal is used as a raw material, there is a tendency that the higher the content of the skin portion, the greater the effect. For example,
In the case of wheat, the efficiency of transformation was 2.0 × 10 ⁷ and 1.2 × 10 ⁷ , respectively. Yes, the effect of bran with a lot of skin was greater than that of lower-grade flour.

A similar tendency can be seen in the comparison between rice bran (bran) and brown rice in the case of rice.

Examples 15 to 21 Table 2 shows the results of extraction using various extraction solvents, using Escherichia coli K12 as a host and plasmid DNA as a vector.
The comparison of the conversion efficiency improvement effect when using pBR322 for transformation was performed.Pure water, NaOH (neutralized extract was neutralized with HCl), and those using an extraction solvent other than acid such as sodium chloride The effect was less than that used. Acetic acid showed a good result when compared with each other. When using salt and citric acid, use the concentrated solution.When using hydrochloric acid, use the concentrated solution after neutralization with caustic soda.The amount of addition is calculated based on the calculated values of sodium chloride, citric acid, and the salt generated by neutralization. The net extract weight subtracted was 1 mg / ml of medium.

Continuation of front page (72) Inventor Miharu Tsutsui 6-26-10 Higashi-Rinkan, Sagamihara-shi, Kanagawa

Claims (10)

(57) [Claims] 1. An active ingredient comprising at least one selected from the group consisting of an acid extract of a grass seed, an acid extract of a cruciferous plant seed, an acid extract of a brown algae plant, and an acid extract of a red algae plant. Bacterial transformation promoter. 2. The transformation promoter according to claim 1, wherein the acid extract of grass seeds is an acid extract of wheat bran, corn hull or rice bran. 3. The transformation promoter according to claim 1, wherein the acid extract of cruciferous plant seeds is an acid extract of rapeseed meal. 4. The transformation promoter according to claim 1, wherein the acid extract of the brown algal plant is an acid extract of hijiki. 5. The transformation promoter according to claim 1, wherein the acid extract of a red algae plant is an acid extract of Ogonori. 6. In the presence of at least one selected from the group consisting of an acid extract of grass seeds, an acid extract of cruciferous seeds, an acid extract of brown algae plants and an acid extract of red algae plants. And transforming the bacterium using the vector. 7. The method according to claim 6, wherein the acid extract of grass seeds is an acid extract of wheat bran, corn hull or rice bran. 8. The method according to claim 6, wherein the acid extract of cruciferous plant seeds is an acid extract of rapeseed meal. 9. The method according to claim 6, wherein the acid extract of the brown algae plant is an acid extract of hijiki. 10. The method according to claim 6, wherein the acid extract of a red algae plant is an acid extract of Ogonori.