JP2729805B2 - 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.

[Prior Art] Conventionally, in gene recombination operations, it has been an important requirement to improve the yield of transformants, and various technical devices have been devised. For example, cell wall degrading enzymes, seed additives such as calcium chloride are used in order to make the cells 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 very small and important 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 abstract]

The present invention provides a bacterial transformation promoter comprising at least one phytic acid or a salt thereof as an active ingredient.

The present invention also provides a method for promoting transformation, comprising transforming a bacterium with a vector in the presence of the above-mentioned transformation promoting agent.

As used herein, the term "transformation" refers to introducing a foreign gene into a target microorganism, for example, a bacterium using a plasmid vector or a phage vector, thereby obtaining a transformed microorganism. .

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 vectorer used.

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

I. Transformation promoter The active ingredient of the transformation promoter of the present invention is phytic acid or at least one of its salts.

As the salt of phytic acid, an alkali metal salt is preferable since it is easily soluble in water, and a sodium salt, a potassium salt, a lithium salt and the like can be used. In addition, calcium magnesium double salts, calcium salts, pentacalcium salts, tetracalcium salts, pentabarium salts, and the like which are widely present in plants can be used. However, those which are hardly soluble in water, such as a pentacalcium salt, are not preferred.

Phytic acid or a salt thereof is 0.2-2.0 mmol /
It is preferable to add it to the medium. 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, there can be mentioned, for example, Aspergillus niger, mustard mold, black mold, Neurospora, Aspergillus, Aspergillus, and blue mold.

Algae Algae include Lugrena, Chlorella, Juremo, Spirulina, Nostmo and the like.

Bacteria are suitable for use in the present invention because they are fast growing and easy to operate. It can be said that yeasts, like bacteria, grow quickly and can be easily transformed.

ii) Pretreatment Step In order to carry out transformation usually, the above bacteria are subjected to some pretreatment to prepare a microorganism in a state in which a foreign gene carried by a 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. The yield of the transformant can be increased.

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, NaCl0.584g, KCl0.186g, 1M MgCl ₂ + 1M
It is preferable to use a rich medium such as MgSO ₄ solution 10 ml glucose 20 mmol /) pH 7.0, but any known medium according to each technique may be used. The amount of the transformation promoter added to the medium is sufficient if 0.2 to 2.0 mmol / active ingredient is added to the medium.

v) Selection of transformant Transformant 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 with a yield of 2 to 10 times that of the conventional method.

Although the cause of the effect of improving the transformation efficiency in the present invention has not been sufficiently investigated, it is considered that the effect other than the mere nutritional or growth promoting effect is involved.

The reason is that in the transformation operation described in each example, the plasmid vector was added to the competent cells, and then ice-cooled-warmed-ice-cooled, and then the medium containing the transformation promoter of the present invention was added, and then the agar medium was added. This is because it takes 30 minutes to 1 hour to spread the transformant, and 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 effect of the extract in the present invention can exhibit high transformation efficiency even with the competent cells degraded by the above-mentioned storage.

〔Example〕

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

Examples 1 to 3 The following experiments were performed using phytic acid (Example 1), calcium phytate (Example 2), and sodium phytate (Example 3) at concentrations of 0.1 to 2.0 mM, respectively, as transformation promoters. Done.

Escherichia coli (K12 strain) in an Erlenmeyer flask with 200 ml of 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 (2,000 x G, 10%).
Minutes), precipitate FSB solution of _{68ml (K Cl7.4g, Mn Cl 2} · 4H 2
_{O8.9g, CaCl 2 · 2H 2 O1.5g} , trichloride hexamine cobalt
0.8g, 1M potassium acetate pH7.510ml, glycerin 100g
The suspension was cooled in ice (15 min), centrifuged (2,000 × G, 10 min) and precipitated.
Suspend in 6 ml of FSB solution, add 0.56 ml of DMSO (dimethyl sulfoxide), cool on ice (5 minutes), and further add 0.56 ml of DMSO
And cool on ice (15 minutes). Escherichia coli becomes competent cells with transformation ability by the above treatment,
This was dispensed in 0.2 ml portions into a microtube and stored at -80 ° C.

Next, the process proceeds to the step of transforming competent cells. First, after thawing the competent cells during storage at -80 ° C. in ice, this plasmid DNApUC19 (Ap ^r, lacZ) added, ice (20 min), then warmed (42 ° C. 45 seconds), After further cooling on ice (for 1 minute), the LB medium containing the above-mentioned transformation promoter of the present invention (each concentration of the transformation promoter of 0.1 to 10%) was added.
2.0 mmol /, 10 g of Difco tryptone, 5 g of Difco yeast extract, 5 g of NaCl in 1, pH 7.5 with NaOH
After culturing at 37 ° C. for 1 hour, a portion thereof was taken, and an agar plate medium for selecting transformants in a petri dish (Difco tryptone 1%, Difco yeast extract 0.5%, NaCl 0. (Ampicillin 50 μg / ml, X-Gal 100 μg / ml, IPTG 47 μg / ml) was added to a 5%, basic medium of pH 7.5), and the number of blue colonies formed by culturing overnight at 37 ° C. was calculated.

The results are shown in Table 1 (Example 1), Table 2 (Example 2), and Table 3 (Example 3).

The transformation efficiency was calculated as the number of transformed colonies / plasmid
It was measured as μg of DNA.

Examples 4 to 6 The effects of the transformant of the present invention using a high concentration of the active ingredient were tested in the same manner as in Examples 1 to 3, except that E. coli JM109 was used for competent cells of Escherichia coli. Example 4 using phytic acid, Example 5 using sodium phytate, and Example 6 using calcium phytate.

 The results are shown in Tables 4 to 6.

Since the competent cells used were different from those of Examples 1 to 3, there was a difference in the numerical value even at the same concentration of the active ingredient of 2 mM, and it was not possible to simply compare, but in the range of 1.0 mM to 2.0 mM from the above data, It can be said that the transformation promoter of the present invention exhibits the highest effect.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Miharu Tsutsui 6-26-10 Higashi-Rinkan, Sagamihara City, Kanagawa Prefecture

Claims (2)

(57) [Claims] 1. A bacterial transformation promoter comprising at least one phytic acid or a salt thereof as an active ingredient. 2. A method for promoting transformation, comprising transforming a bacterium with a vector in the presence of the agent for promoting transformation according to (1).