JPH02207791A - Transformation of bacterium - Google Patents
Transformation of bacteriumInfo
- Publication number
- JPH02207791A JPH02207791A JP1028088A JP2808889A JPH02207791A JP H02207791 A JPH02207791 A JP H02207791A JP 1028088 A JP1028088 A JP 1028088A JP 2808889 A JP2808889 A JP 2808889A JP H02207791 A JPH02207791 A JP H02207791A
- Authority
- JP
- Japan
- Prior art keywords
- dna
- cells
- cell wall
- bacterium
- pulse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000894006 Bacteria Species 0.000 title abstract description 10
- 230000009466 transformation Effects 0.000 title description 4
- 238000000034 method Methods 0.000 claims abstract description 10
- 210000004027 cell Anatomy 0.000 claims abstract description 9
- 241000186226 Corynebacterium glutamicum Species 0.000 claims abstract description 8
- 108020004414 DNA Proteins 0.000 claims abstract 12
- 102000053602 DNA Human genes 0.000 claims abstract 6
- 230000001131 transforming effect Effects 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 4
- 230000002068 genetic effect Effects 0.000 claims description 2
- 241000186146 Brevibacterium Species 0.000 claims 1
- 210000004748 cultured cell Anatomy 0.000 claims 1
- 210000002421 cell wall Anatomy 0.000 abstract description 6
- 238000007796 conventional method Methods 0.000 abstract description 5
- 210000000170 cell membrane Anatomy 0.000 abstract description 2
- 230000035699 permeability Effects 0.000 abstract description 2
- 238000010361 transduction Methods 0.000 abstract 1
- 230000026683 transduction Effects 0.000 abstract 1
- 210000001938 protoplast Anatomy 0.000 description 7
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000011426 transformation method Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000005684 electric field Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 229910052792 caesium Inorganic materials 0.000 description 1
- TVFDJXOCXUVLDH-UHFFFAOYSA-N caesium atom Chemical compound [Cs] TVFDJXOCXUVLDH-UHFFFAOYSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002298 density-gradient ultracentrifugation Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野〉
本発明は、アミノ酸などの有用物質の発酵生産に用いら
れているブレビバクテリウム・ラクトファーメンタムに
プラスミドのようなりＮＡを取り込ませることにより、
その遺伝形質を変換する形質転換法に関するものである
。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention is directed to the production of plasmid-like NA by incorporating NA into Brevibacterium lactofermentum, which is used for the fermentation production of useful substances such as amino acids.
This invention relates to a transformation method for transforming the genetic traits.
〈従来の技術〉
ブレビバクテリウム・ラクトファーメンタムの形質転換
法として、従来はＤＮＡを取り込ませる細胞の細胞壁を
細胞壁融解酵素リゾチームにより除去してプロトプラス
ト化し、プロトプラストとＤＮＡの共存下にポリエチレ
ングリコールを加えることにより、プロトプラストにＤ
ＮＡを取り込ませ、その後プロトプラストを適当な薬剤
を含む再生培地上で再生させる事により形質転換体を取
得する、プロトプラスト法が用いられていた。<Conventional technology> The conventional method for transforming Brevibacterium lactofermentum is to remove the cell wall of the cell into which DNA is to be taken up using a cell wall-melting enzyme, lysozyme, to form a protoplast, and then add polyethylene glycol to the coexistence of the protoplast and DNA. By this, D
A protoplast method has been used in which transformants are obtained by incorporating NA and then regenerating the protoplasts on a regeneration medium containing an appropriate drug.
（昭６１−１４９０８２、昭５７−１８６４９２等があ
る）〈発明が解決しようとしている問題点〉従来用いら
れていたプロトプラスト法には、菌株ごとのプロトプラ
スト化及び再生の最適条件が変動すること、プロトプラ
スト化及び再生を含めると全操作に１週間以上を要する
こと、及び操作が煩雑であることなどの問題点がある。(149082/1982, 186492/1982, etc.) <Problems to be solved by the invention> The protoplast method used in the past has the following problems: There are problems in that the entire operation takes more than one week, including conversion and reproduction, and the operation is complicated.
本発明はこれら問題点を解決し、更にプロトプラスト法
よりはるかに高い形質転換効率で形質転換が行える形質
転換法である。The present invention is a transformation method that solves these problems and further enables transformation with much higher transformation efficiency than the protoplast method.
〈発明が解決しようとしている手段）
本発明者らは、上記問題点を解決する手段として、プロ
トプラスト法に代わる形質転換法として電気パルスを用
いたブレビバクテリウム・ラクトファーメンタムの形質
転換法を開発した。<Means to be Solved by the Invention) As a means to solve the above problems, the present inventors have developed a method for transforming Brevibacterium lactofermentum using electric pulses as a transformation method to replace the protoplast method. did.
電気パルスを用いた形質転換法の原理は、受容菌とＤＮ
Ａの混在下で高電圧パルスをかけることにより、受容菌
の細胞膜及び細胞壁の透過性を一時的に高め、外来ＤＮ
Ａ分子を導入するものである。この方法によれば、細胞
壁除去などの操作無しに導入が可能で、従来法に比べ、
極めて簡便である。また、全操作が約２〜５日と極めて
迅速である。更に、導入効率に関して、１ｏｐｇ〜ｌｎ
ｇのＤＮＡを用いて、最高でμｇＤＮＡ当り約１０８個
の形質転換体が得られ、従来法の約１０００倍の効率で
の導入が可能である。また、同一の条件で多くの変異株
の形質転換が可能であり、高い一般性も持っている。The principle of the transformation method using electric pulses is that the recipient bacteria and DNA
By applying a high voltage pulse in the presence of A, the permeability of the cell membrane and cell wall of the recipient bacteria is temporarily increased, and foreign DNA is removed.
This is to introduce molecule A. According to this method, introduction is possible without operations such as cell wall removal, and compared to conventional methods,
It is extremely simple. Also, the entire operation is extremely quick, approximately 2-5 days. Furthermore, regarding the introduction efficiency, 1opg~ln
Using this method, a maximum of about 10 8 transformants can be obtained per μg DNA, allowing introduction with an efficiency approximately 1000 times higher than that of conventional methods. Furthermore, it is possible to transform many mutant strains under the same conditions, and has high generality.
〈実施例） 以下、実施例に基づき、本発明の詳細な説明する。<Example) Hereinafter, the present invention will be described in detail based on Examples.
実施例 ブレビバクテリウム・ラクトファーメンタムＡ
Ｊ１５１１株のｐＡＪ４３による形質転換（１）プラス
ミドＤＮＡの調製
導入するプラスミドＤＮＡとして、ｐＡＪ６５５ （昭
５８−１９２９００参照）由来のＰＡＪ４３を用いた。Example Brevibacterium lactofermentum A
Transformation of strain J1511 with pAJ43 (1) Preparation of plasmid DNA PAJ43 derived from pAJ655 (see 1982-192900) was used as the plasmid DNA to be introduced.
このプラスミドは宿主にクロラムフェニコール耐性を付
与する。プラスミドＤＮＡは常法に従って調製した後、
セシウムクロライド−エチジウムブロマイド密度勾配超
遠心にて精製した。This plasmid confers chloramphenicol resistance to the host. After preparing plasmid DNA according to a conventional method,
It was purified by cesium chloride-ethidium bromide density gradient ultracentrifugation.
（２） ＤＮＡ受容菌の調製
ＤＮＡ受容菌にはブレビバクテリウム・ラクトファーメ
ンタムＡＪ１５１１株を用いた。受容菌の培養に用いる
培地は微生物が増殖できる培地であればよく、例えば栄
養培地Ｍ−ＣＭ２Ｇ（グルコース５ｇ、ポリペプトン１
０ｇ、酵母エキス１０ ｇ、 ＮａＣ４２５ｇ、ＤＬ−
メチオニン０．２ ｇを純水１１に含み、ｐＨ７，２に
調製した培地）などが用いられる。この培地に微生物を
接種し、３１．５°Ｃにて振盪培養した。比色計によっ
て６６０ｒ＋ｍにおける吸光度（ＯＤ）を測定し、対数
増殖中期（ＯＤ＝０．５〜０．８）に細胞を集菌し、蒸
留水にて３回洗浄したものを使用した。(2) Preparation of DNA recipient bacterium Brevibacterium lactofermentum strain AJ1511 was used as the DNA recipient bacterium. The medium used for culturing the recipient bacteria may be any medium that allows microorganisms to grow, such as nutrient medium M-CM2G (glucose 5g, polypeptone 1
0g, yeast extract 10g, NaC425g, DL-
A medium containing 0.2 g of methionine in pure water 11 and adjusted to pH 7.2 is used. Microorganisms were inoculated into this medium and cultured with shaking at 31.5°C. The absorbance (OD) at 660 r+m was measured using a colorimeter, and the cells were harvested at the mid-logarithmic growth phase (OD=0.5 to 0.8), washed three times with distilled water, and used.
（３）電気パルスの印加
電気パルス発生装置として島津製細胞融合装置５Ｓ）Ｉ
−１型、及び付属のハイパワーユニッ） ｌ（Ｐ［Ｉ−
１型を用いた。遺伝子導入チャンバーとしては、付属の
ＦＴＣ−１１型及びＰＴＣ−１２型を用いた。これらに
より、０〜５６ｋｖ／ｃ１１の電場強度、０〜５００μ
５８Ｃのパルス持続時間の直流、矩形の電気パルスを発
生することができる。(3) Application of electric pulses Shimadzu cell fusion device 5S)I as an electric pulse generator
-1 type and attached high power unit) l(P[I-
Type 1 was used. The attached FTC-11 type and PTC-12 type were used as gene introduction chambers. With these, electric field strength of 0 to 56kv/c11, 0 to 500μ
Direct current, rectangular electrical pulses with a pulse duration of 58C can be generated.
調製した受容菌体と導入するＤＮＡ溶液を混合後、チャ
ンバーに入れた。菌濃度はｌ×１０９〜５ Ｘ １０
ｌ０ｃｆｕ／ ｍｌ、 ＤＮＡ濃度はｌｎｇ／ｍｆ〜１
μｇ／ｍｌとなるよう調製した。この溶液に、電場強度
６〜２０ｋＶ／ｃｍ、パルス持続時間１００〜５００μ
ｓｅｃの電気パルスを印加した。パルス印加は４°Ｃに
て行った。パルス印加後、栄養培地に懸濁し、３１．５
°Ｃにて０−１６時間培養した後形質転換体の選択を行
った。After mixing the prepared recipient bacterial cells and the DNA solution to be introduced, they were placed in a chamber. Bacterial concentration is l x 109 ~ 5 x 10
l0cfu/ml, DNA concentration is lng/mf~1
The concentration was adjusted to be μg/ml. This solution was applied with an electric field strength of 6 to 20 kV/cm and a pulse duration of 100 to 500μ.
An electrical pulse of sec was applied. Pulse application was performed at 4°C. After pulse application, suspend in nutrient medium, 31.5
After culturing at °C for 0-16 hours, transformants were selected.
（４）形質転換体の選択
形質転換体は供与ＤＮＡに由来する遺伝子が、菌に付与
する形質について選択することによって取得できた。形
質転換体の選択は適当な薬剤を含む栄養培地、または最
小培地のプレートにて行った。ＡＪ１５１１株をｐＡＪ
４３で形質転換する場合、パルス印加後、栄養培地にて
培養した菌体を５〜１０μｇ／ｍβのクロラムフェニコ
ールを含むＭ−ＣＭ２Ｇ培地プレート（寒天１．５％を
含む）に塗布し、３１．５°Ｃにて１〜２日培養して形
質転換体を取得した。クロラムフェニコールに自然突然
変異で耐性となる株の出現頻度は１０−７以下であるの
で形質転換体の取得には問題とならなかった。(4) Selection of transformants Transformants could be obtained by selecting for traits imparted to the bacteria by genes derived from donor DNA. Selection of transformants was performed on plates of nutrient medium or minimal medium containing appropriate drugs. AJ1511 strain was pAJ
When transforming with 43, after pulse application, the bacterial cells cultured in a nutrient medium are applied to an M-CM2G medium plate (containing 1.5% agar) containing 5 to 10 μg/mβ of chloramphenicol, Transformants were obtained by culturing at 31.5°C for 1 to 2 days. Since the frequency of occurrence of strains resistant to chloramphenicol due to spontaneous mutation was less than 10-7, there was no problem in obtaining transformants.
上記の方法でμｇＤＮＡ当り１０７〜１０８個の頻度で
形質転換体を取得することができた。By the above method, transformants could be obtained at a frequency of 107 to 108 per μg DNA.
Claims (1)
生物にデオキシリボ核酸（ＤＮＡ）を取り込ませるに当
たり、培養細胞とＤＮＡとを溶液中に共存させた状態で
直流の矩形波電気パルスを加える。これにより、細胞に
ＤＮＡを取り込ませ、供与ＤＮＡ由来の遺伝形質を獲得
した株を取得する事を特徴としたブレビバクテリウム・
ラクトファーメンタムに属する微生物の形質転換法。In order to incorporate deoxyribonucleic acid (DNA) into a microorganism belonging to Brevibacterium lactofermentum, a direct current rectangular wave electric pulse is applied while the cultured cells and DNA coexist in a solution. As a result, Brevibacterium is characterized by incorporating DNA into cells and obtaining a strain that has acquired genetic traits derived from the donor DNA.
A method for transforming microorganisms belonging to Lactofermentum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1028088A JPH02207791A (en) | 1989-02-07 | 1989-02-07 | Transformation of bacterium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1028088A JPH02207791A (en) | 1989-02-07 | 1989-02-07 | Transformation of bacterium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02207791A true JPH02207791A (en) | 1990-08-17 |
Family
ID=12239029
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1028088A Pending JPH02207791A (en) | 1989-02-07 | 1989-02-07 | Transformation of bacterium |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02207791A (en) |
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-
1989
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