JPS5856678A - Novel lysozyme-sensitive microorganism - Google Patents

Abstract

PURPOSE:To obtain protoplast of bacterium, without pretreatment with an agent such as antibiotic, etc. during culturing, only by treating lysozyme-sensitive bacteria belonging to Corynebacterium genus or Brevibacterium genus with lysozyme. CONSTITUTION:A lysozyme-insensitive strain belonging to Corynebacterium genus or Brevibacterium genus is subjected to the mutagenic treatment, and a strain which cannot be grown in a medium containing lysozyme at an extremely low concentration (<about 25mug/ml) to allow the complete growth of the parent strain and nevertheless can be grown in a lysozyme-free medium. The objective lysozyme-sensitive bacteria belonging to Corynebacterium genus or Breviacterium genus [e.g. Corynebacterium glutamicum, L-15 (FERM-P No.5946), Corynebacterium herculis L-103 (FERM-P No.5947), brevibacterium devaricatum L-204 (FERM-P No.5948), Brevibacterium lactofermentum L-312 (FERM-P No.5949), etc.] can be obtained by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel lysozyme-sensitive mold organism, a method for the preparation of proteinaceous bacteria using a cough fungus, and a method for transforming the mold organism.

The present invention will be explained in detail below.

The present invention provides a method for preparing protoplasts and a method for transforming microorganisms using the novel Ijiit lysozyme-sensitive organisms and the Krugae organisms.

The novel lysotame-susceptible strain κ of the present invention belongs to the genus Corynebacterium ▲ or the genus Plebibacterium spp.
Microorganisms showing susceptibility to Norizochii ▲k at concentrations less than 77,/l are found. The cell wall of this organism is simply removed by lysozyme treatment without pretreatment with antibiotics or other drugs during culture, and it becomes glotograst under high conditions. Integrate DIAK (DMA) vector in a test tube and insert it into organism #11.
The genetic engineering fishing technique that we will introduce has recently received a lot of attention. Transformed Qin cells obtained by genetic engineering techniques can also be used as a means of producing heterologous DIJ▲, which can be obtained by producing autonomous Il. It is also a rich flea that confers valuable properties to cells by allowing the true species DI▲ to exist in cells. Most of the deaths in this field are carried out in systems that host the large intestine, but there are also industrially useful organisms other than the large intestine, such as amylase, antibiotics, etc. Attempts to establish ``recombinant DMA techniques'' have been unsuccessful in the production of Hosaso, which is the raw material such as Hososo, and yeast, which is the raw Doso of Iszou alcohol.

In genetic engineering techniques, it is necessary to analyze a hybrid plasmid containing a single vector plus plasmid or a hybrid plasmid incorporating a different @DI▲. First you have to Mal the #total. That break is 1 in order to stop the new development of DI▲.
Since it is necessary to perform it with a uniform condition, it is not possible to use IaIs such as ultrasound and French press, and it is usually used for cell &
This is done using a method using 11M-cumulative.

Recombination of Genesis 1) N▲ In the Cao Ei where research is being done, there are 111i # books that will serve one purpose, and they are used.

In many #im such as large #1 hlk medium hay soda, small soda pongee b
Cells can be easily dissolved and removed by lysozyme, but there are also fine particles that can be easily dissolved and removed by lysozyme. For such cells*b age degradability 0@cypress, the cells are exposed to a high concentration of amino acids such as dalicin and antibiotics such as penicillin during culture to make the cells sensitive to lysotame. A method has been adopted to do so. Glutamic acid, lysine, etc., belonging to the genus Corynebacterium and Plebipacterium, are used for industrial production of many tons.
In case of 81 cells, overloaded and single molds are easily decomposed by lysotame, so in these mats, when an antibiotic such as Nicilin is applied during culture, lysotame is applied to I1 cells.
The throat and mouth to be treated #lI#IlIl! Eunpo was used #i. However, this method is not complicated, and the ability of the protoplasts produced by treatment with rhizonarum to return to normal cells (4% viability) is low. There are drawbacks such as.

Recombinant DM in useful microorganisms such as Corynebacterium and Prepibacterium
In order to establish the Aa method, I have been searching for methods for removing these cell walls.

As one of the methods, the present applicant has proposed special 1! We have investigated the cell wall removal method by Lysochye using Lysochye susceptible strains of Corynepacterium and Glevibacterium species disclosed in Publication J7. In both cases, the cells were not removed by lysozyme without pretreatment with penicillin or the like as a lysotame-non-sgt strain. As a result of further investigation, we found that Ikkyu, who is hypersensitive to lysozyme and has a level of susceptibility to lysozyme, as described in the Cough Publications@, has cell walls that are lysozyme-resistant without being pretreated with penicillin, etc. 1# I found out that it will be connected. Furthermore, the present inventors found that when using a zebra such as ζO, it is possible to extract putosmids from inside the cells, and because the protoplasts produced by Linnaam Asahi are superior in nasal formation, DlmA [using protoplasts] [ The present invention was completed by discovering the ability to efficiently transform the shape of the object.

The lysozyme-sensitive strain of the present invention is of the genus Corynepacterium t.
9 is Previbacterium Norizozyme non-susceptible strain (e.g. wild strain) Mae is Lynchii ▲ Use the low-susceptibility strain as the parent strain,
It is obtained through the zeta f change Ji4 casting process. Conventional methods such as extracorporeal IIIWK injection, radiation irradiation, and spinant treatment are used for the healing of mutant m-guides. To select one strain that is sensitive to lysotame, such as the imitative organism of the present invention (hereinafter, the lysozyme-sensitive taste of this strain can be referred to as a lysozyme-sensitive strain) from the following strains that have been sieved through the I4 strain: The Arashi strain cannot grow on a medium containing lysozyme (II-11μI/Ichika-), which is a falsely low-chain monomer that is added to 7c. good.

As a specific example of the mimic organism of the present invention, Coriopacterium curtamicum-21-104 strain (imitator @ deposited mass★
issue! free! ) was fermented using the parent strain L-/! r strain (m
Engineering contract lJ! Rinsango! ? -47, L-/03 strain derived from Corynebacterium haakieris ▲'
, Previbacterium deiparicum ▲TCO/da0
L-10-strain (A Koken Kikokusei No. jf#I), which was introduced with 20 as one stock, and Previbacterium lactobacterium 7 amentum▲TEOCIJtjJt-9 L-J, which was led as a yearly leave. / Kokyuu (Kyukoi#Sokurekuyusiokyogojtade)
can be given. These rizotame hypersusceptible strains are threatened with mold biotechnology and death, and the fungal research laboratory Iit mentioned above.
It has a ukejo-arrigo number attached to it. In addition, America's No. 1 American
Type Culture - Collection Depression ▲Too▲J/
rJJ, 3111, J/144, J/It7 and J
It has been deposited as III.

In addition, 2 which was named Corynebacterium glutamicum
The 21-/04 strain was isolated from soil in 91 cases, and its bacterial characteristics are as follows. The study of Ichiji @ga is “Manu”
alofMiC+'QbiQ1QgiCalMetho
ca'bytheBocityof7uoerican
Bacte-riOIOgiiltQOmitt@@o
r1$aCt4riOl%i(:IslT@Cbll
11 by the method described in qub (l de j7).

■・Excessive cell shape a7~/. OX/. It has an elliptical or rod-like shape of 5 to 10 microns, but depending on the culture conditions, multiple cells are connected in a chain-like or square-shaped wrinkle pattern, exhibiting nine different pleomorphisms. It does not produce spores due to its guhumu and well-linked properties.

Growth characteristics on agar-rich medium: On an agar medium, the O-rate cluster is circular, with 61 peaks, and pale yellow in color. Growth on the slant is also Ik* color and nonia1. When cultured on a large medium, it grows in the upper part of the cellar, and grows slightly in the deeper part. In S4-culture, it grows slowly and sediments in a constant pattern.

Quantity / stop coffee /) m & 2 411 [t'! Jj~j7°C but 44
．． Grows faintly at 2℃, J) I) J {Two main pHs are 7~t, but it is still viable even in piti~ta, 3) #ml feverish, 4I) aerobic, j) Does not liquefy gelatin , t) Does not degrade and metabolize casein, 7) Non-indole production, l) Cata 2-7 positive, ? ) - Primary non-tetratonic, io) f Produces acid from glucose, fructose, 1-nonose, maltose, but produces acid from xylose, gasictose,
Does not produce Fil1 from lactose and glycerol, ii) Biotin hydrophilicity, /λ) Produces large amounts of glutamic acid in biotin/quantitative media, l3) Produces lactic acid and α-ketoglutaric acid rapidly in biotene-containing media. .

The above results are based on J. Gen. ▲ppLIJicrobi
OL, 7J, 27F-10-/(/147), which is compared with the sea bream Sosho (◆), and corresponds extremely well to Corynebacterium glutamicum. Therefore, here j-1
Strain 04 is a strain identified as Corynebactericium glutamicus ▲.

A specific example of the method for obtaining the lysozyme Chao-sensitive mutant strain of the present invention from lysozyme-insensitive Coriibacterium and Previbacterium strains will be described below. The Arashi strain is inoculated onto a nutrient medium and cultured with shaking at JO to Jj'C. In the middle of the ridge propagation period, cultivation was stopped and the stage was harvested.
Wash with normal saline solution (pMjJ
~to), analogy is M/Ko Trisumate Shun III
Select the mackerel so that there are approximately /X/0' to /X/0' cells/- in the liquid (vd50). This slurry has an elephant Mill λ0
Nitroguanidine was added to give a concentration of 0 to 200μ11/QC, and the mixture was left standing at 10-Jj'C for Jo minutes to 1 hour.

Collect by centrifugation, wash the sodium chloride with the same slow negative solution, suspend in saline solution, dilute 1 ton with saline solution, and transfer a certain amount to a nutrient agar medium. NB agar medium (MB/.
1) Spread on the medium containing agar). The colonies are incubated at 1-3°C for 1 to 3 days, and the resulting colonies are plated on a large nutrient medium, a nutrient agar medium containing lysochy▲ of 2-2 times a day, and a K replica.

After incubating the replica plate for 1 to 3 days at ~Jj°C, it was grown on a trophoblast medium.
gain

All of the above-mentioned IL strains are thus treated as sauteme super IIIk susceptible strains.

For lysotame-susceptible and lysotame-sensitive cells, cells in the logarithmic expansion phase were placed on a NB agar medium containing lysotame and incubated at 30°C. After culturing, it can be determined by observing the canned growth condition of Cao. As a measure), I showed the group nine Ayu-mai, which was compared to each stock.

Takaaki Moto's lysotheme ▲ Super Jllll Hotarusha Hidenori strain cannot be grown on bb agar medium containing lysotheme coj~yojμ17/M like Tsumugi, but it can be obtained by selecting I4, but → An introduction! Noodles that cannot be grown green on BIB bulb medium containing 00 to 00 μI/d of Corynebacterium ▲ and Plevibaquarium mrsm Norizochyi ▲ Susceptible reward JIj strain No. Gorinzyme-00 to Yume 00 μI/d What you get by selecting stocks 9.
There is a totality of nine companies that are realistically drawn. To the apple,
Those money wI Hiro Ikkyu's skinny Rizodeme'##
IIIk's holiday is due to the above-mentioned Rizote five malicious degrees, and it is said that tO0μi/l is
Regardless of whether it is low j- or 4, linozyme sensitivity of Motoya's imitation use @ / 4 to 3 μi / w ne appetizer j--l2λ
It is clearly different from the lysozyme-sensitive Ikkyu and the linonarum-sensitive mcoj~tiroooμl/1 described in the Publication No. 7. In addition, based on the fact that #i cells are easily and slowly removed by Besacilin, the following +1 is given.
! However, because UK does not have these characteristics, the hypersensitive strain HIRIZONA~
It is clear that the strain is different from the mu-susceptible strain.

The usefulness of the mimic organism of the present invention, which is removed by 8J& with lysozyme, is based on: i) excellent regeneration of protoplasts from which cells have been removed;
The last point is to make efficient transformation by DI▲ possible by means of the process, and c) to extract a portion of DI▲ such as psymids from cells. This will be explained in detail below.

l) Transformation of protoplasts from the fungal organism of the present invention with Dll▲ using a skeleton protoplastic. carried out by

The medium can be anything that can be grown on a pedestal, for example, 14Bt nutrient stone is used. Add -1k to the medium and add 37
The Mmk soil is collected by shaking culture at ℃ and in the middle of the row phase or the healthy phase 1. Next, the cells were sifted in a solution containing lysotame with a tactile characteristic of a~10~/d in an excessively strained medium. - For example, f38M medium [glucose op
, (NH,),BO,/Ojl% Marginal volume 31% yeast extract/ml,lUi,}O,/j. uR, -41.0441
．． l●80j・7HJO/0147, Mn80+-see~
4M200. JIl@, Zn80, -71, OQter)
, cuso, -tn. oo. pq, Na, B, O, -i
om, oo. o deso, (N}I,),MO7(J,,-
4C1400.0#Q, Bioten 30 μl 1 thiamine hydrochloride/lIf in pure water/I, pHZJK adjusted, 9 medium] σhiro M axis, 0.07 MMI in 1-fold diluted solution
OI.・! Including HJO, p! 17.0~t. jK adjusted medium PFM'ii can be used. JO~3
By ζ and K, which react at 7°C, normal cells are transformed into two-layered cells, and the extent of this is confirmed by inspection. The amount of time it takes for most cells to become C-tonorasted varies from Ikkyu to Ikkyu, but under the above conditions O. j～1
This is the time barrier. After the action of lysozyme, normal cells that survive without bursting to death under the hypotonic conditions in the zeta potency solution can be subjected to lysozyme treatment and can be reduced to less than 9/0-w of the initial normal cells.

Norotoplasts that undergo v4M in the same way as seeds have the ability to regenerate colonies on Takajia agar medium.

^ Take it as a big melting pot, for example, RCGF medium L glucose jI, casein hydrolysate Jil% yeast extract! l%KkoHPO41J. ! I,KHJPQ@/.
jI, MICl2-tB200. Dream/I, Fe80, -
7M, IC/0147, MrlB-D. I14'~IumaOkoAi, znl*s7kx, υO. 2 inches, (NH41
) AMO70J41-Reference H, L + 0.0 Reference ~, Biotin iosls Thiamine Hanawa [Tanko 2, Cono-Citrate Disodium/3! l, polyvinylpyrrolidone (molecular weight/0,0
00) JO11k fine water/l, pm7.2fc copper moxibustion medium] can be used. ktCL} Protograst Niu in PjQ area is treated with Ta body and lysotheme treatment ms
Depending on the degree of salt crossing the threshold yc, the blood will be strong, but the lysozyme treatment B will stop the first incision, and the effect of 20 to 70 will be 4.
There are differences in the number of culture days among the Fi cocoon strains, as evidenced by the emergence of Nyu colonies. What is the size of the h* case? ~7 days.

Transformation of protoplasts produced from imitators with the above-mentioned characteristics by D-drilling can be carried out using polyethylene glycol (PI(}) or polyvinyl alcohol (PV▲).
) and 21 gold 1sll# ions coexist under hypertonic conditions. The transformed strain consists of the protoplast h
Mr. Oka and Mr. Oka inoculated protoplasts on ROGP layer @ Enji agar medium and obtained them as normal cells. It is also possible to make a contribution based on the % similar cedar quality, which gives +1 to the gene that is transferred to the DN▲. This tracing selection can be done at the same time as S stop on high gI & ball large hinoki ground.
Or once #! Tracing value K? 4. After being allowed to live, the collar cells'
It may also be carried out on a low temperature agar medium with a thickness of 1.5 mm.

When I broke down this method and converted the stock using the normal 1 system DM ▲, the cedar quality convertible stock regenerated once again at l0~lθ
He is served with a degree of chain.

A specific example of the transformation of shadow quality will be described in the example below.

D) Plasmid from the transformed strain D) Culture in a medium in which a single strain of J▲ can grow, such as NBJ@, and collect.
After Il, the lysozyme J-t action causes the am to be resolved. From bath-1 % e.g. BiochimBiophya
．． ▲Sigfusmit't for commonly used diarrhea as described in eta, JIJ vol. 17-vol. 43 (/de 7j)
- You can make a ringbun temple in Guiyi.

11 of Zotame to use, the target Ikkyu will be in the evening ＠ Q / - / 0 teacher / 1 once.

Glutamine and lysine are now used in many industrial industrial preparations for Corinone {Cuterium and Brevibacterium. Other imitations such as Ai no San from gE4II+ are useful Kazusada's birth Il! By cloning the gene involved in c or a section thereof, and increasing the biosynthetic system based on the expansion of the genetic information, we will increase the age of Arikawa, and furthermore, clone the genes of animals and plants,
Useful peptides are generated from these genetic information! A7 ability to pray for raw mg using lIl,
& Higashijou is very useful. The linoteme supersensitive organisms of the present invention are the 7-rasid-like DM▲ eruption branch from cells that are progenitors using recombinant DNA technology, and the negative transformation of Hoki no Sugi by DN▲. To effectively improve the
k-cho hase KL, seven days ago koriyo batatelicum II4s? and νby M offering to the genus Brevipacterium D) 1▲The application of the technique is made to become a tanikan.

The present invention contemplates that the microorganism of the present invention is a recombinant DMAtm.
ζ is useful as a safe host in the law.

The establishment of a safe host (II main)-vector system in the recombinant DM▲ method was first advocated by K. Sabaichi. Conditions for a safe host include that it has very low pathogenicity, that it does not parasitize humans, and that it dies continuously when released into natural burial mounds. Daizenso, which was first used in the recombinant DI▲ technique, is originally a human vertebrate, and there is some debate about its safety. As for strain 12 K, it was found that its parasiticity and susceptibility were significantly reduced, and it was fixed as a 'B1 host. This 5112 strain was administered to Jinjun, and on the day of the joint ship, 90 mO# was observed, and the yield was reported to be around 11 (Scientific Xzoe Vol. 8).
111-394 true hlo cattle).

If the recombinant DI▲ technique is to be expanded to produce organisms such as Ami1's enemies, spores, and antibiotics, it is likely that many of these valuable organisms have been isolated from Doi and have low parasitic potential for the human body. However, it is conceivable that some species can withstand the conditions in the human digestive tract and do not proliferate, but are harvested alive.

In order to further increase the safety of the isolated strain, it is conceivable that κ could be artificially given the property of dying under conditions within the gastrointestinal tract. B2 hosts are being given a fragile property that allows them to die even if they are left under a Hiroshima burial mound.

When the lysozyme-sensitive organism K of the present invention was tested for survival in the gastrointestinal tract of animals, it was found that it easily settled and died in the gastrointestinal tract of one animal. That is, aay
Type Zen Max (weight 19 IT person 1) one review 2 GT animals *!
Administer a mouthful of the following mold extracts of approximately a to sxto', and measure the number remaining in the intestine and the carbon dioxide excreted in bird faeces by diluting each 10-element homogenate into an appropriate amount of diluted MBsF plate medium. I decided to do so. As shown in the 21st RK, the lysozyme-sensitive strain #1 of the present invention is significantly killed and eliminated compared to the ``nozoe'' strain κ, and the number of surviving carbon atoms in the noodles is also significantly lower, and the lysozyme-sensitive strain #1 of the present invention is resistant to the drug. When I looked at the nature of the habitat, I found out that Hashira's antibiotics are sensitive to K.
L9.

In other words, approximately 10 cells in the logarithmic growth phase are doubled in the phase of approximately 10 cells in the logarithmic growth phase.
The susceptibility of the bacteria of the present invention was tested by inoculating the K-bottom on a MB cheret medium containing a certain amount of the drug and incubating it at 30°C for 2 days. As shown on page 3,
It was found to be extremely sensitive to Beniciri 7G, rifanbicillin, etc. This suggests that it is easier to sterilize and sterilize than the Sagi strain with various drugs, and the recombinant DNA
It also shows that it has the effect of alleviating the increase in drug resistance when using opiate-resistant grass ξd as a vector for A.

As mentioned above, the fact that the orizotame-sensitive organisms of the present invention are fatal in the gastrointestinal tract of animals, and are also sensitive to the aphrodisiac agent, is due to the fact that the organism is a recombinant DNA. This indicates that it has favorable properties as a host fungal organism.

夾jllifFlj/. Lysozyme hypersensitivity ^ Kyushu no Shizo Corynebacterium gluta, Mikumu JJj-/04
, Corynebacterium herculis ▲TOO/JI4
t, Previva Ryu▲・Deiparikatsumu▲TOO/4
10 Ov and Brevibacterium 2 K. 7 amentum ▲TOC/J4JJf'kiB medium (9 medium containing powdered pui d λoti, one mother extract Jl in pure water/IK, adjusted to 1 pH), Culture with shaking at 30°C. At half the logarithmic growth phase, the culture was stopped *Il, and after cleaning with physiological saline, M/Jθ tris malate & working a (pa & 0)
Imfl/j to approximately JX/O'HJ cell/1.

In this Teng Tao kick, 1 # eaves result thorn ≠ 00μi/lm v
Add C nitrosogubunidine and release λ31, TJO fraction. By Hatoshin Bunichi Iku-L. lk! In one step, the enemy will be purified, and the enemy will be immersed in saline water, diluted with salt water for a short time, and the resonator of τin K1 and lλIμj//l ▲Word 44 Km hooks up with the big ball.

The colony was enlarged for λ days at JO°C, and the resulting colony was transferred to a 14llt large cypress site containing a 11μII/'Ichinori/team of the Song Dynasty. Lenorikahira &
1rJO℃ for one day, MB store, student,
Grains that do not grow on agar medium containing Linonarum are obtained as five different strains susceptible to Linonarum.

Central example, Hon'ei 91 imitation Shokiri f) Cell transfer disintegration by lysozyme and Nyu corynepacteric ▲ and glutamic ▲ cocoj-/04,5 of the living relative protoplasts
Linebacterium herculis ▲'XOO/Jt41
, Previbacterium deivalika ▲▲TOO/$0
λO, Previbacterium●Lact7Arme/Tam▲T
QO/J4jj and the Lysote hypersusceptible mutants prepared from them in Example 1 are cultured in NB medium in a shaking oven at 0°C. Determine the @ original degree (OD) at ≦tOnm using a Masaru Tanaka colorimeter, and
At the time of D modification, dilute it appropriately with negative saline solution and place a certain amount in MB bulb large medium to determine the initial normal cell count. Collect cells from and increase sales by RCGk'Wi
#/wtv lysotame to ttra (pnzi) about io
Sprinkle noodles on the cells/-, then repel them at 30°C.
React slowly and considerately.

70 minutes on JOOX&, 12 minutes on floor, FGL, 11th to PFM Nanchi, 101st centrifugal purification, Hiroyuki Dong's F1 Naji. 7r) The #i solution was treated with llIl1 line. Take one part of this and inoculate it with i1cGF medium and apply a certain amount to kROGP rim large medium, and the other part should be diluted with appropriate K in raw coffee negative mass water and peeled. Transfer tM to a large medium and culture at 30°C.
Respectively iIII tension condition, uk expression condition teo:yo=-engraving gI
The number of SJwA bacteria (cry') was determined. The number of colonies generated under hypotonic conditions is culture! -I was added on day 12 (the number does not increase even if cultured further). ＾The number of 421 apricot colonies produced at the peak site will not increase any further.7
It was decided on the first day. These results are presented in J.J.

In any case, even if lysozyme t is applied, only camphor-shaped cells are produced in imitation, and the generation of glotopst in Eibutsu cannot be detected. In addition, the number of colonies carved in the hypotension region was not significantly different from that before lysozyme treatment. Ten thousand,
The female lysotame-susceptible strain is lysozyme #il! In the hypotonic conditions, the MN fissures of the first instar are converted into protoplasts, and the MN fissures of the four tomatograsts remain intact (considered to be non-crotoplast cells resistant to osmotic shock). Mine, lysozyme treatment m donation play description l0-″～/O-
It was 6. These Noto 1 lasts had a colonil formation in the case of ^^, and the Niu efficiency was 0~70% compared to the lysozyme treatment K one une. 1m-1's
My first heart was -111 Jn, and I was stuck on the ground last night with a constant small brain.

OkellJL This invention value Ikuichi's Nretoplast plasmid pOG4tDmu▲ by Kataga Tenko/) 1 Lasmid pco
Sesame sales book
CG4I- is l) K extracted from the 1@91 somatic body of Corynepakdelium glutamicum JJj-JjO (Mukuro 1kenonzuukeshoyogoko ItaJtago), which is a possessed somatic body of OG41, as follows. Bunichishimaru. Corynebacterium glutamicum λko!
Eleven! Cultivate 0 in NB crucible in j@ItL nine types! 1, inoculated with 88MtlC of San001 semi-synthetic medium and shaken at 10℃J@
nourish mjj! Measure the absorbance (OD) at 44elnmlc with an ice water light oven colorimetric needle. t,. ODO. Penicillin G was added to the culture at a concentration of 0.7%/d at JK Nakakushima. Further increase *1 sweetfish at 30℃ and OD.
Keep it raw until it reaches about σp.

From 坩**, I received a lesson from T, and T breath b Aya dog liquid LO. (77M
Tris (hydroxymethyl) aminomemene (Tris),
0.001M Ethelenediamine/Sifanken disodium (]
After washing with liDT▲)0.02Atkiacz:pkiiO), lysozyme beast (λjfb'/EieasO.l
MM&Ol, 0. t) jM Tris, 0. llqi/-lysotame pplito)/OmK suspension at J7°C for 4 hours.
Allow to react for I-time. The reaction solution is jMNaOjJlaj
,0.1MEDT▲(Xlli#.j)Q4sj,V-
f# consisting of 9 μm of rawalyl sulfate W sodium and 0.7 MIA & CI! m4A dajljf: Add the noodles, gently stir, and then put in ice water for 7! Give it time. Transfer the 11# whole cucumber to a centrifuge, centrifuge for 1 minute at A*II00xJI, and collect the supernatant fluid. To this, weight daily rate IO%
In addition to a considerable amount of PECk & 000t, the turtle was mixed with water and placed in ice water after thawing. After 1 hour, centrifuge at 100x1 for 10 minutes to collect one pellet. T58 slow coffin.
Add the pellets quietly to 81F! ! Interpretation-(ρ-&
:)/． m/AI for j & fm enazicum bromide
L/s This is Ic cesium [Additionally, it quietly disintegrates and meets [/, j10]. This @y7IOJOυOX
JI, tr'c -1 o'clock threshold concentricity 911k-f. this@
Due to centrifugal centrifugation, the joint was closed and the 1c fractured DMA
The v of the middle and lower part of the pigeon core tube is to emit a black 1r outside the bean paste! They are emerging as a band with a high degree of f.

This band is excavated from the syringe using a syringe, and pOG411 is isolated. Then add a static volume of Inglobil alcohol solution [
Capacity per day 0 Is Inglovir Alcohol% IO'l
Ethidium bromide is extracted and removed by treatment once with a Tt8 slow g Ik solution (containing the amount of cesium chloride dissolved in the K saturated bath), and then dialyzed against a Tl8 slow solution. /j μg of plasmid poaa were obtained.

The 7-sumid pOG≠ is a molecule of approximately 1 tamegadalton, a gene that is resistant to str 7' tomycin and 1 or subectenomycin. BeaRl, AHindLP in the Q-Q molecule
ut. It is a Shinkai group 7 sumid which has the cutting Wr site by l and himu aj5 respectively nda, a, Pm6 and 41.

J) Plasmid pco*osugiji [&. Pleasant trait transfer is
For example, I used the 1-nozozyme-treated m cells that I had prepared in the first place. The protograst wI solution or one was placed in a small test tube and centrifuged at 200xy for I minutes, and the Tb. M.O.
k@叡(/01f,Mgnesium chloride.JOuJMcalcium chloride,jOLM}IJsu(hydroxymethyl)
Aminomethane (Tris), 4AOOm. M sucrose, pH
7. j) 7ajK Suspension and centrifugation. O. to the precipitated protoplasts. Added Volume l vT8MO Yukakuyu. Shake gently and stir the noodles. This is J times the above 7kWII
pOG$D# mixed with 1 degree T8haC-Kuei and l pair/K
Add A 猷σim (o.λμ#l) MA word) and mix, and after a long time, 10% P8 (hJh
The semi-OL version including the Oυ0 meeting is added to the Yurufikan Kaede Soj゛ru. 3 minutes, ROGm'4 ground λ-t-mm, -cj0
0Xl-(j minute FI4) Pigeon/V minute trick on top tx-y
Pinch the fins and a'. Fallen Grotov “Last y7.tQ
After being confused by HAAP expansion, Kaede increase tC in HAAP cultivation.
■'llt (L, -5t amount spectinomycin reference QO
ROG containing μV11t is grown in a cell culture medium K.

In rounding, know the number of colony cedar formations and planes at the time of lBj, R
A fixed amount of the highly diluted solution is also run on the CGF section large medium. The number of colonies developed after culturing at JO℃ for t days is #j
● Take a colony generated on ROGF medium containing spectinomycin, smear it on MB Hachi medium containing streptomycin (/JLJIAI/d), and grow it at 30°C to obtain a streptomycin-resistant form of I+2. Examined.

I came across a 4t pill that showed cross-resistance, and used the brute force method to make 7 russian trimmed pills. It was shown with a box stopper next to the outside of Nire et al.

A spectinomycin-resistant strain grown in a lysozyme-insensitive strain treated with lysozyme did not show cross-resistance to streftomycin;
Although Iwl-like kappa glasmid tjP was used, the presence of POG was not detected. On the other hand, spectenomycin-resistant strains that emerged after lysozyme treatment of lysozyme-hypersensitive bacterial strains had strepto-isin cross-resistance, and busismid isolated from these strains was digested with IIIi@enzyme HincL. After 1 callose 1 IL electrophoresis, 6 L) k4A# fragments were identical to pCa-.

A single example of a plasmid from a microorganism carrying grasmid was shown to my younger brother I. Corynebacterium glutamicum L/j/poop. Corynebacterium herculis L/01/pOG41
L. Plevipacterium tabericatum L104'/
poe≠ and Plevipacterium lacto 7 amentum Lj/co/PCG cultured in MB medium.
- in 111B medium and incubated at JO ℃ for 1 hour with shaking.

The bacterial cells were collected from the culture medium gI solution, thawed with T≦8 slow solution, and then treated with lysochyli
Add 1 drop of Iv to jM Tris, QIN/-Linchy A:pMt)io, and allow reaction to occur at 37°C. Reaction solution K Hatmaaz Yuhiro, cj. jt gate ID1゛▲(p111
1) 0.4m, u%9 uryl sodium phosphate and Q7Ml
Firstly, add a layer of 11 LOj of ginseng, mix gently and then place in ice water for κ/J hours. Transfer the entire amount of the liquid to a centrifuge tube, centrifuge at 4°C for 40 minutes at 00xlO, and remove the supernatant. To this, the weight percentage l0 one phase Todoroki 0-
Add PIGtoot, gently dissolve, and then place in ice water. After one hour, collect the pellet by centrifuging at 100XI"C for 0 minutes. Add 1 amwii solution to redissolve the pellet, and then add Q/d ethidium butemide. To this. Cesium chloride was added and dissolved to adjust the density to 7.7 tO.This solution was subjected to a density gradient gradient under the same conditions as those used in Ishifi JK.
Collect fractions of 1q. The fractionated solution was extracted and removed using Inglovir Alcohol ▲ in the same machine as in Example J to remove promide, and then dialyzed against Tl811 titanium solution.

In this way, from Isure Nori Zozyme Tsutomu sensitive stocks that hold POG$ / J-10 voice I pOGI! DI▲ can be obtained.

-403- −404=

Claims (4)

[Claims] (1) The genus Corynebacterium or the genus Plevipacterium κ, Ko! Novel lysochye-susceptible organisms showing susceptibility to lysochye at concentrations less than μ11/1. (2) The axillary organism is Corynebacterium glutamicum,
Paragraph S of the claim that extends to ζ, which is a fungal organism different from Corynebacterium no. 0@Seiichi. (3) The mold organism is Corynebacterium glutamicum L.
-/! , Corynebacterium norchieris L-/O
J. Plevipacderium daivarikatsu▲L-20# and Brevi I {cuteriu▲・Lactofamentum L-
The mold organism according to claim 1, wherein ζ, which is a microorganism selected from J/J, is an acoustic mold. (4) Corynebacterium ▲ belongs to the genus Previbacterium, and exhibits lysozyme κ sensitivity of less than JI μ77/I. It is believed that KIJ zozyme can be used to generate protoplasts of scissor mold organisms. Preparation method for microorganisms of the genus Corynebacterium ▲ and Plevipacterium, which are used as fungi. (5) Corynebacterium ▲ Iris 19 is Plevibacterium ▲ Genus K1l4
The method is characterized in that lysozyme is applied to lysozyme-sensitive molds exhibiting an altered lysozyme K sensitivity of less than Jjsl/1, and deoxyribonucleic acid (IJ) is injected into the resulting protoplasts, thereby regenerating cell protograsts. Corynebacterium genus Tangjiu is a method for transforming Corynebacterium spp.