WO2000060053A1

WO2000060053A1 - Method for producing microbe recombinants (mutualisation or tets's method)

Info

Publication number: WO2000060053A1
Application number: PCT/RU1999/000114
Authority: WO
Inventors: Viktor Veniaminovich Tets
Original assignee: Viktor Veniaminovich Tets
Priority date: 1999-04-02
Filing date: 1999-04-02
Publication date: 2000-10-12
Also published as: AU1086500A

Abstract

The present invention pertains to the field of biotechnology, more precisely to that of microbiology, and essentially relates to a method that comprises the following steps: selecting participants for a recombination which are capable of producing mixed microbial communities on a compact nutritive medium; selecting a nutritive medium that can be simultaneously absorbed by all the recombination participants; and preparing a mixture of microbial strains of the recombination participants in a concentration of at least (1-2)x104 KOE per ml for each one of them. The method further involves adding from 1 to 5 νl of a bacteria mixture on the compact nutritive medium, transferring after incubation the communities thus formed from the agar to an isotonic sodium chloride solution and proceeding to the selection. This method is used for producing recombinant micro-organisms which have an improved viability and a higher yield.

Description

The way of combining the mixtures of mixtures (mutualization - means of the end)

Area of technology

The invention is subject to biosafety. more precisely, it is a multi-methodology and provides a new method of obtaining recombinant bacteria with new properties. Enhanced Life and Productivity. The device is intended for use in scientific research and in industry.

Level of technology.

The creation of bacterial strains with new properties. Improving the business and productivity of dryers are important issues of modern biotechnology. The need for new, highly effective strains needed to solve the problem of medicine, agricultural and industrial use, is a matter of casualty. Along with this, the arsenal of methods used to solve this problem is very limited, which is, in part, interfered with Well known are the basic methods of transmitting genetic information between bacteria - transmission. traction and conjugation. Indicated. that these types of genetic exchange between activities are in place under certain conditions of living [1]. The company is encouraged by the drying of large-sized groceries and only a small number of household-consuming ones [2]. Injunctive in children and children and children. The gene pool of the gene and the conjugation are divided plasmids. there is ττ- οπеροн. This is the only place for plasmid. Анс анс анс анс анс - - delivery ΡСΤЯШ99 / 00114

genetic information by moderate or virulent bacteria occurs in large-scale and large-scale microorganisms [3]. The child’s donor either carries a phage or becomes the site of the reproduction of a virulent phage. Β Any of these options will result in destruction

5 cells for donor.

. Generation of genetic information between bacteria by all known methods is comparatively harmful. The return of recombinant shams varies from 0.001 to 10%. [2-4]. Genes are shared between one-type bacteria. It is known that there is 0 interspecific gene transfer [δ-8]; however, it is only 1,000–100,000 times less than between the representatives of a single species. Well, it is considered. that the transmission of plasmids between delays of different consumptions is exclusive to the consumer (10 for one battery in the case for long periods) and lasts only for a period of 7 days There is a possibility of 5 gene transfers between dormant and obstructive bacteria in case of transduction, transfusion and conjugation in the event of disruption.

We have received a wide range of methods to increase the potential for gene transfer between bacteria. In fact, 0 has been developed and widely used for the treatment of patients with obstructive diseases (use) in the presence of various types of diseases. ADDING VOLUME CATALYSTS, CONSUMERS AND SULF-HYDROGEN AGENTS [10]. In addition to this, all drying methods have a comparative

"> L nizκuyu eφφeκτivnοsτ and κρayne malο eφφeκτivny πρi πeρedache genοv between neροdsτvennymi miκροορganizmami. Κροme τοgο οτsντsτvuyuτ univeρsalnye sπetsialnye meτοdy. Ποvyshayushie zhiznesποsοδnοsτ and προduκτivnοsτ ρazlichnyχ baκτeρy in οπρedelennyχ uslοviyaχ suschesτvοvannya

The closest we can offer to you is that there are a number of different types of identification methods that are used to obtain the best results. τ Ε ϋ ма ϋ ϋ ϋ ϋ [1 1 - chapter 8]. In this method for gene transfer, intestinal injuries, unsuitable for transduction and conjugation, can be used. With the method, the following actions are taken - the selection of the recipient and the donor, the cultivation of the recipient and the donor

5 in a good environment (usually different - optimal for everyone

. πaρτneρa) selection DΗΚ dοnορa, οslablenie zaschiτnyχ svοysτv ρetsiπienτa πuτem vοzdeysτviya on egο shτamm χimichesκimi πρeπaρaτami and (or) χοlοdοm, introduction οbρabοτannuyu sρedu ρetsiπienτa DΗΚ dοnορa, inκubatsiyu mixture πρimeneniem ledyanοy and vοdyanοy baths, nagρeva and 0 tsenτρiφugiροvaniya, seeding shτammοv, iχ analysis, finding bacteria for previously selected attributes and their replication. For gene transfer, this requires special treatment of the cells of the process, which contains clean calcium and the input to the separator. This method has been selected as a spare part.

15 The written method is one of the best modern methods of production of recombinants and, at the same time, is widely used in scientific and industrial applications. Οdnaκο, uchiτyvaya znachiτelnuyu ποτρeδnοsτ in sοvρemennyχ uslοviyaχ in usκορenii and ρasshiρenii τρansφορmatsii δaκτeρy, sποsοb προτοτiπ not vο all udοvleτvορyaeτ 0 uchenyχ and προizvοdsτvenniκοv due - slοzhnοsτi προtseduρy, οgρanichennyχ vοzmοzhnοsτey ρeκοmbinatsii (vοzmοzhnyχ dοnοροv and ρetsiπienτοv) οτsuτsτviya ποvysheniya zhiznesποsοbnοsτi ρeκοmbinanτοv, iχ προduκτivnοsτi and PRODUCTIVITY.

Ή Objective of the Invention.

The objective of the invention is the elimination of the risk of the radiation from the medical team. The increase in its universality (expansion of the possibility of recombination). Boosting the life of the business community. ι ι () PRODUCTIVITY AND PRODUCTIVITY. Other sovyasami - the task of the invention is the development of new - more than a simple and versatile - the method of obtaining industrial products that is commercially viable for business SUMMARY OF THE INVENTION

The posed problem was solved by the fact that, in the well-known method of receiving recombinants of the bacteria, the suggested one was the selection and the initial extension of the participants of the treatment. sowing the strain for selective environments, their analysis, the selection of bacteria for the previously selected signs and their removal, significant changes were made, and the name changes the participation of the Republic of Belarus. sποsοbnyχ οbρazοvyvaτ mixed miκροbnye sοοbshesτva on πlοτnοy πiτaτelnοy sρede for vyρashivanιχya shτammοv vybiρayuτ πiτaτelnye sρedy, πρiemlimye οdnοvρemennο for vseχ uchasτniκοv ρeκοmbinatsii, πρigοτοvlyayuτ mixture miκροbnyχ shτammοv uchasτniκοv ρeκοmbinashιi κοntsenτρatsii at least (1-2) χ10 ⁴ ΚΟΕ in κazhdοgο ml. then put 1-δ of a mixture of bacteria on a healthy diet. and after incubation, it is transferred to the communal part of the agar from the agrarian industry and produces slurry. A short then sown on selective media for the treatment of recombinants for a given parameter (ΚΟΕ - the largest unit). In other words, the task posed is solved by opening up new, simpler and more convenient means of transmitting genes between non-consuming and non-consuming devices. Risk of gambling is subject to a violation of the law. Delivering superior life and product availability for used products and for consuming identified by bacteria. non-antagonistic properties may include six Particular services that provide different types of strains, such as inappropriate and community-based microorganisms. For the preparation of mixed mixtures, a mixture of strains of such methods is prepared. to

5 minimum usable amount of each of them exceeded 20

- The maximum number of units (ΚΟΕ). Bacteria of different strains are inflicted either in a mixture or separately in any investigation. Аче As a part of a healthy environment, it is used to ensure that all the used in this experience is secured. After sowing, cups 0 are incubated at a temperature that is optimal for these bacteria (from 20 ° C to 41 ° C) for 24-72 hours. After the inoculation, the large community is transported from the agar to the industrial disinfectant. Suspension is prepared, which is seeded on selective media that cause differentiation of the bacteria to the detected symptoms. 5 Addition to this, in this case, the number of participants in the recombination may be more than two, and this is due to the loss of participation.

DISCUSSION OF INVENTION 0

The dryness of the invention is ensured by the following two examples of the receipt of recombinant strains with an increase in the business life of the company. 25 Proceedings I. Transitional genetic genetics between α and α, corresponding to βιιθ. The used strains:

ΙΧсΗегιс / ιια сοϊι ΑΒ П57. Ρ-. 5 and ρΕ44. ϊηg. Ιey. ρгοΑ. ΗΪ5. ϊЫ. agg. ΙасΥ. αaϊΚ. ι () yeah. chuϊ, tϊϊ. ϊзχЗЗ. gr δ З Ι. 5gηΚ

The well-known strain Ε.сυ / ι ΑΒ 1,157 has a comparatively well-known gene. stable to strontium, not present in the gene pool of ρρπ genes ΛШ99 / 00П4

κοnyugatsii (devoid πlazmid, nesuschiχ ϊga-οπeροn) not in uchasτvueτ κachesτve dοnορa genοv πρi τρansduκtsii (not imeeτ προφagοv) and being gρamοτρitsaτelnymi not πeρedaeτ οτ κleτκi κ genes in κleτκe χοde τρasφορmatsii without sπetsialnοy οbρabοτκi κleτοκ.

ΕссИегШα сοϊϊ ΜΟΙбδδ, ρзΡΙ, ρΒΚ322, + Ρ2ο1ά, ΑгηЯ.

Shτamm Ε.sοN ΜΟΙbδδ τaκzhe imeeτ sρavniτelnο izvesτny genοτiπ, usτοychiv κ amπitsillinu not vysτuπaeτ in κachesτve dοnορa genοv πρi κοnyugatsii (devoid πlazmid, nesuschiχ Ιga-οπeροn) not in uchasτvueτ κachesτve dοnορa genοv πρi τρansduκtsii (not imeeτ προφagοv) and being gρamοτρitsaτelnymi , does not transfer genes from the carcass to the carcass in the process of transfiguration without special treatment.

Bacteria of the strains selected for the work were repaired in a period of 24 hours at 37 ° С. The resulting suspensions of the two strains were mixed in equal amounts (1: 1). The mixture was inoculated on meat agar. For this bacteriological needle with a thickening at the end, we threw a suspension cable with a volume of 0.1-0.2 m and put on agar. At the same time, a 10 cm cup Petri can be applied to 10-15 such droplets. After drying, the cups were incubated at a temperature of 37 ° C for 24 hours. Οδρazοvavshiesya on agaρe mixed miκροbnye sοοδshesτva removed from agaρa, ρesusπendiροvali, gοτοvili ρazlichnye ρazvedeniya in izοτοnichesκοm ρasτvορe χlορida naτρiya and plated on diφφeρentsialnye sρedy, sοdeρzhashie anτimiκροbnye πρeπaρaτy πο οτdelnοsτi - myasοπeπτοnny agaρ with amπitsilillinοm (Yumκg / ml) sο sτρeπτοmιshiοnοm (Yumκg / ml) and Simultaneously with two of these preparations in the indicated accents. In the last environment, there were only a few recombinants who were stable, both stabilistin and ampicillin. The original strains could only have occurred in the environment with one of the used anti-malware. To evaluate the number of communities, the resulting dilutions were sown in parallel on meat-grown agar, not containing antibiotics. ΡСΤ / ΙШ99 / 00114

Received recombinants:

The number of recombinants of the ήαskeαsIια community (ΜΟΑΒ and ΑΒΜΟ) - ΑгηΚ, δтΚ,

4 gains in the environment with two anti-optics was 5x10 ΚΟΕ / ml

Of these, ΚΟΕ in 1 ml of the community, which were obtained on a medium without any anti-doses, was 4 x 10.

Taking into account the properties of the strains chosen for the combination, the latter could not take into account the knowledge of the industrial processes involved in this type of processing devices.

After the rssseva mixed mixed wars, we have received strains that have been hindered by an increase in the use of life-threatening conditions.

Ταblιschα 1.Χαρακteρistiκα ροstα isχοdnyχ shtαmmοβ Ε.sοI and shtαmmοβ, ιyuluchennyχ ηοsle ραsseβα smeshαnnοgο miκροbnοgο sοοbschestβα ηοsle 24 χ chαsοβοgο κultiβιιροβαnιιya.

What is it? The strains received from the СС are identical in terms of the size of the larger size compared to the original ones. It is important to note that there are known methods. For one of the strains, one performs the role of the gene donor, and the other recipient I. χ (recipient). In the case of the claimed method, the gene transfer process takes place. in the case of a member state, both in the gene pool and in the share of the patient are significant, they are significantly more efficient and productive. In this way, they are more well-received and universal. From the information provided, it also follows that, at the same time, the stability of the antibiotics succeeds in concurrently and inadequately unacceptable or otherwise inaccurate or otherwise

Primary 2 Transmitted genetic information between the states of the α, which is related to the beta. The used strains are: сζСИЕήСЫα соϊι ΑΒП57. Ρ-, δиρΕ44, ϊηг, Ιei, ρгοΑ, Ыδ, ЫЫ, agh§, ΙасΥ, §а1Κ, aha, χуϊ, тϊϊ, 15x33, rρδΙ-31, δтΚ.

Βybρanny shτamm Ε.sοP ΑΒP57 imeeτ sρavniτelnο izvesτny genοτiπ, usτοychiv κ sτρeπτοmitsinu not vysτuπaeτ in κachesτve dοnορa genοv πρi κοnyugatsii (devoid πlazmid, nesuschiχ Ιga-οπeροn) not in uchasτvueτ κachesτve dοnορa genοv πρi τρansduκtsii (not imeeτ προφagοv) and being gρamοτρitsaτelnymi , does not transmit genes from the battery to the battery in the process without special processing of the battery.

Μιсгососссιιз Ιиϊеιιз νΤ18. (the genotype is unknown, sensitive to the testimycin, produces a yellow pigment).

Bacteria of the strains selected for the work were repaired in the Boulevard of the Russian Federation for 24 hours at 37С, and at 48 hours for 37С. The resulting suspensions of the two strains were mixed in equal amounts (1: 1). The mixture was inoculated on meat agar. For this δacteriological needle with a thickening at the end of δ, they drop the suspension cable with a volume of 0, 1-0.2 mcl and put it on the agar. At a time of 10 cm, a cup may also be applied to 10-15 such droplets. After drying, the cups were incubated at a temperature of 37 ° C for 48 hours. The mixed mixed minerals on the agar were removed from the agar, they were removed from the agar. Prepared various dilutions in the domestic market for acid and sowed on a differential medium containing an antimicrobial preparation (ml) (ml). Ηa ΡСΤΛШ99 / 00П4

this environment could have grown only for small intestine sticks and recuperators of the mycobacterium, which are resistant to medicine. For οtsenκi number ΚΟΕ sοοδschesτv ποluche ^'nnye ρazvedeniya πaρallelnο plated on myasοπeπτοnny agaρ not sοdeρzhaschy anτibiοτiκa. On the other hand, the Holy Father and the Holy Father are distinctly distinguishable by pigmentation.

The number of chemicals in v. 18, stable to potency of 1x10 ⁴ ΚΟΕ / ml

In total, ΚΟΕ in 1 ml of the community, obtained on a medium without antibiotics,

8 δylο 6x10.

Taking into account the properties of the strains chosen for the combination, the latter could not take into account the knowledge of the unknown for the process of the sale of industrial products.

After russev mixed mixed wars, we received strains, quicker accidents caused by increased accidents.

Ταblιschα 2.Χαρακteρistιικα ροstsι isχοdnyχ shtsιmmοβ and shtαmmοβ. ηο irradiated ηριι ραссеβе СΜС ηο after 24χ hours of cultivation and ροβαniya.

How to do it. The strains received from the СС are identical in terms of the size of the larger size in comparison with the best strains. Facilitants are significantly more viable and productive. It is important to note that there are known methods. For one of the strains, one performs the role of the gene donor. and the other recipient ΡСΤΛШ99 / 00П4

10

(recipient), in the case of a claimed method of transmitting genes, the share of a member of the international community is in the process of being incurred by a third party.

In this way, they are more well-received and universal. 5 _. From the information provided, it also follows that, at the same time, the stability of the antibiotics succeeds in concurrently and inadequately unacceptable or otherwise inaccurate or otherwise

0 Intended use

EXAMPLES 1 AND 2 | It allows for the efficient generation of genes between independent and non-essential bacteria. In order to receive the best recombinant strains with new features, as well as strains, 5 possessing an increased business life in the given conditions. At this point, information is transmitted, and, at the same time, all participants carry out the business and the benefit of the patient and the recipient.

Obviously, that way, they are only better, more convenient and universal.

The latter indicates the possibility and usefulness of the use of 0 of this method in biotechnology and medicine for the preparation of strains with new properties.

The above facts prove the practical dryness of the declared method and the achievement of the tasks obtained by the solution:

25 a new one has been developed. effective and universal way of receiving the results of the process. and an increase in the given conditions of productivity of strains (vital activity).

In general, in our opinion, the stated method satisfies all the requirements of the invention. unobvious and

"ο προνι industrial πρname. eleven

Literature.

Ι. Belyakov Β. D., Golubev D. B., Kaminsky G. D., Τec Β.Β. Self-regulation of steam systems. Medicine, Leningrad, 1987, 1 16-123. 5 2 Bρown Β. Genetics of bacteria. Μ., Гedgiz. 1968, 446 p.

Z. Miramov E.Ya., Domusadsky I. ,., Industrial product: options, restrictions, impairments. Μ. ΟΗΤИΤЭИ, Μиκροbiοπροм 1981, 43 p.

4. Bill J. Experiments in molecular genetics Μ, Μиρ, 1976. δ.νаη νΗеϊ Ρ., Βοуеη Α., ΟΙаηзάοгГГΝ. Οη ϊητegδρеϊеδ ηηηηϊϊηηδ: :еге::: ϊе са са саδδ 10 10 10 10 10 10 10 10 10 са са саереререререререререререререререр с с с с с с с с сϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋϋ Αηη.Ιηδϊ.Ρаδϊеиг (ΜϊсгοЫοΙ). 1988, 139: 493-

496. b.Μаζοάϊег Ρ., Ϋаνϊеδ ^τ . Ηеηе ϊгаηδГег

άϊδϊаηϊϊу гϊаϊеά ϊасϊегϊа. Αηη and.

Κеν. Ηеηеϊ. 1991, 25: 147-171.

7. Ρгοδϊ Ι_.8. Βасϊегϊаϊ сοη) и§аϊϊοη: еνегуЬοάу ^' δ άοϊη ϊϊ. Saη.ΙΜϊsgoyuΙ. 1992, 15 38: 1091-1096.

8. ϊеϊ 5.Ρ., ΜекаΙаηοδ 11 Μοάиϊаϊюη οГ οοπζοηϊаΙ §еГе ϊгаηδГег ш ϊаϊЬοееηϊс Басϊепа бу ιη νϊνο δϊεηаϊδ. Ceϊϊ. 1996, 87: 795-798.

9.5 aiηάeg ΙΚ., Μοgeaη ΙΑЖ,

S, Κаϊϊ Ρ.С., Сагϊег 1.?., Ριскиρ

Κ. \ Υ., .ΙοηеδΙΟ., Δаиηάегδ ν.Α. ΗΟηηϊϊ а аρρρρρρρδδδδδδδδϊϊϊϊϊϊ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0ϊ 0ϊ 0 0 0 0 0 0 0 0 0 0 0 0ϊ 0 0ϊ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0δδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδδ. Ιη ΒΒϊϊπππϊϊ ηδδδδδ ϊϊϊ е е еδδδδδδδδδδδδδδδδδδδδδ Εάϊιеά bу

ΙS. Ρgu, Μ. Yow. Sagtaη aηά Ηаϊϊ, Lοηάοη. 1990, 3-21.

Y. Canaan D. Methods of transfiguration Ε.СОН. Β κn. Bowing DΗΚ.

Μ and ρ. Russia, 1988, 140-174.

1 1. ϊаηϊаϊϊδ Τ .. ΡгϊϊδсЬ Ε.Ε. δшбгοοк. Μοϊесиϊаг сϊοηϊηе. Сοϊά δρгϊη ΗагЬοг 5 1-аб. 1982, chapter 8 - προτοτиπ.

"0

Claims

12

ΟΡΜΟΡΜΟΡΜΑΑ ΑΟΟΡΕΤΕΗΡΕΤΕΗ.

Ι. The method of obtaining recombinants of mixtures (mutation is a means of saving), which implies the choice of and the primary

5 vyρaschivanie uchasτniκοv ρeκοmbinatsii, inκubatsiyu mixture seeding ρeκοmbinanτοv on seleκτivnye sρedy, οτbορ ρeκοmbinanτοv πο zaρanee vybρannym πρiznaκam, ΟΤLICHΑYUSCHIYSYA ΤΕΜ, CHΤΟ, vyδiρayuτ uchasτniκοv ρeκοmδinatsii of miκροορganizmοv, w sποsοbnyχ οbρazοvyvaτ mixed miκροδnye sοοδschesτva on πlοτnοy πiτaτelnοy sρede for vyρaschivaniya shτammοv vyδiρayuτ πiτaτelnye sρedy, suitable for all participants of the combination, use a mixture of microstring of participation in the combination of at least

15 (1-2) χ10 in ⁴ ml ΚΟΕ κazhdοgο, zaτem nanοsyaτ 1-5 mκl mixture baκτeρy on πlοτnuyu πiτaτelnuyu sρedu and ποsle inκuδatsii - πeρenοsyaτ οbρazοvavsheesya sοοbschesτvο with agaρa in izοτοnichesκy ρasτvορ χlορida naτρiya and izgοτavlivayuτ susπenziyu, κοτορuyu zaτe.m on vysevayuτ selective media for environmental products

20 πο given preset.

2. The case of π 1, which is different from the fact that the number of participants in the resolution is more than two.

Z. Spirosδπππ1 and 2, differing in volume. that all reciprocating agents sow in parallel to different selective media, in each of 5, they automatically dispense with different supplies.