GB2109408A - Method for cultivating a micro-organism containing a recombinant plasmid - Google Patents
Method for cultivating a micro-organism containing a recombinant plasmid Download PDFInfo
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- GB2109408A GB2109408A GB08231516A GB8231516A GB2109408A GB 2109408 A GB2109408 A GB 2109408A GB 08231516 A GB08231516 A GB 08231516A GB 8231516 A GB8231516 A GB 8231516A GB 2109408 A GB2109408 A GB 2109408A
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Abstract
A selective method for cultivating a micro-organism having a recombinant plasmid comprises incorporating a phage deoxyribonucleic acid fragment coding for a protein necessary for the maintenance of the lysogenic state of a lysogenic phage into a plasmid which is replicatable in a host micro-organism, then the resulting recombinant plasmid is introduced into the micro-organism in which a lysogenic phage having mutation in the deoxyribonucleic acid region coding for a protein necessary for the maintenance of the lysogenic state of said lysogenic phage is in a lysogenic state, and finally the micro-organism is cultivated under conditions where the formation of said lysogenic phage is induced. Thus, when a gene fragment coding for a valuable product is further incorporated into the foregoing replicatable plasmid carrying a suppressor gene and a micro-organism having the resulting plasmid is cultivated, only the micro-organism having the plasmid can selectively be grown, leading to an increase in productivity in fermentation of valuable products in genetic engineering.
Description
SPECIFICATION
Method for cultivating a micro-organism containing a recombinant plasmid
The present invention relates to a selective culture method for cultivating a micro-organism containing a recombinant plasmid. More specifically, it relates to a method in which a phage deoxyribonucleic acid fragment coding for a protein necessary for the maintenance of the iysogenic state of a lysogenic phage is incorporated into a plasmid which is replicatable in a host micro-organism, and then the recombinant plasmid is introduced into the microorganism in which a lysogenic phage having mutation in the deoxyribonucleic acid region coding for a protein necessary for the maintenance of the lysogenic state of a phage is in a lysogenic state, followed by cultivating the micro-organism under conditions where the formation of said lysogenic phage having mutation is induced.
In recent years, attempts have been made to breed new micro-organisms through the incorporation of a deoxyribonucleic acid (DNA) coding for the products into the micro-organisms. it is know, however, that recombinant plasmids having a DNA fragment coding for valuable products are generally unstable and liable to be eliminated from micro-organisms into which they have been introduced. Theretore, the unstableness of plasmids in a micro-organism leads to a reduction in productivity when the microorganism is cultivated on an industrial scale to produce valuable products in recombinant DNA tech- nology.
For the resolution of this problem, several methods have been reported in which there is emp loyed a selective medium which permits the growth of a micro-organism containing a plasmid but prevents the growth of a micro-organism from which a plasinid has been removed (Japanese patent first publication No. 15696/1981, Japanese patent first publication No. 156591/1980). In most of the methods, a micro-organism to be cultivated contains a plasmid carrying a gene coding for resistance to at least one specific antibiotic and the culture medium contains said at least one antibiotic, with flie result that only the micro-organism holding the plasmid can growin the medium.In industrial production, however, the methods in which an antibiotic is added in a culture medium suffer from a disadvantage that large amounts oS the antibiotic are consumed and troublesome purification processes are required ; moreover, the residual antibiotic in waste water can cause the v-i ida spread of antibiotic 5-esis- tant micro-organisme in natural environments.
Thus the present nventors have made intensive investigations to develop a selective culture mothod for cultivating a micro-organism containing a hydrid plasmid. As a result, the present inventors have succeeded in establishing an alternative selective method for cultivating only a micro-organism having a recombinant plasmid; in the method, a DNA fragment coding for a repressor of a lysogenic phags is incorporated into a plasmid replicatable in a host micro-organism, and the recombinant plasmid is introduced into a micro-organism in which a lysogenic phage having mutation in a repressor gene is in a lysogenic state, and then the micro organism is cultivated under conditions where the formation of said lysogenic phage having mutation is induced.
According to the method of the present invention, in the cultivation of a micro-organism having a plasmid, since the expression of a repressor gene incorporated into the plasmid can repress the induction of a phage which, having mutation in a repressor gene, lies been lysogenized on a chromosone in a host rnicro-organism, a micro-organism having said clasmid can grow, whereas one from which said plasmid has been removed will die as a result of the induction of the phage having mutation in a repressor gene; therefore, cells living in the culture medium have all the plasmid.
By using the foregoing method, micro-organisms can be cultivated more inexpensively and readily than by methods in which an antibiotic is added to the culture medium; moreover, in the former method, there are fewer problems in working-up treatments such as purification than in the latter methods.
In addition, .t is notable that micro-organisms grow much faster in the present method 'han in methods using an antibiotic. Furthermore, it is one of advantageous eatures of the present method that there can be used any culture medium in which a micro-organism can grow.
Therefore, when a gene fragment coding for a val uable product is further incorporated into the foregoing replicatable plasmid carrying a suppressor gene and a micro-organism having the resulting plasmid is cultivated, only the micro-organism having the plasmid can selectively be grown; thus the reduction in productivity can be prevented in a fermentative production of valuable products.
The presentmethodis applicable to the cultivation of any micro-organism having a Iysogenic phage. As a combination of a micro-organism and a lysogenic phaga to which the present method can be applied, there are, for example, Escherichia cell and its phages # 80, @ @ imm @@, etc., Bacillus subtilis and its lysogenic phages p11, # 105, etc., and Nocardia
Mediterranei ATCC 13685 and its lysogenic phages ss, v. etc.Thepresent method is considered applicable also to micro-organisms having their lysogenic phages which vfill be found in the future.
Ropressor gene mutants of lysogenic phages, such as repressor deficient mutants and temperature-sensitive mutants can readily be obtained by mutagenesis with ultravioiet rays or chemical mutagens such as There have already been roported isolation 0 repressor deficient mutants [A.D. Kaiser, Virology, 3, 42 (1957)] and temperature-sensitive mutants [M. Lieb,
Journal of Molecular Eiology, 16, 149 (1966)].
The purification of particles of lysogenic phages and the extraction of DNA can be carried out by CsC density gradient centrifugation and a phenol extraction method. [F. Frankel, Proceedings of the National
Academy of Sciencies, U.S.A., 49, 366 (1963)].
A repressor gene of a lysogenic phage can be
incorporated into a plasmid by joining a phage DNA to a plasmid DNA with a DNA ligase after the treatment of the both with a restriction endonuclease.
The restriction endonuclease may be any one which can cleave a plasmid, but is preferably one which does not cleave the repressor gene of the lysogenic phage into fragments. There may be used as a vector in the process of the present invention any plasmid which is replicatable in cells of a micro-organism; examples are pBR322 and pSC101 of Escherichia call, pU3110 and pTP5 of Bacitius subtllis, and pSC101 and pSC2 of Streptomyces coelicolor. All known transformation techniques can be used to introduce the hybrid plasmid composed of a replicatable vector and a repressor gene into a host micro-organism, although the transformation efficiency may be variable for each technique. For example, there can be used with
Escherichia coli an incorporation of a plasmid DNA into cells treated with calcium chloride [F.Boliver et al., Gene,2, (1977)], with Bacillus subtilis a method in which a recombinant plasmid is introduced into competent cells or protoplast cells in the presence of polyethylene glycol [C. Anagnostopoulos et al., Journal of Bacteriology, 81, 741 (1961); S. Chang et al., Molecular General Genetics, 168, (1979)], and with actinomycetes a transformations of a protoplast with a plasmid DNA in the presence of polyethylene glycol[M. J. Bibb et al., Nature,274, 398 (1978)].
The transformants containing the hybrid plasmid carrying a phage repressor gene fragment can readily be selected because they show resistance to a repressor deficient lysogenic phage and sensitivity to a virulent mutant of the lysogenic phage.
When a plasmid from such a transformant is introduced into a micro-organism in which a
lysogenic phage having mutation in a repressor gene is in a lysogenic state and the micro-organism
is cultivated under conditions where the formation of said lysogenic phage having mutation is induced, only cells containing the plasmid will grow selectively. Consequently, when a DNA fragment coding for useful gene products is further incorporated into the plasmid a micro-organism having the plasmid is cultivated, stable and high productivity of the useful product can be expected.
As conditions for inducing the formation of the phage having mutation in a repressor gene, with repressor deficient strains, any cultivating condi tionsfavourableforthegrowth of the microorganism containing the recombinant plasmid can be adopted; on the other hand, with temperature sensitive strains, a temperature condition must be adopted where the micro-organism can grow but the repressor of the lysogenic phage having mutation becomes inactive.
Thus in the cultivation of the latter strains, any cultivating conditions encouraging the growth of the micro-organism containing the recombinant plasmid can be adopted as long as the cultivating temperature is controlled to be 30 C or above, preferably 35 C C or above, at which temperatures does the rep- ressor of the lysogenic phage become inactive.
For example, when the host micro-organism is
Escherichia coli, the temperature conditon is 3C 45 C because the repressors of the mutated phage will not function at 35 C or above and the Escherichia coli will not grow at atemperature higher than 45'C.
To explain the present invention in more detail, the following examples are given.
Example 1
A plasmid having a DNA fragment coding for a repressor gene of a lysogenic phage a80 (Cl gene) was used to transform Escherichia call, a lysogen of a e80 repressor mutant sensitive to temperature. By cultivating the micro-organism at a high temperature, only cells having the plasmid can selectively be grown.
(1) Isolation of lysogenic phage 80 mutants.
By multiplying a a80 phage ion a host Escherichia coli W3110, a a80 lysate (6 x 10'0 PFU/ml) was prepared. The phage particles were collected by centrifugation (25000xg, 60 minutes) and resuspended in a 10 mM of MgSO4. The suspension was irradiated with a UV lamp set at a distance of 50 cm from the suspension for 6 minutes. Under these conditions, the survival rate of the phage was 0.7%. Escherichia coli W3110 which had previously been irradiated with ultraviolet rays for a short time (30 seconds) was infected with the foregoing phage, and allowed to form plaques in soft agar. A repressor deficient strain (hereinafter referred to as o80 Cl) was isolated as phages forming clear plaques.A 80 repressor mutant sensitive to temperature (80 CITs) was isolated as phages which form turbid plaques at a low temperature and whose lysogen induces phages by the treatment at a high temperature (35 ~ 45 C).
(2) Cloning of o80 Cl gene
A a80 lysate (6 x 106 PFU/ml) was prepared by infecting Escherichia coli W3110 with a 80 phage.
The phage particles were collected by centrifugation (25000xg, 60 minutes) and purified by CSCI density gradient centrifugation.
From the phage particles, #80 DNA was extracted with phenol. The plasmid pBR322 carrying genes resistant to tetracycline and ampicillin was used as a vector for cloning. The plasmid pBR32 was prepared according to a method reported by D. B.Clewell [Journal of Bacteriology, 110, 667 (1972}, Escherichia call W3110 having the plasmid pBR322 [hereinafter referred to as W3110 (PBR322)] was cultivated at 3PC in the following culture medium: the Culture medium was prepared by dissolving 0.5% casamino acid, 0.2% glucose, and 0.1% yeast extract into an inorganic salt medium (6g of Na2HPO4, 39 of KH2PO4, 0.5g NaCI, 1g NH4CI), and the medium was sterilized at 120 C under the atmospheric pressure for 20 minutes; to the resulting medium was added 10 ml each of 0.01M CaCl2and0.1M MgSO4thathad been sterilized under the same conditions. When
Escherichia coli (PBR322) had grown to reach the logarithmic growth phase, chloramphenicol was added to attain a final concentration of 170 g/ml ; thereafter, the cultivation was further carried out at 37 C for 17 hours. After collection, the cells were lysed by a lysozyme-Brij 58-deoxycholate treatment.
The resulting lysate was centrigued (48000xg, 30 minutes) to obtain the supernatant. The plasmid pBR32T DNA was purified from the supernatant by
extraction with phenol and precipitation with ethanol followed by CsC - equilibrated density gradient centrifugation.
DNA chains of 0.9 g of a80 DNA and 0.7 g of pBR32 DNA were cleaved by subjecting them to the action of EcoRI, a kind of restriction endonuclease, at 3TCfor 3 hours.
After incubation at 65 C for 5 minutes, the DNA fragments were ligated with T4 DNA ligase at 10 C for 19 hours in the presence of ATP and dithiothreitol. DNA was precipitated by the addition of a twice volume of ethanol to the reaction mixture, and then recovered by centrifugation.
The DNA ligated was added to a suspension of competent W31 10 cells prepared by a treatment with 0.1M CaCI2. After incubation at C for an hour, the cells were subjected to a heat pulse at 42 C for 75 seconds. To the reaction mixture was added an LB medium (described in J. H. Miller, Experiments in
Molecular Genetics, P.433: Cold Spring Habor
Laboratory, 1972), and the resulting mixture was incubated at 37 C for 2 hours.
To this culture broth was added a80 Cl to give an moi 1 and the mixture was incubated for additional minutes. The cells were inoculated into an LB agar
medium containing 20 g/ml ampicillin and 15 Fg/ml of tetracycline. On incubation of the cells at 37 C overnight, 47 colonies were formed. Every colony isolated was confirmed to have resistance to the both drugs, resistance to a80 CI, and sensitivilityto a80 Vir.The restriction enzyme analysis of the plasmid of these transformants revealed that all the transformants contain a 80 repressor gene inserted into an EcoR1 site of pBR322. A plasmid having a fragment of 1.7 Md CI repressor gene incorporated at the EcoR1 site of PBR322 is designated PKN319. The
PKN319 was purified according to the same method as described above.
(3) The stability of plasmid PKN319
The phage a80 Clts was lysogenized into
Escherichia coli W3110 at a low temperature (30 C) to prepare a lysogen W3110 (80 Clts). A suspension of competent cells of W3110 (80 Clts) was prepared by a treatment of the lysogen with 0.1 CaCI2. The plasmid KN319 was added to the suspension, and the resulting mixture was allowed to stand at 0 C for 1 hour. After an LB medium had been added to the cell suspension, the cells were incubated at 33 C for 135 minutes. The resulting culture broth was inoculated into an LB agar medium containing 20 yg/ml of amplicillin and 15 ,ug/ml of tetracycline.The inoculum was incubated at 33 C overnight to prepare a transformant W31 10 (a80 Clts) (PKN319) [FERM BP-69].
The transformant W3110 (#80 Clts) (PKN319 was inoculated into an LB agar medium and cultivated at for a day. The cells grown on the medium was mixed uniformly. An aliquot of the cells was again inoculated into a new LB agar medium and incubated at 43 C for a day; this procedure was repeated 3 times. During the foregoing incubations, each 100 colonies was taken from the grown cells and tested for the presence of a plasmid; the presence of a plasmid can be examined by observing the growth ofthe colonies in an LB agar medium containing 15 ,ag/ml of tetracycline and 20 g/ml of ampicillin. The same procedure was followed with W31101 (pMN319) and W3110 (pBR322) as controls.
As Table 1 clearly shows, the plasmid contained in W31 10 (a80 Clts) will not be lost in a prolonged cultivation. Thus, according to the method of the present invention, micro-organisms having a plasmid can selectively be cultivated.
Table 1
Proportion of cells containing a placmid (%) Number of subculture Micor-organism 0 1 2 3 4 W3110 (PBR322) 100 98 86 61 23 W3110 (PKN319) 100 83 46 4 0 W3110 (#80 Clts) (PKN319) 100 99 98 100 100 The microorganisms mentioned herein are depo
sited at The Fermentation Research Institute, 1-3
Higashi 1 Chome Yatabe-Machi, Tsukuba-Gun
Ibaraki-Ken 305, Japan. Date of deposit under
Budapest Treaty November 2, 1981. Receipt No.
Ferm. BP-69.
Claims (8)
1. A method for cultivating a micro-organism
having an recombinant plasmid which comprises
incorporating a phage deoxyribonucleic acid frag mentcoding for a protein necessaryforthe mainte
nance of the lysogenic state of a lysogenic phage
into a plasmid which is replicatable in a host micro
organism; introducing the resulting recombinant
plasmid into the micro-organism in which a
lysogenic phage having mutation in the deox
yribonucleic acid region coding for a protein neces
sary for the maintenance of the elysogenic state of a
lysogenic phage is in a lysogenic state; and cultivat
ing the micro-organism under conditions where the
formation of said lysogenic phage having mutation is induced.
2. The method according to Claim 1 wherein the
micro-organism is a bacterium.
3. The method according to Claim 2 wherein the bacterium is Escherichia coli.
4. The method according to Claim 2 or Claim 3 wherein the lysogenic phage is 80.
5. The method according to Claim 1, Claim 2,
Claim 3, or Claim 4 wherein the protein necessary for the maintenance of the lysogenic state of a lysogenic phage is temperature-sensitive.
6. The method according to Claim 5 wherein the conditions for cultivating the micro-organism containing the recombinant plasmid include a temperature condition where the micro-organism can grow but the lysogenic phage having mutation becomes inactive.
7. The method according to Claim 5 and Claim 6 wherein a temperature condition for cultivating the micro-organism containing the recombinant plas mid is 30"C C or above.
8. A method for cultivating a micro-organism having a recombinant plasmid substantially as described in the examples disclosed herein.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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JP56178724A JPS5878589A (en) | 1981-11-06 | 1981-11-06 | Cultivation method of microorganism containing plasmid |
Publications (2)
Publication Number | Publication Date |
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GB2109408A true GB2109408A (en) | 1983-06-02 |
GB2109408B GB2109408B (en) | 1985-03-20 |
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GB08231516A Expired GB2109408B (en) | 1981-11-06 | 1982-11-04 | Method for cultivating a micro-organism containing a recombinant plasmid |
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GB (1) | GB2109408B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0080848A2 (en) * | 1981-11-27 | 1983-06-08 | Eli Lilly And Company | Stabilizing & selecting cells |
US4650761A (en) * | 1981-11-27 | 1987-03-17 | Eli Lilly And Company | Method for stabilizing and selecting recombinant DNA containing host cell |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4506013A (en) * | 1980-10-03 | 1985-03-19 | Eli Lilly And Company | Stabilizing and selecting recombinant DNA host cells |
-
1981
- 1981-11-06 JP JP56178724A patent/JPS5878589A/en active Pending
-
1982
- 1982-11-04 GB GB08231516A patent/GB2109408B/en not_active Expired
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0080848A2 (en) * | 1981-11-27 | 1983-06-08 | Eli Lilly And Company | Stabilizing & selecting cells |
EP0080848A3 (en) * | 1981-11-27 | 1985-01-16 | Eli Lilly And Company | Stabilizing & selecting cells |
US4650761A (en) * | 1981-11-27 | 1987-03-17 | Eli Lilly And Company | Method for stabilizing and selecting recombinant DNA containing host cell |
Also Published As
Publication number | Publication date |
---|---|
GB2109408B (en) | 1985-03-20 |
JPS5878589A (en) | 1983-05-12 |
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PCNP | Patent ceased through non-payment of renewal fee |