DK159282B - PROCEDURE FOR STABILIZING EXTRA-CHROMOSOMAL ELEMENTS IN A GROWTH BACTERIA IN CULTURE, TRANSFORMED BACTERIES AND A PROCEDURE FOR MANUFACTURING A DESIRED PRODUCT IN SUCH TRANSFORMED BACTERIES - Google Patents
PROCEDURE FOR STABILIZING EXTRA-CHROMOSOMAL ELEMENTS IN A GROWTH BACTERIA IN CULTURE, TRANSFORMED BACTERIES AND A PROCEDURE FOR MANUFACTURING A DESIRED PRODUCT IN SUCH TRANSFORMED BACTERIES Download PDFInfo
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Denne opfindelse angår en fremgangsmåde til stabilisering af extra-kromosomale elementer i en værtsbakterie under dyrkning, transformerede bakterier samt en fremgangsmåde til fremstilling af et ønsket produkt i sådanne transformerede bakte-5 rier.This invention relates to a method for stabilizing extrachromosomal elements in a host bacterium during culture, transformed bacteria, and a method for producing a desired product in such transformed bacteria.
Mikroorganismer, der indeholder extra-kromosomale genetiske elementer, her eksemplificeret ved plasmider, er i almindelighed ustabile forstået på den måde, at plasmidet ofte kan tabes irreversibelt. Denne ustabilitet er særlig udpræget, hvis plas-10 midet ikke hidrører fra værtsorganismen, fordi det f.eks. indeholder gener fra andre organismer eller er konstrueret ved gensplejsning.Microorganisms containing extra-chromosomal genetic elements, exemplified here by plasmids, are generally unstable in that the plasmid can often be irreversibly dropped. This instability is particularly pronounced if the plasmid does not originate from the host organism, because it e.g. contains genes from other organisms or is engineered by genetic engineering.
For at forøge plasmidstabilitet er antibiotika eller andre bioaktive forbindelser, overfor hvilke plasmidet, men ikke 15 kromosomet udviser resistens, sædvanligvis sat til mediet anvendt til dyrkning af mikroorganismen. I sådan et medium vil kun de celler, der bevarer plasmidet med genet for antibiotisk resistens, formere sig. Den væsentlige ulempe ved denne metode, er at den kræver dyrkning i stor skala af antibiotisk resistente mikro-20 organismer, tilsætning af dyre antibiotika til vækstmediet med eventuel uheldig effekt på omgivelserne og efterfølgende ekstensiv oprensning for at fjerne antibiotikaet fra det ønskede produkt.To enhance plasmid stability, antibiotics or other bioactive compounds against which the plasmid, but not the chromosome, exhibit resistance are usually added to the medium used for culture of the microorganism. In such a medium, only those cells that retain the plasmid with the antibiotic resistance gene will multiply. The major disadvantage of this method is that it requires large scale cultivation of antibiotic resistant micro-organisms, addition of expensive antibiotics to the growth medium with possible adverse effect on the environment and subsequent extensive purification to remove the antibiotic from the desired product.
Komplementering af en auxotropisk mutation af værts-25 kromosomet er en anden kendt metode til stabilisering af plasmider. Denne metode lægger imidlertid alvorlige restriktioner på sammensætningen af dyrkningsmediet og kræver dyrkning i et vækstmedium, som ikke indeholder det næringsstof, som værtsorganismen kræver, hvorved mulighederne for at forbedre produktiviteten 30 begrænses.Complementation of an auxotropic mutation of the host chromosome is another known method for stabilizing plasmids. However, this method places severe restrictions on the composition of the culture medium and requires cultivation in a growth medium which does not contain the nutrient required by the host organism, thereby limiting the potential for improving productivity.
Formålet med den foreliggende opfindelse er at tilvejebringe en fremgangsmåde til stabilisering af extra-kromosomale elementer i transformerede bakterier uden nødvendigheden af at bruge antibiotika og uden at lægge alvorlige begrænsninger på 35 sammensætningen af vækstmediet.The object of the present invention is to provide a method for stabilizing extra-chromosomal elements in transformed bacteria without the need to use antibiotics and without imposing severe restrictions on the composition of the growth medium.
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Det er et yderligere formål med den foreliggende opfindelse at tilvejebringe transformerede bakterier, der indeholder sådanne stabiliserede extra-kromosomale elementer, samt en fremgangsmåde til at fremstille ønskede produkter i transformerede 5 bakterier uden nødvendigheden af at bruge antibiotika eller at lægge specielle restriktioner på sammensætningen af vækstmediet.It is a further object of the present invention to provide transformed bacteria containing such stabilized extra-chromosomal elements, as well as a method for producing desired products in transformed bacteria without the need to use antibiotics or to impose special restrictions on the composition of the growth medium. .
Opfindelsen beror på den erkendelse, at extra-kromosomale elementer kan bevares i værtsbakterier under dyrkningen i sædvanlige medier, hvis de extra-kromosomale elementer indeholder 10 en bestemt funktion, der under de givne betingelser er et krav for normal vækst af værten.The invention is based on the recognition that extra-chromosomal elements can be retained in host bacteria during culture in conventional media if the extra-chromosomal elements contain a specific function that under the given conditions is a requirement for normal growth of the host.
Når en værtsbakterie med en mutation, en deletion eller en anden defekt i et gen, der koder for en funktion, der under de givne betingelser er nødvendig for normal vækst af værten, trans-15 formeres med f.eks. et plasmid, der indeholder et gen, der koder for denne funktion, vil kun sådanne celler, der er transformeret med og bevarer plasmidet, overleve, fordi værtskravet kun i sådanne celler suppleres af plasmidet. Permanent plasmidbevaring sikres imidlertid kun, hvis overførsel af genetisk information 20 fra plasmid til kromosom ikke kan finde sted, og hvis graden af spontan mutation af værtsbakterien til en mutant uden et sådant krav, er uden betydning.When a host bacterium having a mutation, deletion or other defect in a gene encoding a function necessary under the given conditions for normal growth of the host is transformed by e.g. a plasmid containing a gene encoding this function will survive only such cells that are transformed with and retain the plasmid because the host requirement in such cells is supplemented only by the plasmid. However, permanent plasmid conservation is assured only if the transfer of genetic information 20 from plasmid to chromosome cannot occur and if the degree of spontaneous mutation of the host bacterium to a mutant without such a requirement is immaterial.
Ifølge et første aspekt af den foreliggende opfindelse tilvejebringes der en fremgangsmåde til stabilisering af extra-25 kromosomale elementer i en værtsbakterie under dyrkning, og denne fremgangsmåde er ejendommelig ved, at der i det extra-kromosomale element indføres en DNA-sekvens, der koder for D,L-alaninrace-mase, og at det extra-kromosomale element, der indeholder nævnte DNA-sekvens, transformeres i værtsbakterien, idet der anvendes en 30 værtsbakterie med en defekt i det kromosomale dal gen for D,L-alaninracemase. Kravet forårsaget af den kromosomale gendefekt i værtsbakterien undertrykkes herved af det extra-kromosomale element og tab af det extra-kromosomale element under dyrkningen er uden betydning.According to a first aspect of the present invention, there is provided a method for stabilizing extra-chromosomal elements in a host bacterium during culture, and this method is characterized by introducing into the extra-chromosomal element a DNA sequence encoding D, L-alanine race, and that the extra-chromosomal element containing said DNA sequence is transformed into the host bacterium, using a host bacterium with a defect in the chromosomal valley gene for D, L-alanine racemase. The requirement caused by the chromosomal gene defect in the host bacterium is thereby suppressed by the extra-chromosomal element and loss of the extra-chromosomal element during cultivation is immaterial.
35 Som anvendt her betyder udtrykket "extra-kromosomale elementer" plasmider, bakteriophager eller ethvert genetisk materiale, der ikke normalt findes i værtsbakterien enten som selv 3As used herein, the term "extra-chromosomal elements" means plasmids, bacteriophages, or any genetic material not normally found in the host bacterium either as itself 3
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stændige molekyler eller integreret i kromosomet. Foretrukne extra-kromosomale elementer er plasmider, men der kan også anvendes bakteriophager eller andre vektorsystemer.constant molecules or integrated into the chromosome. Preferred extra-chromosomal elements are plasmids, but bacteriophages or other vector systems can also be used.
Ifølge et yderligere aspekt af den foreliggende opfin-5 delse tilvejebringes der en transformeret bakterie, der er ejendommelig ved, at den er afledt fra en værtsbakterie, der har en defekt i det kromosomale dal gen for D,L-alaninracemase, samt at den indeholder et extra-kromosomalt element, der indeholder en DNA-sekvens, der koder for D,L-alaninracemase.According to a further aspect of the present invention there is provided a transformed bacterium which is characterized in that it is derived from a host bacterium which has a defect in the chromosomal valley gene for D, L-alanine racemase and contains an extra-chromosomal element containing a DNA sequence encoding D, L-alanine racemase.
10 Den foreliggende opfindelse tilvejebringer desuden en fremgangsmåde til fremstilling af ønskede produkter (f.eks. DNA, RNA, peptider eller proteiner) i transformerede bakterier, og denne fremgangsmåde er ejendommelig ved, at en bakterie, der har en defekt i det kromosomale dal gen og indeholder et extra-kromo-15 somalt element, der indeholder en DNA-sekvens, der koder for D,L-alaninracemase, samt en DNA-sekvens, der koder for det ønskede produkt, dyrkes i et egnet dyrkningsmedium, hvorpå det ønskede produkt udvindes fra dyrkningsmediet.The present invention further provides a method for producing desired products (e.g., DNA, RNA, peptides or proteins) in transformed bacteria, and this method is characterized in that a bacterium that has a defect in the chromosomal valley gene and contains an extra-chromosomal element containing a DNA sequence encoding D, L-alanine racemase, and a DNA sequence encoding the desired product is grown in a suitable culture medium upon which the desired product is extracted from the culture medium.
Egnede værtsbakterier hører til Bacillus- eller 20 Enterobacteriaceae-arter, f.eks. Bacillus subtilis og Escherichia coli, skønt andre egnede bakterier vil være indlysende for fagmanden.Suitable host bacteria belong to Bacillus or Enterobacteriaceae species, e.g. Bacillus subtilis and Escherichia coli, although other suitable bacteria will be obvious to those skilled in the art.
I beskrivelsen anvendes følgende udtryk: dal gen: genet for D,L-alaninracemase 25 dal+ gen: et funktionelt gen (vildtype) for D,L- alaninracemase dal-1 gen: et gen med en mutation i D,L-alaninracemasegenet, der normalt bevirker krav for eksternt D-alanin dal vært: en vært med en mutation i dal genet (der normalt 30 kræver ekstern tilførsel af D-alanin for vækst dal+ vært: en vært med et vildtype dal gen “j**In the specification, the following terms are used: dal gene: the gene for D, L-alanine racemase 25 dal + gene: a functional gene (wild type) for D, L-alanine racemase dal-1 gene: a gene with a mutation in the D, L-alanine racemase gene that normally requires external D-alanine dal host host: a host with a mutation in the dal gene (which normally requires external delivery of D-alanine for growth dal + host: a host with a wild-type dal gene “j **
Dal vært: en vært uden krav for eksternt D-alaninValley host: a host with no external D-alanine requirement
Dal vært: en vært med krav for eksternt D-alaninValley host: a host with requirements for external D-alanine
RR
Cam : chloramphenicolresistens 35 Kan : kanamycinresistensCam: chloramphenicol resistance 35 Can: kanamycin resistance
AmpR; ampicillinresistensAmp; ampicillin resistance
o Ro R
bla: gen for beta-lactamase, der forårsager Amp 4bla: gene for beta-lactamase causing Amp 4
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cat: gen for chloramphenicolacetyltransferase, der forårsager Cam amyM: gen for en maltogen amylase 5 For de fleste bakterier afhænger evnen til at vokse og dele sig af en velbevaret celleafgrænsning (d.v.s. cellemembraner, cellevægge og beslægtede strukturer). Sprængning eller nedbrydning af celleafgrænsningen fører sædvanligvis til opløsning eller ophøring af vækst.cat: chloramphenicol acetyltransferase gene causing Cam amyM: gene for a maltogenic amylase 5 For most bacteria, the ability to grow and divide depends on a well-preserved cell delineation (i.e., cell membranes, cell walls, and related structures). Explosion or degradation of the cell boundary usually leads to dissolution or cessation of growth.
10 En af de komponenter, der er nødvendige for en stabil celleafgrænsning i Bacillus subtilis og i de fleste andre bakterier, er D-alanin. D-alanin er en essentiel del af cellevæggen, i hvilken den tjener til at tværbinde polysaccharidkæder og således giver cellen den nødvendige stivhed. D-alanin findes ikke i de 15 fleste sædvanlige vækstmedier, og mange bakterier omfattende B. subtilis og E. coli syntetiserer denne aminosyre ud fra L-alanin ved hjælp af enzymet D,L-alaninracemase og kræver ikke en ekstern kilde for D-alanin for vækst. Nogle mutanter behøver en ekstern tilførsel af D-alanin for vækst, f.eks. dal-1 mutanter af B.10 One of the components necessary for stable cell delineation in Bacillus subtilis and in most other bacteria is D-alanine. D-alanine is an essential part of the cell wall in which it serves to crosslink polysaccharide chains and thus provides the cell with the necessary rigidity. D-alanine is not found in most common growth media, and many bacteria comprising B. subtilis and E. coli synthesize this amino acid from L-alanine by the enzyme D, L-alanine racemase and do not require an external source of D-alanine. for growth. Some mutants need an external supply of D-alanine for growth, e.g. dal-1 mutants of B.
20 subtilis, i hvilke D,L-alaninracemasegenet er blevet beskadiget på grund af mutationen (Freese et al., Proc.Natl.Acad.Sci.20 subtilis in which the D, L-alanine racemase gene has been damaged due to the mutation (Freese et al., Proc.Natl.Acad.Sci.
51:1164 - 72, 1964 og Dul et al., J.Bacteriol. 15: 1212-14, 1973).51: 1164-72, 1964 and Dul et al., J. Bacteriol. 15: 1212-14, 1973).
Ifølge den foreliggende opfindelse er det blevet vist, 25 at plasmider, der indeholder et funktionelt gen for D,L-alanin-racemase sikrer deres bevarelse i en dal vært, hvis dal+ genet ikke kan overføres fra plasmid til kromosom, og hvis hyppigheden af spontan mutation af værten til en Dal+ phenotype er uden betydning, samt hvis eksternt D-alanin ikke er tilgængeligt for 30 cellen.According to the present invention, it has been shown that plasmids containing a functional gene for D, L-alanine racemase ensure their conservation in a dal host if the dal + gene cannot be transferred from plasmid to chromosome and if the frequency of spontaneous mutation of the host to a Dal + phenotype is of no significance, as well as if external D-alanine is not available to the cell.
Ved indsætning af et gen for et ønsket produkt i et passende dal+ plasmid og dyrkning af plasmidet i en passende dal-vært, vil således dal+ plasmidet bevares i cellepopulationen under vækst, hvorved der sikres høje udbytter af det ønskede pro-35 dukt, der udtrykkes under den autonome replikation af plasmidet.Thus, by inserting a gene for a desired product into a suitable dal + plasmid and culturing the plasmid in a suitable dal host, the dal + plasmid will be conserved in the cell population during growth, thereby ensuring high yields of the desired product expressed. during the autonomous replication of the plasmid.
Da D-alanin ikke findes i mange sædvanlige vækstmedier, lægges der ingen restriktioner på dyrkningsmediet.Since D-alanine is not found in many usual growth media, no restrictions are placed on the culture medium.
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Den foreliggende opfindelsen tilvejebringer således en bekvem metode til stabilisering af extra-kromosomale elementer, der bærer gener for ønskede produkter, under dyrkningen.Thus, the present invention provides a convenient method for stabilizing extra-chromosomal elements carrying genes for desired products during cultivation.
For at sikre at graden af spontan mutation af 5 værtsorganismen til en Dal+ phenotype er insignifikant, kan det være nødvendigt at fjerne en del af dal genet. Ved kombination af en sådan vært, der indeholder en deletion af en del af dal genet + + nødvendigt for udtrykkelse af Dal phenotypen, med et dal plasmid, der indeholder hele dal+ genet og segmenter, der er homologe 10 til det DNA, der flankerer begge sider af deletionen på kromosomet, kan overførsel af dal+ allelet fra plasmid til kromosom finde sted ved homolog overkrydsning. For at undgå en sådan homolog overkrydsning blev der konstrueret en vært, i hvilken dal deletionen fortsatte ind i segmenter, der flankerer dal genet på 15 kromosomet. Der blev også konstrueret et plasmid, der indeholdt et funktionelt dal+ gen, men ikke DNA homologt til det DNA, der flankerer begge sider af deletionen på kromosomet. Kombinationen af dette plasmid med den førnævnte dal vært udgør det foretrukne vært-vektor-p ar.To ensure that the degree of spontaneous mutation of the host organism to a Dal + phenotype is insignificant, it may be necessary to remove part of the dal gene. By combining such a host containing a deletion of a portion of the dal gene + + necessary for expression of the Dal phenotype, with a dal plasmid containing the entire dal + gene and segments homologous to the DNA flanking both sides of the deletion on the chromosome, transfer of the dal + allele from plasmid to chromosome can occur by homologous crossover. To avoid such a homologous crossover, a host was constructed in which the dal deletion continued into segments flanking the dal gene on the chromosome. A plasmid also containing a functional dal + gene was constructed but not DNA homologous to the DNA flanking both sides of the deletion on the chromosome. The combination of this plasmid with the aforementioned valley host constitutes the preferred host vector pair.
20 Som et alternativ kan dal* allel overførsel undgås ved anvendelse af en rekombinantdeficient vært eller ved at anvende et extra-kromosomalt dal+ gen,.der har ingen eller ringe DNA-homologi med værtsbakteriens kromosom, dal genet fas fortrinsvis fra en B. subtilis stamme som forklaret i yderligere detaljer i 25 det følgende.. Foruden dal+ genet bør plasmidet også indeholde en sekvens for plasmidreplikation, f.eks. replikationsfunktioner fra højkopiplasmidet pUBllO for replikation i Bacillae eller andre grampositive bakterier eller replikationsfunktionen fra pBR322 for replikation i Enterobacteriaceae eller andre gram-negative 30 bakterier.Alternatively, dal * allelic transfer can be avoided by using a recombinant-deficient host or by using an extra-chromosomal dal + gene, which has no or little DNA homology to the host bacterium's chromosome, the dal is preferably phased from a B. subtilis strain as explained in further detail in the following. In addition to the dal + gene, the plasmid should also contain a sequence of plasmid replication, e.g. replication functions of the high-copy plasmid pUB110 for replication in Bacillae or other Gram-positive bacteria or the replication function of pBR322 for replication in Enterobacteriaceae or other gram-negative bacteria.
Produktion af et ønsket produkt ifølge den foreliggende opfindelse vil blive illustreret ved produktion af en maltogen amylase fra en Bacillus stamme (NCIB 11837) udtrykt ved dens egen promoter. Andre eksempler på ønskede produkter, der kan fremstil-35 les ifølge den foreliggende opfindelse, er andre amylaser, amylo-glycosidaser, pullulanaser, proteaser, lipaser, hormoner og andre slags enzymer eller'eukaryote proteiner og peptider.Production of a desired product according to the present invention will be illustrated by the production of a maltogenic amylase from a Bacillus strain (NCIB 11837) expressed by its own promoter. Other examples of desired products that can be prepared according to the present invention are other amylases, amylo-glycosidases, pullulanases, proteases, lipases, hormones and other kinds of enzymes or eukaryotic proteins and peptides.
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Opfindelsen vil blive forklaret yderligere under henvisning til tegningen, på hvilken fig. 1 viser konstruktion af plasmiderne pDN691, pDN770, pDN820 og pDNl050, 5 fig. 2 viser konstruktionen af pDN1122, fig. 3 viser konstruktionen af plasmiderne pDN1090, pDNll20, pDNl222 og pDNl277 og et kort for restriktionsenzymsites på pDNlOOO, fig. 4 viser konstruktionen af plasmiderne pDNll30 og 10 pDNl290, fig. 5 viser konstruktionen af plasmid pDNl274, og fig. 6 viser konstruktionen af plasmid pDNl800.The invention will be further explained with reference to the drawing, in which 1 shows the construction of plasmids pDN691, pDN770, pDN820 and pDN1050; 2 shows the construction of pDN1122; FIG. Figure 3 shows the construction of plasmids pDN1090, pDN1120, pDN1222 and pDN12277 and a restriction enzyme sites map on pDN1010. 4 shows the construction of plasmids pDN1130 and 10 pDN1290; 5 shows the construction of plasmid pDN1274, and fig. 6 shows the construction of plasmid pDN1800.
dal+ genet blev opnået fra DN497, der er et derivat af 15 Bacillus subtilis 168 (Spizizen, Proc.Natl.Acad.Sci. 44, 1072 -78, 1958). Kromosomal DNA blev fuldstændig fordøjet med passende restriktionsenzymer og splejset med et plasmid pDN691, der udviser resistens overfor chloramphenicol og kanamycin og er i stand til at replikere i B. subtilis. Det splejsede DNA blev overført i 20 en D-alaninkrævende B. subtilis dal-1, idet der selekteres for komplementeringen af D-alaninkravet. En Dal+ transformant, derThe dal + gene was obtained from DN497, a derivative of Bacillus subtilis 168 (Spizizen, Proc.Natl.Acad.Sci. 44, 1072 -78, 1958). Chromosomal DNA was completely digested with appropriate restriction enzymes and spliced with a plasmid pDN691 which exhibits resistance to chloramphenicol and kanamycin and is able to replicate in B. subtilis. The spliced DNA was transferred into a D-alanine-requiring B. subtilis dal-1, selecting for the complementation of the D-alanine requirement. A Dal + transformant that
OISLAND
samtidig er blevet Cam (chloramphenicolresistent) indeholdt et rekombinant plasmid afledt fra pDN691, på hvilket Dal+- og CamR-pheriotyperne er forbundet. Sandsynligvis på grund af homolog 25 rekombination mellem kromosom og plasmid kunne kun ubetydelige mængder af plasmidet påvises i transformanten.at the same time, Cam (chloramphenicol resistant) has contained a recombinant plasmid derived from pDN691 to which the Dal + and CamR pheriotypes are linked. Probably due to homologous recombination between chromosome and plasmid, only negligible amounts of the plasmid could be detected in the transformant.
Den foreliggende opfindelse tillader dyrkning af transformerede bakterier.i antibiotikafri medier. Det er imidlertid underforstået, at dette ikke er en betingelse for opfindelsens 30 gennemførlighed kun en konsekvens deraf. Hvis det skulle blive nyttigt eller foretrukket at dyrke de transformerede bakterier ifølge opfindelsen i nærvær af antibiotika, kan dette naturligvis gøres.The present invention allows the cultivation of transformed bacteria in antibiotic-free media. However, it is to be understood that this is not a condition of the feasibility of the invention but a consequence thereof. Of course, if it is to be useful or preferred to grow the transformed bacteria of the invention in the presence of antibiotics, this can be done.
For at undgå rekombination og efterfølgende integration 35 af plasmidet i kromosomet blev plasmid fremstillet fra denne Dal+ R — og Cam transformant og transformeret ind i en stamme DN733 dal recE , der kræver D-alanin og ikke har tilbøjelighed til rekom- 7To avoid recombination and subsequent integration of the plasmid into the chromosome, plasmid was prepared from this Dal + R and Cam transformant and transformed into a strain DN733 dal recE requiring D-alanine and not prone to recombination.
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bination, idet der selekteredes for Dal+. Transformanterne blev “I* R Rbination, selecting for Dal +. The transformants became “I * R R
samtidig Dal Cam og Kan (kanamycinresistente). recE-genet er et gen, der .er til stede i normale B. subtilis stammer. Det udtrykker et enzym, der tillader genetisk rekombination mellem 5 homologe (dvs. ens eller næsten ens) DNA-sekvenser. (Dubnau D. et al., J.Bacteriol. 117 (1974), 488 - 493).at the same time Dal Cam and Kan (kanamycin resistant). The recE gene is a gene present in normal B. subtilis strains. It expresses an enzyme that allows genetic recombination between 5 homologous (i.e., similar or nearly identical) DNA sequences. (Dubnau D. et al., J. Bacteriol. 117 (1974), 488-493).
Transformanterne in.deholdt en lille mængde af et plasmid på ca. 16 kb. Fra dette plasmid blev et 2,0 kb Clal-Sphl-fragment, der udviser en Dal+ phenotype klonet på et 2,6 kb 10 fragment på plasmid pDN820: Cam i stamme DN608: dal , idet der selekteres for Cam Dal , hvorved der blev opnået et rekombinant plasmid pDNlOOO på 4,6 kb. Dette plasmid kunne imidlertid erstat-te dal-1 mutationen på kromosomet af .rekombinationsdygtige stammer (f.eks. DN608) med dal+ allelet ved homolog rekombination, 15 hvorpå selekteringen af en Dal phenotype uden samtidig Cam selektering ikke mere vil sikre bevarelsen af plasmidet.The transformants contained a small amount of a plasmid of ca. 16 kb. From this plasmid, a 2.0 kb Clal-Sphl fragment exhibiting a Dal + phenotype was cloned onto a 2.6 kb 10 fragment on plasmid pDN820: Cam in strain DN608: dal, selecting for Cam Dal, obtained a 4.6 kb recombinant plasmid pDN1000. However, this plasmid could replace the dal-1 mutation on the chromosome of recombinant strains (eg DN608) with the dal + allele by homologous recombination, whereby the selection of a Dal phenotype without simultaneous Cam selection will no longer ensure the preservation of the plasmid.
For at forebygge overførsel af dal+ allelet til kromosomet ved homolog rekombination og efterfølgende tab af plasmid og for at formindske frekvensen af mutationer, der forårsager en 20 Dal+ phenotype, blev der udført en deletion i både dal genet i værten og i et nabostillet segment i værtskromosomet: det klonede dal gen blev skåret med restriktionsenzymer EcoRl og EcoR5 og derpå fordøjet med exonuclease Bal31. Den fordøjede blanding blevTo prevent transmission of the dal + allele to the chromosome by homologous recombination and subsequent loss of plasmid and to reduce the frequency of mutations causing a 20 dal + phenotype, a deletion was performed in both the dal gene in the host and in a neighboring segment of the host chromosome. : The cloned valley gene was cut with restriction enzymes EcoRl and EcoR5 and then digested with exonuclease Bal31. The digested mixture became
splejset og transformeret ind i DN608 dal , idet der blev selek-Rspliced and transformed into the DN608 valley, becoming Selek-R
25 teret for Cam . Transformanter, der indeholdt plasmider med dele- _ tioner i dal genet, blev identificeret som Dal Cam . For at konstruere en værtsstamme med den passende deletion i dal genet, blev en dal+ vært transformeret med en af deletionsplasmiderne, R _ p pDN1274 Cam dal . Idet der selekteredes for Cam , var ca. 0,1% 30 af transformanterne Dal- antagelig på grund af erstatningen af det kromosomale dal+ gen med dal deletionen fra pDNl274 ved homolog rekombination. Efter spontant tab af pDNl274 under dyrkningen i nærværelse af D-alanin og i fravær af chloramphenicol forblev den plasmidfrie stamme DN1280 Dal-. Southern blotting 35 analyse af chromosomal DNA fra DN1280 viste, at den faktisk indeholdt den forventede dal deletion. Denne dal- deletionsvært, stamme DN1280, blev transformeret med et dal+ plasmid (f.eks.25 for Cam. Transformants containing plasmids with deletions in the dal gene were identified as Dal Cam. To construct a host strain with the appropriate deletion in the dal gene, a dal + host was transformed with one of the deletion plasmids, R _ p pDN1274 Cam dal. When selected for Cam, approx. 0.1% 30 of the transformants are Dal- probably due to the replacement of the chromosomal dal + gene with the dal deletion from pDN1274 by homologous recombination. Following spontaneous loss of pDN1274 during culture in the presence of D-alanine and in the absence of chloramphenicol, the plasmid-free strain DN1280 Dal-. Southern blotting analysis of chromosomal DNA from DN1280 revealed that it actually contained the expected dal deletion. This valley deletion host, strain DN1280, was transformed with a dal + plasmid (e.g.
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pDNl090), der indeholder både hele dal+.genet og segmenter homologe til det DNA, der flankerer begge sider af deletionen på kromosomet. Overførsel af dal+ genet fra plasmid til kromosom ved homolog rekombination kunne derfor finde sted, og værten kunne *Φ* o 5 omdannes i dal . Bevarelse af plasmidet var saledes ikke længere en betingelse for en Dal+ phenotype, og plasmidet blev hyppigt tabt. Et dal plasmid pDN1277, indeholdende hele det funktionelle dal+ gen, men ikke DNA homologt til det DNA, der flankerer deletionen i værtskromosomet som forklaret ovenfor, blev derpå kon-10 strueret. Overførsel af dal+ fra plasmid til kromosom ved homolog rekombination kunne følgelig ikke finde sted efter transformation af dal deletionsværten ved pDNl277. DN1280 er således en velegnet dal vært i D-alaninfrie medier for dal plasmider, der ikke indeholder DNA omfattende EcoR5 sitet, der normalt flankerer dal 15 genet på kromosomet, men som er blevet fjernet i DN1280.pDN1090) containing both the whole dal + gene and segments homologous to the DNA flanking both sides of the deletion on the chromosome. Therefore, the transfer of the dal + gene from plasmid to the chromosome by homologous recombination could take place and the host could be transformed into dal. Thus, preservation of the plasmid was no longer a condition of a Dal + phenotype and the plasmid was frequently lost. A dal plasmid pDN1277, containing the entire functional dal + gene but not DNA homologous to the DNA flanking the deletion in the host chromosome as explained above, was then constructed. Consequently, transfer of dal + from plasmid to chromosome by homologous recombination could not occur after transformation of the dal deletion host by pDN1277. Thus, DN1280 is a suitable dal host in D-alanine-free media for dal plasmids that do not contain DNA comprising the EcoR5 site, which normally flanks the dal 15 gene on the chromosome but has been removed in DN1280.
For at sikre, at klonede gener af videnskabelig eller kommerciel interesse, virkelig kan bevares på plasmider i D-alaninfrie medier ved hjælp af det ovennævnte værtsvektorsystem, blev genet for den maltogene amylase fra Bacillus C599 (NCIB 20 11837) overført til et dal+ plasmid. To plasmider pDNH30 og pDNl290 blev konstrueret. Begge plasmider indeholder replika-tionsfunktionerne fra plasmid pUBlllO og funktionelle dal og amyM gener. Ingen af plasmiderne udviser resistens mod nogen antibiotika. Plasmiderne pDNH30 og pDNl290 blev transformeret i den 25 ovennævnte B. subtilis stamme DN1280. Stamme DN1280 indeholder som nævnt ovenfor en kromosomal deletion, der omfatter både en del af dal genet nødvendig for udtrykkelse af Dal+ phenotypen og et tilstødende segment, der ikke kræves for Dal+ phenotypen, hvad enten det er en del af dal genet senso strictu eller ej. Denne 30 sidste del af deletionen er ikke indeholdt i pDN1290. Følgelig kan dal genet ikke overføres fra pDN1290 til kromosomet ved dobbelhomolog rekombination. Plasmid pDNH30 indeholder på den anden side både hele segmentet, der er fjernet på kromosomet, og tilstødende segmenter. Følgelig kan overførsel af dal+ genet fra 35 plasmid til kromosom og efterfølgende plasmidtab finde sted.To ensure that cloned genes of scientific or commercial interest can really be conserved on plasmids in D-alanine-free media by the above host vector system, the gene for the Bacillus C599 maltogenic amylase (NCIB 20 11837) was transferred to a dal + plasmid. Two plasmids pDNH30 and pDNl290 were constructed. Both plasmids contain the replication functions of plasmid pUB110 and functional dal and amyM genes. None of the plasmids exhibit resistance to any antibiotics. The plasmids pDNH30 and pDNl290 were transformed into the aforementioned B. subtilis strain DN1280. Strain DN1280, as mentioned above, contains a chromosomal deletion that includes both a portion of the dal gene required for expression of the Dal + phenotype and an adjacent segment not required for the Dal + phenotype, whether or not part of the dal gene senso strictu. This last 30 part of the deletion is not contained in pDN1290. Accordingly, the dal gene cannot be transferred from pDN1290 to the chromosome by double homologous recombination. Plasmid pDNH30, on the other hand, contains both the entire segment removed on the chromosome and adjacent segments. Accordingly, transfer of the dal + gene from 35 plasmid to chromosome and subsequent plasmid loss can occur.
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De opnåede transformerede stammer DN1297 (=DN1280 pDNll30) og DN1300 (=DN1280 pDNl290) blev testet for plasmidsta-bilitet under dyrkningen (jfr. eksempel 7). Under dyrkning af DN1297 skete der homolog overkrydsning, der gendannede dal'1' genet 5 på kromosomet, og både plasmidet og Amy+ phenotypen blev hyppigt tabt.The transformed strains obtained DN1297 (= DN1280 pDN1130) and DN1300 (= DN1280 pDN1290) were tested for plasmid stability during culture (cf. Example 7). During the cultivation of DN1297, homologous crossover occurred, which restored the dal'1 'gene 5 on the chromosome, and both the plasmid and Amy + phenotype were frequently lost.
Ved dyrkning af DN1300 blev der ikke observeret noget tab af plasmid- Amy+ phenotype under dyrkningen i et medium uden D-alanin. Hvis der blev sat D-alanin til mediet, tabte cellerne 10 imidlertid ofte plasmidet og blev Amy Dal”. Stabil bevarelse af et plasmid, der indeholder et gen, der ikke hidrører fra værtsorganismen, af kommerciel interesse i antibiotikafrit medium blev således demonstreret.When culturing DN1300, no loss of plasmid Amy + phenotype was observed during culture in a medium without D-alanine. However, if D-alanine was added to the medium, cells 10 often lost the plasmid and became Amy Dal ”. Thus, stable conservation of a plasmid containing a gene not derived from the host organism of commercial interest in antibiotic-free medium was demonstrated.
For at demonstrere at plasmider også kan bevares i 15 gram-negative organismer ved undertrykkelse af et D-alaninkrav, blev dal genet fra B. subtilis klonet på et plasmid i en D-alaninkrævende Escherichia coli mutant og vist at kunne komplementere kravet.To demonstrate that plasmids can also be conserved in 15 gram-negative organisms by suppressing a D-alanine requirement, the dal gene from B. subtilis was cloned onto a plasmid in a D-alanine-requiring Escherichia coli mutant and shown to complement the requirement.
Opfindelsen skal forklares yderligere ved hjælp af de 20 efterfølgende eksempler.The invention will be further explained by the following 20 examples.
Fremstilling af plasmider og kromosomalt DNA og transformering af Bacillus subtilis og E. coli skete ifølge de følgende generelle procedurer. Fordøjelse med restriktionsenzymerne, Bal31 nucleasebehandling, oligo-DNA-linker indføring og splejs-25 ning med T4-ligase af DNA blev udført med enzymer fra New England Biolabs under de af forhandlerne anbefalede betingelser.Production of plasmids and chromosomal DNA and transformation of Bacillus subtilis and E. coli were performed according to the following general procedures. Digestion with the restriction enzymes, Bal31 nuclease treatment, oligo-DNA linker insertion and splicing with T4 ligase of DNA was performed with enzymes from New England Biolabs under the conditions recommended by the dealers.
StammerOriginates
Alle stammer var derivater af Bacillus subtilis 168 30 (Spizizen, Proc.Natl.Acad.Sci., 44: 1072-78, 1958) RUB200: aroI906, amyE07, amyR2 blev opnået fra Dr. Frank Young,All strains were derivatives of Bacillus subtilis 168 (Spizizen, Proc.Natl.Acad.Sci., 44: 1072-78, 1958) RUB200: aroI906, amyE07, amyR2 was obtained from Dr. Frank Young,
University of Rochester, New York. SL438:trpC2 (sporulerings- og protease-deficient) blev opnået fra Dr. Kim Hardy, Biogen, 4"University of Rochester, New York. SL438: trpC2 (sporulation and protease deficient) was obtained from Dr. Kim Hardy, Biogen, 4 "
Geneve. DN497: amyE07, amyR2 er en aro transformant af RUB200 35 med kromosomal DNA fra SL438, QB1133: aroI906, metB5, sacA321, amyE var fra Dr. Georges Rapoport, IRBM, Paris. QB1130: dal, metB5, sacA331, amyE' blev opnået fra Bacillus Genetic Stock 10Geneva. DN497: amyE07, amyR2 is an aro transformant of RUB200 35 with chromosomal DNA from SL438, QB1133: aroI906, metB5, sacA321, amyE was from Dr. Georges Rapoport, IRBM, Paris. QB1130: valley, metB5, sacA331, amyE 'was obtained from Bacillus Genetic Stock 10
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Centret, Columbus, Ohio. DN608: dal-1, metB, sacA, amyE var en aro+, dal-1 transformant af QB1133 med kromosomal DNA fra QB1130.Center, Columbus, Ohio. DN608: dal-1, metB, sacA, amyE was an aro +, dal-1 transformant of QB1133 with chromosomal DNA from QB1130.
MT120: leuB6, recE4 r m blev opnået fra Dr. Teruo Tanaka, - -----— mmMT120: leuB6, recE4 r m was obtained from Dr. Teruo Tanaka, - -----— mm
Mitsubishi-Kasei Institute of Life Sciences, Tokyo. DN773: dal-1, 5 amyE, recE, sacA var en met+ recE transformant af DN608 med kromosomal DNA fra MT120. DN606 er DN608 transformeret med plasmid pUBllO.Mitsubishi-Kasei Institute of Life Sciences, Tokyo. DN773: dal-1,5 amyE, recE, sacA was a met + recE transformant of DN608 with chromosomal DNA from MT120. DN606 is DN608 transformed with plasmid pUB110.
Escherichia coli stamme TKL10: thr-1 leuB6 codAl trp64 pyrFlOl his-108 thyA6 argG66 ilvA634 thi-1 alr-1 deoCl lacYl 10 tonA21 tsx95 supE44 (Wijsman, Genet.Res., Camb. 20: 269-77, 1972) blev opnået fra Dr. Barbara Bachmann E. coli Genetic Stock Center Connecticut, U.S.A. (CGSC 5466).Escherichia coli strain TKL10: thr-1 leuB6 codAl trp64 pyrFlOl his-108 thyA6 argG66 ilvA634 thi-1 alr-1 deoCl lacYl 10 tonA21 tsx95 supE44 (Wijsman, Genet.Res., Camb. 20: 269-77, 1972) was obtained. from Dr. Barbara Bachmann E. coli Genetic Stock Center Connecticut, U.S.A. (CGSC 5466).
Plasmider 15 pUC9 på 2,7 kb udviser resistens mod ampicillin og blev _ opnået fra pBR322 (Vieira et al., Gene 19: 259 - 68, 1982) pBR322 på 4,4 kb udviser resistens mod ampicillin og tetracyclin (Bolivar et al., Gene 2: 95 - 113, 1977).Plasmids 15 pUC9 of 2.7 kb exhibit resistance to ampicillin and were obtained from pBR322 (Vieira et al., Gene 19: 259-68, 1982) pBR322 of 4.4 kb exhibits resistance to ampicillin and tetracycline (Bolivar et al. , Gene 2: 95 - 113, 1977).
Plasmider pUBllO og pBD64 (Gryczan et al., J.Plasmids pUB110 and pBD64 (Gryczan et al., J.
20 Bacteriol. 134: 318 - 329, 1978, og Gryczan et al., J. Bacteriol. 141: 246 - 53, 1980) blev henholdsvis isoleret fra B. subtilis stamme BD366 og BD624. pUBllO og pBD64-udviser begge resistens mod kanamycin og pBD64 også mod chloramphenicol. B. subtilis stammer BD366 og BD624 kan fås fra Bacillus Genetic Stock Center, 25 Columbus, Ohio, USA (stamme nr. BGSC 1E6 og 1E22). Plasmid pDN452 på 7,6 kb udvoser resistens mod chloramphenicol og kanamycin og indeholder det strukturelle gen for en maltogen amylase fra Bacillus subtilis NCIB 11837. Konstruktionen af pDN452 er beskrevet i publiceret EP patent ansøgning nr. 120.693.Bacteriol. 134: 318-329, 1978, and Gryczan et al., J. Bacteriol. 141: 246-53, 1980) were isolated from B. subtilis strain BD366 and BD624, respectively. pUB110 and pBD64 both exhibit resistance to kanamycin and pBD64 also to chloramphenicol. B. subtilis strains BD366 and BD624 are available from Bacillus Genetic Stock Center, 25 Columbus, Ohio, USA (strain Nos. BGSC 1E6 and 1E22). 7.6 kb plasmid pDN452 exerts resistance to chloramphenicol and kanamycin and contains the structural gene for a maltogenic amylase from Bacillus subtilis NCIB 11837. The construction of pDN452 is described in published European Patent Application No. 120,693.
30 I._Transformering af B. subtilis30 I._Transformation of B. subtilis
Kompetente Bacillus subtilis celler blev fremstillet ifølge Yasbin et al (J. Bacteriol. 21: 296 - 304, 1975). Cellerne blev derpå høstet ved centrifugering(7000 rpm, 3 min.), resuspen-35 deret i 1/10 rumfang af supernatanten indeholdende 20% glycerol, frosset i flydende nitrogen og opbevaret ved -70°C. Til transformering blev frosne celler optøet ved 42°C og blandet med 1 rum- 11Competent Bacillus subtilis cells were prepared according to Yasbin et al (J. Bacteriol. 21: 296-304, 1975). The cells were then harvested by centrifugation (7000 rpm, 3 min), resuspended in 1/10 volume of the supernatant containing 20% glycerol, frozen in liquid nitrogen and stored at -70 ° C. For transformation, frozen cells were thawed at 42 ° C and mixed with 1 room-11
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fang puffer (Spizizen's minimal medium (Spizizen, Proc.Natl.Acad. Sci. USA 44: 1072 - 78, 1958)) med 0,4% glucose, 0,04 M MgCl2 og 0,002 m EGTA). DNA blev tilsat, og blandingen blev inkuberet under omrystning ved 37°C i 20 minutter. Cellerne blev derpå 5 udspredt på egnede selekteringsmedier.capture buffer (Spizizen's minimal medium (Spizizen, Proc.Natl.Acad. Sci. USA 44: 1072-78, 1958)) with 0.4% glucose, 0.04 M MgCl 2 and 0.002 m EGTA). DNA was added and the mixture was incubated with shaking at 37 ° C for 20 minutes. The cells were then spread on suitable selection media.
II. _Transformering af E. coliII. Transformation of E. coli
En kultur af E. coli K-12 stamme nr. 802, der var opbevaret natten over i LB (10 g Bactotrypton, 5 g Bacto gærekstrakt 10 og 10 g NaCl per liter vand, pH 7,0) blev fortyndet 100 gange i 5 00 ml LB og dyrket ved 3 7°C til OD^q = 0,4. Kulturen blev afkølet, anbragt 15 minutter på is, centrifugeret i 15 minutter ved 3000 rpm (i en Sorvall GS3 rotor), resuspenderet i 200 ml kold 0,1 M CaCl2, anbragt på is i 20 minutter, centrifugeret i 10 15 minutter ved 3000 rpm, resuspenderet i 5 ml kold 0,1 M CaCl2 °9 anbragt på is i 20 timer. Derpå blev der tilsat kold glycerol til et indhold på 10%, og prøver blev frosset i flydende nitrogen og opbevaret ved -70°C. Frosne celler blev optøet på is, DNA blev tilsat, blandingen blev inkuberet i 45 minutter på is, i 2 minut-20 ter ved 37°C og blev derpå udspredt på et egnet selektionsmedium.A culture of E. coli K-12 strain # 802 stored overnight in LB (10 g Bactotrypton, 5 g Bacto yeast extract 10 and 10 g NaCl per liter of water, pH 7.0) was diluted 100 times in 5 00 ml of LB and grown at 37 ° C to OD ^ q = 0.4. The culture was cooled, placed 15 minutes on ice, centrifuged for 15 minutes at 3000 rpm (in a Sorvall GS3 rotor), resuspended in 200 ml cold 0.1 M CaCl2, placed on ice for 20 minutes, centrifuged for 10 15 minutes at 3000 rpm, resuspended in 5 ml of cold 0.1 M CaCl 2 ° 9 placed on ice for 20 hours. Then, cold glycerol was added to a content of 10% and samples were frozen in liquid nitrogen and stored at -70 ° C. Frozen cells were thawed on ice, DNA was added, the mixture was incubated for 45 minutes on ice, for 2 minutes at 37 ° C and then spread on a suitable selection medium.
III. _Fremstilling af plasmider fra E. coli E. coli blev dyrket natten Over i 250 ml LB, 0,4% glucose og et passende antibiotikum. Cellerne blev høstet ved 25 centrifugering og resuspenderet i 4 ml puffer 1 (0,025 M Tris-HCl, pH = 8,0, 0,01 M EDTA, 0,05 M glucose, 2 mg/ml lysozym). Suspensionen blev inkuberet ved 0°C i 15 minutter og derpå blandet med 8 ml puffer 2 (0,2 M NaOH, 1% SDS). Derpå blev der tilsat 6 ml puffer 3 (3M NaAcetat, pH = 4,8), og blandingen blev holdt 30 ved 0°C i 60 minutter efterfulgt af centrifugering i 20 minutter ved 19000 rpm (ca. 45000 g i Sorvall SS34 rotor). Supernatanten blev udfældet med 0,6 rumfang kold isopropanol og resuspenderet i 1,2 ml 5TE (0,05 M Tris-HCl, pH = 8,0, 0,005 M EDTA), plus 20 μΐ kogt RNase A (Boehringer) (2 mg/ml). 30 minutter senere blev 35 opløsningen udspredt på toppen af 4,0 ml puffer 4 (80 g CsCl plus 56 ml 5TE) og 0,1 ml EtBr (10 mg/ml ethidium bromid) i et VTi65III. Production of plasmids from E. coli E. coli was grown overnight in 250 ml of LB, 0.4% glucose and an appropriate antibiotic. Cells were harvested by centrifugation and resuspended in 4 ml of buffer 1 (0.025 M Tris-HCl, pH = 8.0, 0.01 M EDTA, 0.05 M glucose, 2 mg / ml lysozyme). The suspension was incubated at 0 ° C for 15 minutes and then mixed with 8 ml of buffer 2 (0.2 M NaOH, 1% SDS). Then 6 ml of buffer 3 (3M NaAcetate, pH = 4.8) was added and the mixture was kept at 0 ° C for 60 min followed by centrifugation for 20 min at 19000 rpm (about 45000 g in Sorvall SS34 rotor). The supernatant was precipitated with 0.6 volumes of cold isopropanol and resuspended in 1.2 ml of 5TE (0.05 M Tris-HCl, pH = 8.0, 0.005 M EDTA), plus 20 μΐ of boiled RNase A (Boehringer) (2 mg / ml). Thirty minutes later, the solution was spread on top of 4.0 ml of buffer 4 (80 g of CsCl plus 56 ml of 5TE) and 0.1 ml of EtBr (10 mg / ml of ethidium bromide) in a VTi65
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12 rør. Blandingen blev derpå centrifugeret ved 45000 rpm i 20 timer. Plasmid blev derpå fjernet fra røret, dialyseret og ekstraheret som beskrevet i sektion VI.12 tubes. The mixture was then centrifuged at 45000 rpm for 20 hours. Plasmid was then removed from the tube, dialyzed and extracted as described in section VI.
5 IV._Fremstilling af plasmider fra B. subtilis5 IV._ Production of plasmids from B. subtilis
Plasmiderne blev fremstillet som beskrevet for E. coli stammer (se sektion III), men med følgende modifikationer. Dyrkning blev foretaget i LB indholdende 0,01 M kaliumphosphat, pH = 7,0 og et passende antibiotikum (f.eks. 6 μg/ml chloramphenicol) 10 og hvis krævet 100 μg/ml D-alanin. Efter høst blev cellerne inkuberet ved 37°C med lysozym. Puffer 2 blev erstattet af en blanding af et rumfang puffer 2 og tre rumfang puffer 5 (0,2 M glycin, 0,2 M NaCl og 1% SDS). De følgende trin var de samme som i III.The plasmids were prepared as described for E. coli strains (see section III) but with the following modifications. Cultivation was done in LB containing 0.01 M potassium phosphate, pH = 7.0 and an appropriate antibiotic (eg 6 µg / ml chloramphenicol) 10 and if required 100 µg / ml D-alanine. After harvest, the cells were incubated at 37 ° C with lysozyme. Buffer 2 was replaced by a mixture of one volume of buffer 2 and three volumes of buffer 5 (0.2 M glycine, 0.2 M NaCl and 1% SDS). The following steps were the same as in III.
15 V. _Fremstilling i lille skala af plasmider fra B. subtilis15 V. Small-scale production of plasmids from B. subtilis
Plasmid fra 5 ml B. subtilis i LB (indeholdende 0,01 M phosphat pH = 7,0 og passende antibiotika og D-alanin hvis krævet) blev fremstillet som beskrevet i sektion IV med undtagelse 20 af, at 1: puffernes rumfang blev reduceret fire gange, 2: 0,5 ml phenol og 0,5 ml chloroform blev tilsat efter puffer 3, 3: efter centrifugering ved 19000 rpm, blev supernatanten udfældet med ethanol, resuspenderet i 400 μΐ puffer 6 (0,05 M Tris-HCl pH = 8,0, 0,1 M NaAcetat), plasmidet blev igen udfældet, resuspenderet 25 i 400 μΐ puffer 6, udfældet, vasket og resuspenderet i 100 μΐ TE (0,01 M Tris- HCl, pH = 8,0, 0,001 M EDTA) med 1 μg/ml kogt RNase A (Boehringer).Plasmid from 5 ml of B. subtilis in LB (containing 0.01 M phosphate pH = 7.0 and appropriate antibiotics and D-alanine if required) was prepared as described in section IV except that 1: the volume of the buffers was reduced four times, 2: 0.5 ml phenol and 0.5 ml chloroform were added after buffer 3, 3: after centrifugation at 19000 rpm, the supernatant was precipitated with ethanol, resuspended in 400 μΐ buffer 6 (0.05 M Tris-HCl pH = 8.0, 0.1 M NaAcetate), the plasmid was again precipitated, resuspended in 400 μΐ buffer 6, precipitated, washed and resuspended in 100 μΐ TE (0.01 M Tris-HCl, pH = 8.0, 0.001 M EDTA) with 1 μg / ml boiled RNase A (Boehringer).
VI. Fremstilling af kromosomalt DNA fra B. subtilis 30 En pille af frosne celler fra ca. 50 ml kultur blevWE. Preparation of B. subtilis chromosomal DNA A pellet of frozen cells from ca. 50 ml of culture was
resuspenderet i 1,1 ml puffer (0,05 M Tris-HCl, pH = 7,4, 0,1 M NaCl, 25% sucrose). 100 μΐ lyzosym (25 mg/ml) og 150 μΐ EDTA (0,5 M, pH = 8,0) blev tilsat. Blandingen blev inkuberet ved 37°C i 30 minutter, og 2 ml 0,2% SDS blev tilsat efterfulgt af inkubering i 35 30 minutter ved 37°C. 1 g CsCl og 0,05 ml EtBr (10 mg/ml) blev tilsat per 0,95 ml blanding, og blandingen blev centrifugeret ved 45000 rpm ved 15°C i 20 timer i en VTi65 rotor (Beckman). DNAresuspended in 1.1 ml buffer (0.05 M Tris-HCl, pH = 7.4, 0.1 M NaCl, 25% sucrose). 100 μΐ lyzosym (25 mg / ml) and 150 μΐ EDTA (0.5 M, pH = 8.0) were added. The mixture was incubated at 37 ° C for 30 minutes and 2 ml of 0.2% SDS was added followed by incubation for 35 30 minutes at 37 ° C. 1 g of CsCl and 0.05 ml of EtBr (10 mg / ml) were added per 0.95 ml of mixture and the mixture was centrifuged at 45000 rpm at 15 ° C for 20 hours in a VTi65 rotor (Beckman). DNA
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13 blev lokaliseret under en langbølge UV lampe og fjernet ved punktering af røret med en nål. EtBr blev ekstraheret med isopro-panol, og opløsningen blev dialyseret i 2 timer mod TEE (0,01 M Tris-HCl, pH = 8,0, 0,01 M EDTA). Opløsningen blev derpå indstil-5 let til 8 ml med TEE og ekstraheret to gange med phenol og en gang chloroform. DNA blev udfældet med 0,1 M NaCl og kold ethanol og opløst i 1 ml TE (0,01 M Tris-HCl, pH = 8,0, 0.001 M EDTA). Opløsningen af kromosomalt DNA blev opbevaret ved 4°C.13 was located under a long wave UV lamp and removed by puncturing the tube with a needle. EtBr was extracted with isopropanol and the solution was dialyzed for 2 hours against TEE (0.01 M Tris-HCl, pH = 8.0, 0.01 M EDTA). The solution was then adjusted to 8 ml with TEE and extracted twice with phenol and once chloroform. DNA was precipitated with 0.1 M NaCl and cold ethanol and dissolved in 1 ml TE (0.01 M Tris-HCl, pH = 8.0, 0.001 M EDTA). The chromosomal DNA solution was stored at 4 ° C.
10 Eksempel 1Example 1
Konstruktion af plasmid pDNl050 (fig. 1)Construction of plasmid pDN1050 (Fig. 1)
Plasmid pBD64 blev skåret med restriktionsenzymer EcoRl og Sphl, og et 3,6 kb fragment blev splejset til et 0,56 kb EcoRl-Sphl fragment fra E. coli plasmid pBR322. Det fremkomne 15 plasmid pDN691 på 4,2 kb udviste chloramphenicol- og kanamycin-resistens. Et 0,4 kb Hind3-BamHl fragment af pDN691 blev erstattet med et 0,02 kb Hind3-BamHl fragment fra E. coli plasmid pUC9. Det fremkomne plasmid pDN720 på 3,8 kb udviste chloramphenicol-og kanamycinresistens.Plasmid pBD64 was cut with restriction enzymes Eco RI and SphI, and a 3.6 kb fragment was spliced to a 0.56 kb Eco RI-SphI fragment from E. coli plasmid pBR322. The resulting 4.2 kb plasmid pDN691 showed chloramphenicol and kanamycin resistance. A 0.4 kb Hind3-BamH1 fragment of pDN691 was replaced with a 0.02 kb Hind3-BamH1 fragment from E. coli plasmid pUC9. The resulting 3.8 kb plasmid pDN720 exhibited chloramphenicol and kanamycin resistance.
20 Et 2,8 kb Ncol-Ncol-fragment af pDN720 blev erstattet med et 1,8 kb Ncol-Ncol-fragment fra pDN770.'pDN770 på 3,6 kb var en spontant deletion af pBD64 og udviste chloramphenicolresi-stens. Det fremkomne plasmid pDN820 på 2,8 kb udviser chlor-amphenicolresistens. pDN820 blev åbnet ved det enkelte HgiAl site 25 og fordøjet med Bal31 i 30 sekunder ved 30°C, hvorved der blev fjernet et fragment på 0,1 kb. De opnåede lineære fragmenter blev splejset med en Bgl2 oligonucleotidlinker fra New England Nuclear (nr. 1001). Plasmid pDNl050 på 2,7 kb indeholdende en Bgl2 linker og udvisende chloramphenicolresistens blev isoleret 30 fra ligeringsblandingen.A 2.8 kb NcoI-NcoI fragment of pDN720 was replaced with a 1.8 kb NcoI-NcoI fragment from pDN770. The 3.6 kb pDN770 was a spontaneous deletion of pBD64 and exhibited chloramphenicol resistance. The resulting 2.8 kb plasmid pDN820 exhibits chlorine-amphenicol resistance. pDN820 was opened at the single HgiAl site 25 and digested with Bal31 for 30 seconds at 30 ° C, removing a 0.1 kb fragment. The obtained linear fragments were spliced with a Bgl2 oligonucleotide linker from New England Nuclear (no. 1001). Plasmid pDN1050 of 2.7 kb containing a Bgl2 linker and exhibiting chloramphenicol resistance was isolated from the ligation mixture.
Eksempel 2Example 2
Konstruktion af plasmid pDN1122 (fig. 2)Construction of plasmid pDN1122 (Fig. 2)
Plasmid pDN452 på 7,6 kb og udvisende chloramphenicol-35 og kanamycinresistens og indeholdende det strukturelle gen amyM for en maltogen amylase fra Bacillus C599 blev fordøjet med Aval, behandlet med exonuclease Bal31, splejset med EcoRl oligonucleo- 147.6 kb plasmid pDN452 and exhibiting chloramphenicol-35 and kanamycin resistance and containing the structural gene amyM of a Bacillus C599 maltogenic amylase was digested with Aval treated with exonuclease Bal31 spliced with EcoRl oligonucleotide 14
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tidlinker (Biolab nr. 1004), fordøjet med EcoRl, splejset med T4-ligase og transformeret i B. subtilis DN497;amyE, idet der R + blev selekteret for Cam . En Amy transformant indeholdt plasmid p520-20 på 6,6 kb. Amylaseudbyttet fra p520-20 var ikke mindre 5 end udbyttet fra pDN452.time linker (Biolab # 1004), digested with EcoRl, spliced with T4 ligase and transformed into B. subtilis DN497; amyE, with R + being selected for Cam. An Amy transformant contained 6.6 kb plasmid p520-20. The amylase yield from p520-20 was not less than the yield from pDN452.
Et 2,7 kb Ncol-Ncol fragment fra p520-20 blev derpå erstattet med et 1,7 kb Ncol-Ncol fragment fra pDN770 (fremstil- ; let som i eksempel 1). Det opnåede plasmid pDN808 på 5,6 kb indeholdt amyM og udviste resistens mod chloramphenicol.A 2.7 kb NcoI-NcoI fragment from p520-20 was then replaced with a 1.7 kb NcoI-NcoI fragment from pDN770 (prepared as in Example 1). The 5.6 kb plasmid pDN808 obtained contained amyM and exhibited resistance to chloramphenicol.
10 Et 2,6 kb EcoRl-Sphl-fragment fra pDN808 blev erstattet med et 2,4 EcoRl-Sphl-fragment fra pDN1050 (fremstillet som beskrevet i eksempel 1). Det fremkomne plasmid pDNH22 på 5,4 kb indeholdt amyM og udviste resistens mod chloramphenicol.A 2.6 kb EcoRl-Sphl fragment from pDN808 was replaced with a 2.4 EcoRl-Sphl fragment from pDN1050 (prepared as described in Example 1). The resulting 5.4 kb plasmid pDNH22 contained amyM and exhibited resistance to chloramphenicol.
15 Eksempel 3Example 3
Kloning af dal genetCloning of the valley gene
Ca. 3 μg kromosomal DNA fra B. subtilis stamme DN497 og R RCa. 3 μg of chromosomal DNA from B. subtilis strain DN497 and R R
1 μΘ plasmid pDN691 Cam Kan blev fuldstændig fordøjet med restriktionsenzymer BamHl og Sphl. Kromosomal og plasmid DNA blev 20 blandet og splejset med T4-ligase og transformeret i stamme “ R + DN606;dal pUB110:Kan . Blandt ca. 200 Dal transformanter var en R + samtidig blevet Cam som en indikation af, at Dal phenotypen var forbundet til et rekombinant plasmid afledt fra pDN691. Plasmider T5 blev fremstillet fra denne Dal Cam transformant og transforme-25 ret ind i stammen DN773 dal recE-, idet der blev selekteret for Dal . Dal transformanter blev samtidig Cam og Kan . Resistensen mod kanamycin er indbygget i kloningsvektoren pDN691. Denne påvirkes ikke af kloningen af dal genet, således at rekombinant-plasmidet, der transformeres ind i DN733 er Dal Cam Kan .1 μΘ of plasmid pDN691 Cam Kan was completely digested with restriction enzymes BamH1 and Sphl. Chromosomal and plasmid DNA were mixed and spliced with T4 ligase and transformed into strain “R + DN606; dal pUB110: Kan. Among the approx. 200 Dal transformants had an R + simultaneously become Cam as an indication that the Dal phenotype was linked to a recombinant plasmid derived from pDN691. Plasmids T5 were prepared from this Dal Cam transformant and transformed into the strain DN773 dal recE-, selecting for Dal. Valley transformants were simultaneously Cam and Kan. The resistance to kanamycin is built into the cloning vector pDN691. This is not affected by the cloning of the dal gene, so that the recombinant plasmid transformed into DN733 is Dal Cam Kan.
30 Transformanterne indeholdt kun en meget lille mængde af et plasmid på ca. 16 kb.The transformants contained only a very small amount of a plasmid of ca. 16 kb.
Fra dette plasmid blev et 2,0 kb Clal-Sphl-fragment, der udviste en Dal+ phenotype, klonet i Clal og Sphl sitet i plasmid pDN820 i stamme DN608;dal til dannelse af et rekombinant 35 plasmid, pDNlOOO på 4,6 kb.From this plasmid, a 2.0 kb Clal-Sphl fragment exhibiting a Dal + phenotype was cloned into the Clal and Sphl site of plasmid pDN820 in strain DN608; dal to form a recombinant 35 plasmid, pDN10OO of 4.6 kb.
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Et kort for restriktionsenzymsites på pDNlOOO er vist i fig. 3. Den transformerede stamme DN1000=DN608 pDNlOOO er blevet deponeret ved The National Collection of Industrial Bacteria (NCIB), Torry Research Station, Aberdeen, Scotland, d. 7. decem-5 ber 1984 og fik referencenummeret NCIB 12029.A restriction enzyme sites map on pDN1000 is shown in FIG. 3. The transformed strain DN1000 = DN608 pDN1000 was deposited at The National Collection of Industrial Bacteria (NCIB), Torry Research Station, Aberdeen, Scotland, on December 7, 1984, and was assigned reference number NCIB 12029.
NCIB, der er et internationalt deponeringsinstitut anerkendt af Budapest traktaten af 1977, sikrer permanensen af depotet og offentlighedens adgang dertil ifølge regel 9 og 11 i denne traktat.NCIB, an international depositary recognized by the Budapest Treaty of 1977, ensures the permanence of the depository and public access thereto in accordance with Rules 9 and 11 of this Treaty.
10 De følgende iagttagelser beviser, at det klonede kromo somale fragment virkelig indeholdt dal genet (som defineret ved dal-1 mutationen) og ikke et andet gen, der er i stand til at undertrykke D-alaninkravet: 1: Lineariseret (ikke-replikativ) plasmid overførte 15 en dal modtager til Dal , men ikke til Cam som en indikation af homolog rekombination mellem dal genet på kromosomet og det klonede kromosomale fragment.10 The following observations prove that the cloned chromo somal fragment did indeed contain the dal gene (as defined by the dal-1 mutation) and not another gene capable of suppressing the D alanine requirement: 1: Linearized (non-replicative) plasmid transferred a dal recipient to dal but not to cam as an indication of homologous recombination between the dal gene on the chromosome and the cloned chromosomal fragment.
2: Ca. 0,2% af plasmider fremstillet fra en værtsbak- 20 terie, der er dal-1 på kromosomet, var dal (dog med et restriktionsenzymmønster, der ikke kunne skelnes fra det oprindelige dal+ plasmid). Disse dal plasmider kunne ikke komplementere den kromosomale dal-1 mutation som en indikation af, at den kromosomale mutation var blevet overført til dal plasmider ved homolog 25 rekombination.2: Approx. 0.2% of plasmids made from a host bacterium dal-1 on the chromosome were dal (though with a restriction enzyme pattern indistinguishable from the original dal + plasmid). These dal plasmids could not complement the chromosomal dal-1 mutation as an indication that the chromosomal mutation had been transferred to dal plasmids by homologous recombination.
3: Ved Southern blotting analyse af kromosomalt B.3: By Southern blotting analysis of chromosomal B.
subtilis DNA blev det demonstreret, at et kromosomalt Clal-Sphl-fragment hybridiserede med et fragment af samme størrelse fra 30 pDNlOOO. Der skete således ikke nogen væsentlig omarrangering af dal genet før dets kloning på pDNlOOO.subtilis DNA, it was demonstrated that a chromosomal Clal-SphI fragment hybridized with a fragment of the same size from 30 pDN1000. Thus, no significant rearrangement of the dal gene occurred prior to its cloning on pDN1000.
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Eksempel 4Example 4
Konstruktion af plasmid pDNll30 (fig. 3 og 4) pDNlOOO (fra eksempel 3) indeholdende dal+ genet blev åbnet ved Clal-sitet, fordøjet med exonuclease Bal31 og splejset 5 med en BamHl oligonucleotidlinker (nr. 1017 fra Biolabs). Det fremkomne plasmid pDN1090 indeholdt dal+ genet og udviste chlor-•amphenicolresistens.Construction of plasmid pDN1130 (Figs. 3 and 4) pDN1000 (from Example 3) containing the dal + gene was opened at the ClaI site, digested with exonuclease Bal31 and spliced 5 with a BamH1 oligonucleotide linker (no. 1017 from Biolabs). The resulting plasmid pDN1090 contained the dal + gene and exhibited chloramphenicol resistance.
pDN1122 (fra eksempel 2) blev skåret med Sphl og Bgl2, og et 4,4 kb fragment blev splejset med et 2,0 kb Sphl-BamHl 10 fragment fra pDN1090.pDN1122 (from Example 2) was cut with Sphl and Bgl2, and a 4.4 kb fragment was spliced with a 2.0 kb Sphl-BamHl 10 fragment from pDN1090.
• + +• + +
Det fremkomne plasmid pDNll30 indeholdt amyM og dal genet, men udviste ikke resistens mod chloramphenicol.The resulting plasmid pDN1130 contained the amyM and dal gene but did not show resistance to chloramphenicol.
Eksempel 5 15 Konstruktion af plasmid pDNl290 (fig. 3 og 4) " " “..... ' ' + £Example 5 Construction of plasmid pDNl290 (Figs. 3 and 4)
Plasmid pDNlllO dal Cam på 4,5 kb blev konstrueret ved exonuclease Bal31 fordøjelse af pDNlOOO fordøjet med Sphl og indføring af både en Bgl2 og en Sacl oligonucleotidlinker (henholdsvis nr. 1001 og nr. 1005 fra Biolabs).Plasmid pDN1010 dal Cam of 4.5 kb was constructed by exonuclease Bal31 digestion of pDN10OO digested with SphI and introduction of both a Bgl2 and a Sacl oligonucleotide linker (# 1001 and # 1005 from Biolabs, respectively).
20 pDNlllO blev åbnet ved det enkelte EcoR5 site, exo nuclease fordøjet med Bal31 og splejset med to Bgl2 oligonu-cleotidlinkere (nr. 1001 fra Biolabs). Fusionen af linker med en plasmidende dannede et Bell site. Det fremkomne plasmid pDNl222 indeholdt dal+ genet og udviste chloramphenicolresistens.20 pDN1110 was opened at the single EcoR5 site, exo nuclease digested with Bal31 and spliced with two Bgl2 oligonucleotide linkers (no. 1001 from Biolabs). The fusion of links with a plasmid forming a Bell site. The resulting plasmid pDN1222 contained the dal + gene and exhibited chloramphenicol resistance.
25 Et 0,7 kb Bcll-Bcll-fragment fra pDNl222 blev splejset med et 3,5 kb BamHl-Bcll fragment fra pDNl090 (fra eksempel 4).A 0.7 kb BcII-BcII fragment from pDN1022 was spliced with a 3.5 kb BamH1-BcII fragment from pDN1090 (from Example 4).
Det fremkomne plasmid pDNl277 på 4,2 kb indeholdt dal+ genet og udviste chlormaphenicolresistens. Plasmid pDNl277 blev transformeret ind i stamme DN1280 (se eksempel 6) og den fremkomne stamme 30 DN1517 (=DN1280 pDNl277) blev deponeret ved National Collection of Industrial Bacteria, Torry Research Station, Aberdeen,The resulting 4.2 kb plasmid pDNl277 contained the dal + gene and exhibited chlormaphenicol resistance. Plasmid pDN1227 was transformed into strain DN1280 (see Example 6) and the resulting strain DN1517 (= DN1280 pDN1227) was deposited at the National Collection of Industrial Bacteria, Torry Research Station, Aberdeen,
Scotland, d. 7. december 1984 og fik referencenummeret NCIB 12030.Scotland, December 7, 1984 and received reference number NCIB 12030.
pDNl090 blev yderligere omdannet til plasmid pDN1120 35 dal Cam på 4,5 kb ved erstatning af et 2,6 kb Sphl-BamHl-frag-ment med et 2,5 kb Sphl-BamHl-fragment fra pDN1050 (fremstillet som beskrevet i eksempel 1).pDN1090 was further converted to plasmid pDN1120 35 dal Cam of 4.5 kb by replacing a 2.6 kb Sphl-BamH1 fragment with a 2.5 kb Sphl-BamH1 fragment from pDN1050 (prepared as described in Example 1 ).
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+ R+ R
PDN1265 amyM Cam blev konstrueret ved Bal31 exo-nucleasefordøjelse af pDNll20 fordøjet med EcoR5 og efterfølgende fordøjelse med Sphl. Et fragment på 2,9 kb blev derpå splejset med Sacl oligonucleotidlinker (Biolabs nr. 1005) og et 2,9 kb 5 fragment fra pDNll30 opnået ved fordøjelse med EcoRl og Bal31 exonucleasebehandling og påfølgende fordøjelse med Sphl.PDN1265 amyM Cam was constructed by Bal31 exo-nuclease digestion of pDN1120 digested with EcoR5 and subsequent digestion with SphI. A 2.9 kb fragment was then spliced with SacI oligonucleotide linker (Biolabs # 1005) and a 2.9 kb fragment from pDN1130 obtained by digestion with Eco RI and Bal31 exonuclease treatment and subsequent digestion with SphI.
Det fremkomne plasmid pDN1265 på 5,8 kb indeholdt amyM+ genet og udviste chloramphenicolresistens.The resulting 5.8 kb plasmid pDN1265 contained the amyM + gene and exhibited chloramphenicol resistance.
pDN1265 blev derpå skåret med Sphl-Bgl2, og et 4,7 kb 10 fragment blev splejset med et 1,6 kb Sphl-Bgl2 fragment fra PDN1277.pDN1265 was then cut with Sphl-Bgl2 and a 4.7 kb 10 fragment was spliced with a 1.6 kb Sphl-Bgl2 fragment from PDN1277.
Det fremkomne plasmid pDNl290 på ca. 6,3 kb indeholdt amyM'1' og dal+ genet, men ikke nogen antibiotisk resistensmarkør.The resulting plasmid pDNl290 of ca. 6.3 kb contained the amyM'1 'and dal + gene, but no antibiotic resistance marker.
15 Eksempel 6Example 6
Konstruktion af en vært med en deletion i dal genet (fig. 5)Construction of a host with a deletion in the valley gene (Fig. 5)
Ved skæring af 5 μg af pDNl090 i det klonede dal+ gen med restriktionsenzymer EcoRl og EcoR5 og påfølgende fordøjelse af fragmenterne med exonuclease Bal31 i 60 sekunder, splejsning 20 og transformering i DN608:dal-l, blev der iagttaget deletioner, 4· der ødelagde Dal phenotypen af plasmidet. Transformering afBy cutting 5 μg of pDN1090 in the cloned dal + gene with restriction enzymes EcoRl and EcoR5 and subsequent digestion of the fragments with exonuclease Bal31 for 60 seconds, splicing 20 and transformation in DN608: dal-l, deletions were observed, 4 · which destroyed Dal the phenotype of the plasmid. Transforming the
RR
stammen DN497 med et af disse deletionsplasmider pDNl274 Cam _ Rstrain DN497 with one of these deletion plasmids pDNl274 Cam _ R
dal og selektering for Cam gav ca. 0,1% transformanter, der varvalley and selection for Cam gave approx. 0.1% transformants that were
Dal , antagelig på grund af erstatning af det kromosomale dal+ 25 gen med dal deletionen fra pDNl274. Efter spontant at have tabt R — pDNl274 blev transformanten Cam , men forblev Dal . Southern blotting analyse af kromosomal DNA af denne stamme DN1280 viste, at den virkelig indeholdt den forventede dal deletion. Frekvensen af mutationer, der kunne forårsage tilbageførsel af denne dal 30 deletionmutant til en Dal phenotype var mindre end 10 . DN1280 indeholdende plasmid pDNl277 = DN1517 (se eksempel 5) blev deponeret.Dal, presumably due to replacement of the chromosomal valley + 25 gene with the dal deletion from pDN1274. After spontaneously losing R - pDNl274, the transformant became Cam, but remained Dal. Southern blotting analysis of chromosomal DNA of this strain DN1280 showed that it really contained the expected valley deletion. The frequency of mutations that could cause the return of this dal 30 deletion mutant to a dal phenotype was less than 10. DN1280 containing plasmid pDNl277 = DN1517 (see Example 5) was deposited.
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Eksempel 7Example 7
Stabilitet af dal+ amyM+ plasmider i en dal" værtStability of dal + amyM + plasmids in a dal 'host
For at demonstrere både stabilitet af vært - plasmid kombinationen DN1300 (=DN1280 pDN1290) og betydningen af fravær 5 af overlappende kromosomale og plasmid DNA segmenter, der flankerer den kromosomale deletion, blev følgende eksperiment foretaget:To demonstrate both the stability of host - plasmid combination DN1300 (= DN1280 pDN1290) and the importance of absence 5 of overlapping chromosomal and plasmid DNA segments flanking the chromosomal deletion, the following experiment was performed:
Enkelte kolonier af stammerne DN1297 (=DN1280 pDNll30) og DN1300 (=DN1280 pDNl290) blev resuspenderet i LB-medium sup-10 pieret med 10 mM kaliumphosphatpuffer pH = 7,0 og 0,2% glucose. Halvdelen af hver resuspension blev podet i det ovennævnte medium, den anden halvdel i identiske medier suppleret med 200 μg/ml D-alanin. Ved fortynding af kulturerne enten 100 eller 1000 gange blev stammerne dyrket et antal generationer ved 37°C under god 15 beluftning enten i nærvær eller fravær af D-alanin i mediet. For . 7 hver fortynding blev ikke mindre end 10 celler overført. Med et interval på 1 dag blev frekvensen af Amy+ kolonier (et total på ca. 100) testet på LB-medieplader med eller uden D-alanin ifølge vækstbetingelserne. Resultaterne er vist i tabel 1. 10 tilfældigt 20 valgte Amy kolonier viste sig ikke at indeholde noget plasmid.Single colonies of strains DN1297 (= DN1280 pDN1130) and DN1300 (= DN1280 pDNl290) were resuspended in LB medium sup-10 pierced with 10 mM potassium phosphate buffer pH = 7.0 and 0.2% glucose. Half of each resuspension was seeded in the above medium, the other half in identical media supplemented with 200 μg / ml D-alanine. By diluting the cultures either 100 or 1000 times, the strains were grown a number of generations at 37 ° C under good aeration either in the presence or absence of D-alanine in the medium. For . 7 each dilution, no less than 10 cells were transferred. At a 1-day interval, the frequency of Amy + colonies (a total of about 100) was tested on LB media plates with or without D-alanine according to the growth conditions. The results are shown in Table 1. Ten randomly selected Amy colonies were found to contain no plasmid.
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Tabel 1Table 1
Frekvens af Amy celler efter vækst i LB medium med og uden D-alanin.Frequency of Amy cells after growth in LB medium with and without D-alanine.
Generationer 5 Stammer Plasmid 51 68 85 __% Amy~ DN1297 PDN1130 2% 20% DN1297 + D-ala1 pDN1130 60% DN1300 PDN1290 0% 0% 0% 10 DN1300 + D-ala1 pDN1290 60% 90% 1 Første 20 generationer i fravær af D-alanin 15 De i tabel 1 viste resultater demonstrerer, at et plas mid pDNl290 er meget ustabilt under ikke-selektive betingelser {tilsætning af D-alanin til vækstmediet), men bliver stabil under vækst i et selektivt medium (uden tilsætning af D-alanin). Det er yderligere blevet demonstreret, at homolog dobbeloverkrydsning, 20 der gendanner dal+ genet på værtskromosomet, og efterfølgende plasmidtab ikke fandt sted i DN1300 (ingen Amy celler efter dyrkning i 85 generationer), hvorimod frekvensen af Amy~ celler var 20% efter 68 generationer af DN1297 dyrket i et D-alaninfrit medium, antagelig på grund af overførsel af dal+ genet fra plas-25 mid til kromosom og efterfølgende plasmidtab.Generations 5 Strains Plasmid 51 68 85 __% Amy ~ DN1297 PDN1130 2% 20% DN1297 + D-ala1 pDN1130 60% DN1300 PDN1290 0% 0% 0% 10 DN1300 + D-ala1 pDN1290 60% 90% 1 First 20 generations in absence of D-alanine 15 The results shown in Table 1 demonstrate that a plasmid pDNl290 is very unstable under non-selective conditions {addition of D-alanine to the growth medium) but becomes stable during growth in a selective medium (without addition of D -alanine). It has been further demonstrated that homologous double crossing, 20 restoring the dal + gene on the host chromosome, and subsequent plasmid loss did not occur in DN1300 (no Amy cells after 85 generations of culture), whereas the frequency of Amy cells was 20% after 68 generations of DN1297 grown in a D-alanine-free medium, presumably due to transfer of the dal + gene from plasmid to chromosome and subsequent plasmid loss.
Eksempel 8Example 8
Konstruktion af plasmid pDN1800 (fig. 6) 30 pDNl284 blev konstrueret ved kombination af det 1,4 kb store Bgl2-Bgl2 fragment fra pDN1222 (se fig. 3) med det 1,5 kb store BamHl-Bgl2 fragment fra pDNl050 (se fig. 3).Construction of plasmid pDN1800 (Fig. 6) 30 pDN1284 was constructed by combining the 1.4 kb Bgl2-Bgl2 fragment from pDN1222 (see Fig. 3) with the 1.5 kb BamH1-Bgl2 fragment from pDN1050 (see Fig. 3).
Fra pDNl284 blev dal+ genet overført på et 1,4 kb Bgl2-Sall fragment til den klassiske E. coli plasmidvektor 35 pBR322, der blev skåret med BamHl og Sall. Rekombinantplasmidet pDNl800:Amp (ampicillinresistent) blev transformeret i E. coli stamme TKL10, idet der blev selekteret for AmpR, til opnåelse af 20From pDN1284, the dal + gene was transferred on a 1.4 kb Bgl2-SalI fragment to the classical E. coli plasmid vector 35 pBR322 cut with BamH1 and SalI. The recombinant plasmid pDN1800: Amp (ampicillin resistant) was transformed into E. coli strain TKL10, selected for AmpR, to give 20
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stamme DN1800:pDNl800. Stamme DN1800 blev deponeret ved NCIB d.strain DN1800: pDNl800. Strain DN1800 was deposited at NCIB d.
22. november 1985 og fik referencenummeret NCIB 12181. Permanen-cen af depotet og offentlighedens adgang hertil er således sikret (se ovenfor). TKL10 udviser en D-alaninkrævende phenotype ved 5 42°C, men kravet undertrykkes i DN1800. pDNl800, der indeholder dal genet fra B. subtilis, er således i stand til at komplementere D-alaninkravet i en E. coli stamme, der har en defekt i alaninracemaseaktiviteten (Wild et al., Molec.Gen.Genet. 198:315-22, 1985).November 22, 1985 and was given the reference number NCIB 12181. The permanence of the repository and the public's access thereto are thus secured (see above). TKL10 exhibits a D-alanine-requiring phenotype at 5 42 ° C, but the requirement is suppressed in DN1800. Thus, pDN1800 containing the dal gene from B. subtilis is capable of complementing the D-alanine requirement in an E. coli strain that has a defect in the alanine racemase activity (Wild et al., Molec.Gen.Genet. 198: 315- 22, 1985).
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