CA1272143A - Dna vector comprising a low copy number and a high copy number origin of replication and their use in recombinant dna technology - Google Patents
Dna vector comprising a low copy number and a high copy number origin of replication and their use in recombinant dna technologyInfo
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Abstract
ABSTRACT
DNA VECTORS AND THEIR USE IN RECOMBINANT DNA TECHNOLOGY.
The invention provides a new class of DNA vectors, each comprising two replication systems; a first origin of replication resulting in a low copy number and stable inheritance of the plasmid, and a second, high copy number, origin of replication at which replication is directly controllable such that, when host cells carrying the vector are propagated under a first set of conditions, replication takes place mainly from the low copy number origin, and that when said cells are propa-gated under a second set of conditions, replication takes place also from the high copy number origin to produce a high yield of gene product. The controllable origin of replication may be under the control of a natural promoter of RNA transcription or a substitute promoter such as the PL promoter or lac promoter.
DNA VECTORS AND THEIR USE IN RECOMBINANT DNA TECHNOLOGY.
The invention provides a new class of DNA vectors, each comprising two replication systems; a first origin of replication resulting in a low copy number and stable inheritance of the plasmid, and a second, high copy number, origin of replication at which replication is directly controllable such that, when host cells carrying the vector are propagated under a first set of conditions, replication takes place mainly from the low copy number origin, and that when said cells are propa-gated under a second set of conditions, replication takes place also from the high copy number origin to produce a high yield of gene product. The controllable origin of replication may be under the control of a natural promoter of RNA transcription or a substitute promoter such as the PL promoter or lac promoter.
Description
DNA VECTORS AND THEIR USE IN RECOMBINANT DNA TECHNOLOGY.
Field of t~le Invention This invention relates to DMA vectors, their production and -their use in recombinan-t DNA techno-logy. The invention also relates -to the control of replication of DNA vectors in the cells of host organisms, and to the production of gene produc-ts.
Bac~ground of the Invention DNA vectors, such as plasmids, are normally circular, extrachromosomal DNA molecules which replica-te autonomously within the cells of host organisms. The cells of many unicellular organisms, including some bacteria, contain naturally-occuring wild-type plasmids which contribute ~arious functions to the host cells 5 SUC~l as antibiotic resistance and fertility. These ~ild--type plasmids and deriva-tives of them are the basic tools of recombinant DNA technology, providing vehicles for the transformation of the cells of host organisms with foreign DNA sequences whichcode for product on,~
within the transformed cells, of corresponding foreign polypeptide and protein products. Thus, in recombinant DNA techniques, plasmids are cu-t open at specific sites using restric-tion enzymes and recombined in vitro with additional DNA sequences,including genes coding for desired foreign products, -to give recombinant plasmids which may be used to transform appropriate host cells.
These recombinant plasmids, similar to the parent plasmids from which they are derived, are capable of autonomous replication within host cells, and on re-3~ plication reproduce no-t only the DNA sequences of the parent plasmid bu-t also the inserted additional DNA
sequences, including the foreign genes. During protein synthesis, transcription and translation of the DNA
sequences of the recombinant plasmids carried within transformed host cells give rise inter alia to the ~,, ~2~21~13
Field of t~le Invention This invention relates to DMA vectors, their production and -their use in recombinan-t DNA techno-logy. The invention also relates -to the control of replication of DNA vectors in the cells of host organisms, and to the production of gene produc-ts.
Bac~ground of the Invention DNA vectors, such as plasmids, are normally circular, extrachromosomal DNA molecules which replica-te autonomously within the cells of host organisms. The cells of many unicellular organisms, including some bacteria, contain naturally-occuring wild-type plasmids which contribute ~arious functions to the host cells 5 SUC~l as antibiotic resistance and fertility. These ~ild--type plasmids and deriva-tives of them are the basic tools of recombinant DNA technology, providing vehicles for the transformation of the cells of host organisms with foreign DNA sequences whichcode for product on,~
within the transformed cells, of corresponding foreign polypeptide and protein products. Thus, in recombinant DNA techniques, plasmids are cu-t open at specific sites using restric-tion enzymes and recombined in vitro with additional DNA sequences,including genes coding for desired foreign products, -to give recombinant plasmids which may be used to transform appropriate host cells.
These recombinant plasmids, similar to the parent plasmids from which they are derived, are capable of autonomous replication within host cells, and on re-3~ plication reproduce no-t only the DNA sequences of the parent plasmid bu-t also the inserted additional DNA
sequences, including the foreign genes. During protein synthesis, transcription and translation of the DNA
sequences of the recombinant plasmids carried within transformed host cells give rise inter alia to the ~,, ~2~21~13
- 2 -synthesis of foreign products corresponding -to the inserted foreign genes.
One factor which affects the yield of synthesised foreign product is the number of copies of the foreign gene which are present within the transformed cells, i.e.
the copy number at which the recombinant plasmid is maintained within the host cells, this being defined normally as the number of copies of the plasmid per host genome. Generally speaking, the higher the copy number of the recombinant plasmid the greater is the yield of foreign product. Both low copy number plasmids, us~lally maintained within host cells at about 1-10 copies per genome, and high copy number plasmids, usually m~intained at from 11 up to severalhundred copies per genome, are known. The copy number of a given wild-type replicon is controlled by DNA sequences surrounding and including a DN~ sequence which defines the origin of replication. Thus hereinafter we refer to high copy number~and low copy number origins of replica-t-on.
High copy number plasmids have been used in re-combinant systems with a view to obtaining good yields of for~ign products. This can lead to undesirable results, however, since many such high copy number plasmids -tend not to be maintained stably within transformed cells and may be lost from the cells before they can be grown to sufficien-t levels to permit bulk production of foreign produc-ts. For example, the foreign produc-t may inhibit propaga-tion of the transformed cells or the high copy number plasmids themselves may be inherently unstable.
One factor which affects the yield of synthesised foreign product is the number of copies of the foreign gene which are present within the transformed cells, i.e.
the copy number at which the recombinant plasmid is maintained within the host cells, this being defined normally as the number of copies of the plasmid per host genome. Generally speaking, the higher the copy number of the recombinant plasmid the greater is the yield of foreign product. Both low copy number plasmids, us~lally maintained within host cells at about 1-10 copies per genome, and high copy number plasmids, usually m~intained at from 11 up to severalhundred copies per genome, are known. The copy number of a given wild-type replicon is controlled by DNA sequences surrounding and including a DN~ sequence which defines the origin of replication. Thus hereinafter we refer to high copy number~and low copy number origins of replica-t-on.
High copy number plasmids have been used in re-combinant systems with a view to obtaining good yields of for~ign products. This can lead to undesirable results, however, since many such high copy number plasmids -tend not to be maintained stably within transformed cells and may be lost from the cells before they can be grown to sufficien-t levels to permit bulk production of foreign produc-ts. For example, the foreign produc-t may inhibit propaga-tion of the transformed cells or the high copy number plasmids themselves may be inherently unstable.
3~ It is known that the copy numbers of some plasmids can be amplified above normal levels by inhibition of protein synthesis; for instance, by addition o-f protein synthesis inhibitors such as chloramphenicol to the fermentation medium. However, protein synthesis is required for production of most gene products, and therefore the inhibitor must be removed before synthesis of foreign gene products can take place. This removal of inhibitor requires complicated manipula-tions and is not always possible.
Various other solutions have beenproposed to over-5 come the problem of s-table maintenance of high copy number plasmids in host cells. For example, in UK
Patent Specification No:1,557,774 it has been proposed to use mutant plasmids having a temperature-dependent plasmid copy number pattern such that the plasmid shows a controlled constant plasmid copy number when host bacteria carrying the plasmid are cultivated at one temperature, but an altered plasmid copy number pattern, allowing a much higher or to-tally uncontrolled copy number, when the host bacteria carrying the plasmid are .15 ~rown at a different temperature. Thus cells may be propagated to desired production size cul-ture at one temp~erature at which the plasmid replicates at low copy number and at which its gene products do not signifi-cantly-inhibit cell growth. The temperature ~y then ~ be altered, greatly increasing the plasmid copy number and also the corresponding production of gene products.
The introduction of copy number temperature dependence in such mu-tant plasmids, however, may introduce a source of instability into the plasmid, and i-t is likely that ~5 these mutant plasmids may be unstable or subject to loss when cells carrying them are propagated over a prolonged period of time.
The replication of plasmids is controlled by nucleotide sequencescontained within the overall DNA
3~ sequence of the plasmid. These sequences include a sequence defining the origin of replication at which DNA replication is initiated and often also associated sequences which control the :initiation of replication at the origin and the copy number at which the plasmid is maintained. For example, certain plasmids, of which ColEl is a typical example, have plasmid replication systems having a number of features in common, These systems comprise a DNA sequence defining an origin of replication and upstream thereof a DNA sequence coding for transcription, in opposing directions 9 of two RNA
species, RNAII and RNAI. The RNAII species provides an RNA primer which forms a complex a-t or near the origin from which DNA synthesis is initiated; -the ~NAI
species interferes with the formation of this initiation comple~, Transcription of the two RNA species is con-trolled by separate promoter sequences associated with the DNA sequences which code for their transcription.
In ~ddit.ion there is a small polypeptide (the rop protein) which is believed to interact with the promoter for RNAII; this polypeptide is not essential for re-plication and its role is unclear. The origin of replication, the RNA coding sequences and associated promoters together provide an internally self-regulated system,.which controls the replication incompatibility ?O and the copy number of these plasmids. Certain other plasmids, exemplified by RI and some Staphyloccocal plasmids, also control replication initiation at the transcriptional level, but by a messenger RNA species whose product provides an initiation factor, probably ~5 a polypeptide, which is involved in DNA replication.
It is an object of the present invention to provide new DNA vectors which have controllable copy number patterns and therebyovercome problems associated with stable maintenance of vectors which replicate at hi~h copy number only, which new vectors will not be subject to the potential instability of previously described mutant plasmids which have temperature-dependent copy number patterns, and which, furthermore, will have the advantage that their copy number can be controlled by agents other than temperature, e.g.
metabolite concentration.
Accordingly, in a first embodiment the invention provides a DNA vector comprising two replication systems;
a first origin of replication resulting in a low copy number and stable inheritance of the vector and a second, high copy number origin of replication at which replica-tion is directly controllable as the result of replacement or alteration by DNA manipulation of the natural vector s~uence(s) which control replication at said origin.
By means of the invention, when host cells carrying the vector are propagated under a first set of conditions, replication takes place mainly, and preferably exclusively, from the low copy number origin, and when the cells are pro~agated under a different set of conditions, replication takes place at high copy number at the second origin.as 1~ well and the production of large amounts of foreign gene products encoded by the vector is induced.
In a preferred embodiment the invention provides that the second, high copy number, origin of replication comprises an origin of replication and an associated DNA
-~ se~uence encoding an RNA species which provides a primer or i~nitiation factor (e.g. a polypeptide) which initiates DN~ replication by formation of a complex at or near the origin of replication, in which transcription of said RNA
species i~5 directly controllable such that, when host ~ells carrying the vector are propagated under selected conditions, replication takes plase at high copy number ~rom the ori~in and the production of large amounts of Eoreign gene products encoded by the vector is initiated.
~he invention also includes a method for the 3~ preparation of a vector according to the first aspect, ~:72~3 - 5a -comprising including in the DNA sequence coding for the second replication system a DNA sequence which per-mits direct control of replication at the second origin.
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Methods for the preparation of vectors according to the flrst aspect of the invention suitably com-prise ligating a first DNA sequence coding for the re-plication system comprising the first origin wi-th a second DNA sequence coding for the secondary replication system. The DNA sequence which permits direct control of replication at the second origin may be incorporated into the second DNA sequence either before or after ligation with the first DNA sequence. Thus the inven-tion further provides methods for the preparation ofvectors according to the second aspect of the inven-tion 9 comprising including in the second DNA sequence a DNA
s~uence which permits control of replication at the second origin by controlling transcription of the RNA species.
~5 The invention further includes a process for the production of a polypeptide, protein or other gene product which comprises transforming host cells with a vector according to the first aspect of the invention, in which said vector contains a gene sequence coding for ~0 production of said polypeptide, proteinor other gene product, propagating said transformed cells under a first set ofconditions at which replication takes place at low copy number mainly, and preferably exclusively, from the first origin, and then propagating said ~5 transformed cells under a second set of conditions at which replication takes place at high copy number also (or exc-lusively) from the second origin and the expression of said polypeptide, protein or other gene product is induced.
By means of the process of the invention, trans-formed cells are propagated to give the large scale culturesrequired for economic production of polypeptide~
protein or other products under conditions where the vector replicates at low copy number and the instability problems associated with high copy number vectors are avoided, followed by propagation under different condi-tions where the vector replicates at high copy number ..~
~ 2~zle~3 wth concomi-tant high yield of polypeptide, pro-tein or other products.
In particular it has been found, when the ex-pression of the gene product is under the control of a promoter which is regulated by cytoplasmic levels of a repressor, that the increase in vector copy number leads to outstripping of the repressor control and high level expression of the gene product. This is so even in the case when the synthesis of the repressor is autoregulated, e.g. when the promoter/repressor system is that of the tryptophan operon.
The control systemswhich are used to con-trol the copy number of the vectors of the invention may comprise anyof the control systems which are known for controll-ing replication (and/or expression) in recombinant DNAtechnology. In particular, the copy number of the controllable origin of replication may be controlled by temperature or one or more metabolites or metabolite analogues. Examples of metabolite-dependent sys~ems which may be used include: tryptophan, lactose, galactose, arabinose or any other metabolite or meta-bolite analogue, the presence, removal or further metabolism of which can be used to activate transcrip-tion from a given promoter.
~5 The controllable replication system used in the vectors of the invention may be derived from high copy number cloning vectors, such as ColEl-like plasmids, e.g. pAT153, NTPl, CloDF13, RSF1030 or P15A, which ha~Je eopy number control systems which involve transcription 3~ o~ RNAII or a similar RNA species which provides a primer which initiates DNA replication by formation of a complex at or near the origin of replication. In addi-tion other plasmid replication origins whose replication is controlled by an mRNA species and/or its product~s~
may provide the controllable replication systems used in the vectors of -the invention. Such replication origins ~7~L3 - 7a -may be obtained from Gram ve bacterialspecies and are exemplified by Rl, R6, R100, RP4, or Gram +ve bac-terial species, which are exemplifiedbypUB100, pC194 and certain other Staphylococcal plasmids. Furthermore, bacteriophage origins may be used for secondary con-trollable origin, e.g. those from ~, T3, T4, T7, M13,~X174, SPPl, SP02 etc.
In an important embodiment of the invention, the controllable replication systems may be prepared from such high copy number cloning vectors by replacement of 1~ the natural promoter, which pr~motes transcription of the RNA species, by a contr~llable promoter, such as the PL promoter, PR prorroter, Pre pr~moter, P'R prornoter, T71ate pr~moters, t~p prorr~ter, tac proToter, lac promoter, gal pr~rnoter~ara promoter or _ecA promoter 5 (the origin OI replication in such a system is tenned a "hybrid origin"). Altematively the natural promo-ter may be used and trans-cription of the RNA species made controllable by incorpc)rating a regulating f~nction, such as an operator sequence, e.g. the lac operator or OL or OR operators of phage lambda~into the replication systems.
The plasmid pMG9 (containing an Xho I linker DNA
sequence inserted as described by K Tatchell e-t al, Cell, Vol 27, pages 25-35, November 1981 (Part 2)) provides a convenient starting material for preparation 15 Of a controllable replication system based on the ColEl replication origin. This plasmid has a unique Xho I
restriction site, close to the start of the sequence coding for transcription of the RNAII species, which we have found may be used for insertion of operator and 20 controllable promoter sequences, e.g. ;tPL, to give a directly controllable replication system. Plasmid pM&9 was deposited at the National Collection of Type Cultures, Central Public Health Laboratory. Colindale Avenue, London NW9 5HT on 24 ~. rch 1983, under NCTC
No.11539, as a culture of cells of E coliK12, strain DHl, containing the plasmid pMG9.
The replication system comprising the first origin of replication may be obtained from any suitable low copy number plasmid. For example, a replication 30 system comprising the pSC101 origin may be used, and the plasmid pHSG415 (T Hashimoto-Gotoh et al~ &ene, 16 (1981) pages 227-235) provides a convenient source for such a replication system. It will be appreciated that pHSG415 has a temperature sensitive replication 35 origin, but that this origin may be replaced by its wild-type temperature-stable counterpart from pSClO1.
pHSG415, however, provided a convenient, temperature ~ 272~L~3 sensiti~e replication origin for use in the exarnples hereinafter described.
Controllable functions may be incorporated into high copy number replication systems, high copy number and low copy number replication systems may be ligated, and foreign genes may be incorporated into the vectors to produce vectors according to the invention using techniques which are known and understood by workers skilled in the recombinant DNA art. The resultant vectors may then be used to transform suitable host cells usina standard procedures -to produce foreign poly-peptide, protein and other products.
The host cells may comprise eucaryotic cells, includir.g yeast cells e.g. ~.cerevisae, or, more usually, bacterial cells, of species such as B.subtilis or, especially, E.coli.
I
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Tne invention is furche} ~escribea vy way of illustration only in the following E.samples, Examples 1-7. These Exan~ples relate to the construction of spacific dual origil~ plasmids according to the invention, and to studies of copy number control, heterologous ~ene e~pression and stability of tllese plasmids. It will be appreciated that the invention is not limited to the specific plasmids and methods described.
Thesa E~amples refer to tne accompanying diaDrams in which:
~ iguro 1 shows plas~id restriction maps and indicates the DNA
~aniplllations which were used to prepare a dual origin plasmid according to th-e il~vention, p2lG411;
Finure 2 is an agarose gel oL DNA isolated fro~. c~ltures of a p~7G411 trans~ormaint of E.coli QY7 ta~en at hourly intervals following a tenperatLre shift from 30C to 42C (lanes 1-7, hours C-6);
Figuro 3 snows plasmid restriction maps and indicates the DNA
~anipulations used to prepare two furtller dual origin plasmids according to ehe invention, p~iGl~9 and p~lG16~;
~ i~ur3 ~l is a graph of copy nu~ber induction following a te~perature shi~t (30C to 42S with subsequent incubation at 37C) of trans-~ormants of ~.coli carrying pr.l&lS9, pMGlbS, pb!G168 and p~ l69;
~ igure 5 shows plasmid restriction maps and i~.dicates tl~e DNA~anipulations used to prepare a metabolite controllable dual origin ~ector according to the present invention, pPIG427;
~72~3 Figure S sho-Ys plasmid restriction maps and indicates the DNi~
~anipulations used to prepare two f~rther metabolite controllable dual origin vectors according to the invention, p~lG415 and p.~.iG416;
Figure 7 is a lG.~o polyacrylamide gel of protein ~ro~ total cell e~tracts prepared from E.coli E103(S) transforrJed by p~lG169 after tenperature shift treat~ent, samples being taken at hourly intervals ~lanes 1-8, hours 0-7);
~ igure 3 is a graph showing incrcase in plas~id copy nu~ber and ~hlorn~pllenicol acetyltransferase activity for cultures of E.coli El03(S) transforrled by pi'>lG169 after teDperature shock treat~ent, and Figure 9 is a 10~ polyacrylamide gel of protein from total cell e~tracts of cultures of p~lG169 transformed E.coli ElU3(S) at hourly intervals over a perio~ of S hours after te~perature snoc~ treatment (lanes 6-l, hours 0-5).
ElV~lPLE 1 Constr~ction of ~ Dual-origin Plasmid ~ith Copy Number under Pl Control (a) Construction of a Dual-origin Plasmid pMG4U4 Plasmid p~Gl5 (Figure l) is a derivative of ~Sa-12 (pBR322 replicon) which has constitutively high copy nunber (approxirnately 300 copies per chromosor.e) due to the insertion of a XhoI linker (CCTCGA~G) into the ori~in region (Table l) (Tatchell et al, 1981)o During construction of ~S~ the ro~ gene was lost by spontaneous deletion extending beyond the ~S~1I site in the tet gene, bllt reLnining the Bami1I site. Tne position of the hoI linker ~vas determined by inserting the l.lkb ~hnI-P~m~1I DNA fragment of p~-!Gl5 into bacteriophage ~.113mp~ and DN~
sequencin~ from the XhoI site by the method of Sanger et al, (1977).
Ins_rtion of the linker was at a point 30 bases do-N~s~raam of the tra1lscriptional start of ~AII (Table l). ~lis ~hoI linker provided a ~iquc clcavn~e site close to the S' end of the ~lAII transcript, such that th~ l.lkb ~hoI-Xa~-lI fragment isolated from p~lGl5 carried a promoter-lcss P'~AII sequence (Figure l). To determine if this seq-~ence could fl~ction as n primer of DNA replication when coupled to another promoter, the l.lkb fra~ment was inserted downsteam of the ~m resistance gene (~rl~) nromoter of pl~s~id pT1S~4lS (Tlashimoto-Gotoh ~ al, 1901) ~Fi:~urc 1). p~SG415 is a stable low copy number Apr~ ~m~ Cn1~ plasmid whoso replicQtion origin is derived from pSClOl; this plasmid replicates at 30~C b~lt not ~t 42C.
Site of Insertion of XhoI Linker near the Origin WILD TYPE S~nUE~'CE
~h'AII
~' CTTGC~-~ACA~A~ CCACCGCTACCM CGGTGGTTT
~555 p`~lG15 SEQUENCE
P~NAII
5' CTTGC~U~C.~A~ ACCACCGCTACC~C~GTCCTCGAGG&&TT
I XhoI Linkei -~55 Conditions for restriction enzyme digestions, DNA ligations, and sg3rose gel cl3ctrophoresis were as described in ;~;ianiatis et al (1982).
Insertion of the XhoI-~amEI frag~.ent fro~ pilG15, coupling the pIomoterless ~'~AII sequenca to the ~mP~ promoter, gave tne dual origin plas~id pr~lG404 (Figur~ l~. Analysis OI the plasmid pilG404 by restriction en7~e dig~stion verifie~ its structure (results not shown), and plasmid r~ yields demonstrated that its copy number was considerably higher than thnt o~ p~iSG415, In addition, pdlG404 was maintained in bac,eria at 4~C
wll~r3ns p}iSG4l5 was lost due to its in3bility to replicate at that ~p3ratur-e, It was conclllded tnat the hybrid 'ColEl origin' w~s functioning in prllG404, da~onstrating that the ~ gene pro~oter could substitute for tne ~NAII promoter, Confirmation of the functioning of the hybrid origin was obtained by digesting p~.G404 with BalI and recircularising the larger fragment .. . . . ..
~2~
co~ltaining onl~ t~e hy~rid ori~in. This ~eneraced a plasr,~id ~L~ which ~vas capablc of antono~ous replication at both 30~ and ~2C, clemonstrating thæt tne hybrid origin was functional.
(b)Construction of Dual-origin p~G411 Plasmid p~lG404 has two origins of replication: the lo~v copy number origin of pSC101 and the 'Col~l' hybrid origin. The pSC101 origin should ensura stable replication of the plasmid at 30C (~shi~oto-Go.oh et ll, l9~1), allosYing the construction of derivati~es with the 'ColEl' hybrid Ol`i~iU driven by a contro]lable pror,~oter. A 0.5~b SalI-~coRl `ra~ment carryin~ .he Pl promoter OI bac~eriopnage ~ was isolated from the plasmid pCT10~0 (Figure 1) and ligated to the 5.5kb EcoRl-XhoI
fr~gment from p~lG404. The ligation mixture wæs used to transform E
coli ~Y7 (l cI~57 defective lysogen) and D~ll at JO~, and the Apr~
.ransfor~ants screened for CmS clones. One of these, prlG411 was chosen ~or further st~dy (Figure 1). Plasmid phG411 transformants of Dl~l and QY7 ~rew well under selective conditions and copy n~mber determinations were m~de ~t a variety of gro~vth temperatures.
~c)Copy Number ~leasurements of phlG411 Plasmid copy num3er ~vas determinecl by tl,vo mothods. One aepended upon tho separation of chromosomal and supercoiled DNA by eaesiu~ chloride eentri~ug~tion~ Tho second method depended upon the separation of Ghromosomal and plasmid DNA by agarose gel electrophoresis. Cells were grown overnight in L-broth containing ampicillin, and diluted one hundred fold into minimal ?,'9 salts medium containing a~picillin. At an OV~60 of 0.~, 2-deo:cyadenosine (200 ~g/ml) thymidine (1 ~g/ml1 and ~3il]-thymidine .
IZ7~ 3 ~ ~g Ci/ml) wera a~ded, and incu~ation continued for 2 hours. Cells wcrc centrifllg-d, ~Yashed and resuspended in an equal volume of 50 ~F,J tris L~Cl 7.~ sucrose (-~/v) and lysozyme (200 ~g/ml). After incubation for minutes at 0C, EDTA y1as added to a concentration of 10 ~I and incubation contined for 10 minu~es, and finally sarkosyl ~0.4~ w/v) ~as sdded. Chromosomal DNA in the lysate was sheared by 6 passages through a 1~ ga~ge needle, and eell debris removed by centriEugation. ~NA
preparations were futher purified by a single phenol and chloroform e~traction and ethanol precipitation. This labelled DNA prepaIation was th~n u~ed for eopy number determination by either method.
For the ngarose gel method, samples were electrophoresed on a 0.7$
a~arose gel (in 0.04 ~i tris acetate, 0.001 ~I EDTA, plI 7.9) for 12 hours at ~0 V. DNA bands were visualised by stai~ins in ethidiu~ b~omide. Tke chromosomal~ and plas~id DNA bands were cut from the gel, dissolved in saturated sodiu~ iodide and the DNA precipitated by the addition of 10~
(wlv) triehloroacetic acid (TCA). Precipitates were collected on GF/C
lihat.~lan filters, ~ashed in 1~ (w/v) TCA, ethanol and then air dried.
~adioactivity was determined by counting in a liquid scintillation counter, ~ or eopy nl~er determinations by caesium chloride ce~trirugation DNA
s~rl~lcS ~vere eentrif~lged to equilibrium in caesius chloride-ethidiu~
bro~ a in a r.ec~man TI50 rotor at 4S,000 rpm for 24 hours. The gradients ~ere fraetionated, and the fractions precipitated witn TCA onto GF/C
~`~hatman filters, washed and processed as described above.
~ 3 PlasmiG GOpy l~r~nbers were determined fro~. pIIGl5, pAT153 and p~iIC411 at 30C, 37C and ~C and the results obtained are given in Table 2.
. . .
Variation of Plasmid Copy N~bar with T~mperat~re PlasmidBacterial Strain Percenta~c of Copies pRr (growth temperature) total DNAChromosome n~;Gl~~101 (37C) 33 309 p~l`l5~ ~E101 (37C) 5.~ 59 ~:G~llQ~'7 t3noc) 0.7 4 ~t~741.' ~Y7 (37C) 13.~ 7 p~lG411 QY7 (42C) 24.~ 143 I~Ieasurenents of p~lG411 copy nt~ber in strain QY7 ~l lyso~en) at various te~ceratures de~onstrated that tne A repressor controls pi~G411 cop~ number.
After Drowtlt at 30C, copy n~ber was estimated at 4 per c~ro~.osome, ~hilst at 37C it had increased to 78 and at 42C it had increased to 1~.3 ~er chror~.oso~e (Table 2).
To de~onstrate that the hybrid 'ColEl' origin in p~'G411 could be s~vitched OII by inactivatino the ~ repressor, a transform~tnt of QY7 was ~ro~vn at 30C~ and then the tempe~ature raised to 42C durin~
o~ponantial ~rowth. The plasmid copy number increase ~as followed by agaroso gcl analysis of D~IA isolated from 1 ml cultures ~Figure 2).
Samplas wera taken at 1 hour intervals over a period of 7 hoursO As prodictod, an increase in pbG~ll copy number was observea over the 7 hour period.
EX~IPLE 2 Constrllction of Dual Origin Plasmids with Ind~cible Copy Number under Pr Control ~)Construction of Dual Origin Plasmids pblG159 and pMG165 Plasmid p~lG411 has two origins of replication, the low copy n~ber origin of pSC101, and the Pl-driven ColEl origin. Since the repressor gene, cIgs7 is resuired for copy number control~ this limits the use of p`l.'G411 to lysogenic bacterial strains~ To overcome this constraint, a du~l-ori~,in plasmid carrying the cIgs7 gene was constructed.
~ irstly, to facilitate subsequent cloning of foreign genes an oligo-nucleotide comprisin~ a SalI and nindIII restriction site was inserted into the a~I site immediately downstream of the hybrid origin in p~ ll; this gave p~rsl53 (Figure 3). The 5.9'~b EcoRl-Ea~II fragnent of p~ 71:;3 w~s purified and ligated to a l.lkb ~col~-Ea~lI fragnent of p~V2 (Queen, 198~), to give pi.lG159. The EcoRl-Za~I fragment of pCQV2 cnrries both Pr and the cIs~7 gene, and on insertion into p~C153, r~places the Pl containing fragment, fusing the ColEl origin RNAIT Seqnence to Pr. Note that the repressor gene is transcribed away fron tne origin IFi~ure 3). To increase plas~id stability (see bclow) when the dual-origin v~ctcrs are maintained at 30C, the pS~101 2.85kb ~incII fragnent ~nrryin~ the ori~in of replication, replication protein and par sequence, ~las introduced into p.,lG159, by replacine the 3.25kb EcoRl-BalI fragment w~ith the pSC101 l~incII fragment with an EcoBl linker at one end. This ~ave ph'&l65 ~Figure 3).
(b)Copy Number Control of Pr Driven Dual ~rigin Plasmids To demonstrnte t~at the 'Col~l' hybrid origin driven 'Dy Pr was controlled by tl1e ter.~per~ture sensitive repressor expressed fro~ ~he cloned cIs~7 gene, p~'GlS9 and plUGl65 were transfo~med into E.coli El03(S).
Copy number determinations at 30C were made as previoasly described from 31~-thymidine labelled cells (Table 3). Copy number induction after temperature sihift to 42C was followed by agarose gel electrophoresis.
To quantitate the copy n~ber change and kinetics of induction in L-brot~, a 'spot' hybridisation method was used.
T.~BLE 3 Variation o~ Plas~id Copy Nu~ber with T3mperaturs Pl~smidB~cterinl Strain Copies p~r Chromosome (g.owth temperature) p~ 9~103(S) (30C) 3-4 p~ 103tS) (30C) 3-4 ~Gl5gElO3(3) (~2C~ ~ 320 p'~ l5~El03(S) (4~C) ~ 248 ~ or the 'spot' hybridisation method 32P-labelled probe DMA was pre~a~ed by n-cl~ translation (-`~aaidtiS et al, l~?). D~A samples were orepared by tne al~aline lysis method (Ish-~orowitz and Rurke, lgSl) from l ml of cu1ture takel1 at various times after the temperature shift. Samples Or thc D~A preparations were treated with P~!ase at 37C for 5 minutes, follo~Yed by incuDation with restriction enzy~e ~a~I for 30 ~inutes, and héat denaturation at lO0C for 3 minutes. Varying amounts of the digested ~IA preparations were spotted onto nitrocellulose filters, which were then dried at 70C, ~.nd subseq~ently hybridised ovcrnight (15 ho~rs) with t'ne den~tured probe Di`~A at 37C in 2 x SSC/50~ for~amide. ~ilters ~vere thell washed twice in 2 x SSCtS0~ formamide and once in 2 x SSC, and then air dried, ~adioactivity was determined by co~ttin~ in a liquid scintillation counter. The values obtained were corrected for cell growth durin~ induction, and the magnitude of the eopy nu~ber change over the uni~dueed value was determined.
~ otll p~i~l5~ and pi.G165 sho~led a rapid copy number induction ~ollowing a t~p~ratuxe shift to 42C and continued incubation at 37C. Uninduced valuas of 3-~ copies per chromosome rising to 90-100 copies after 2 hours inlluetion, and up to 300-400 eopies after 4-5 hours induction were obtained (Fi~u~e 4), clearly de~onstrating the eontrol of copy n~ber e~erted by the eloned Pr promoter and cIgs7 gene.
EXA~LE 3 Construction of Dual Origin Plas~ids with Inducible Copy N~nber under Ptrp Co~trol The t~o ~bove Esatnples of dual origin plas~nids with eontrollable eopy nl~ber, emI)loyed the use of temperature as the indueing agent, this ~sample desGri'~es 'clle use of a ,metaboii~e to control copy nu~b~r. '~he 6.2~b llindIII-E~lII fra&ment of p~iG411 was ligated to the 0~65kb ~indfII-~a~I fra&~ent o~ pCT54 (Figure 5) using standard burfers and techniques ('~!nniatis et al, 1982) to give pMG426. This plas~id was unstable even under a~pieillin seleetion and witll tryptophan present in the medium (whicn should repress transeripcion fro~ Ptrp). It ~as concluded that the instability was due to the inability to repress completely transcriptin ~IOm Ptrp, tnereby giving a high copy nl~mber. To decrease transcription tllrough the origin, a transcriptional ,erminator ~as inserted behYeen the Ptrp nlld the 'ColE1' origin sequence. Such terminators reuuce levels o.
transcripLion appro~i~ately 10-fold. ~ l~Obp AluI fragment from bncteriopll3~e T7 DNA, carr-~ing the early transcription terminator was lignted to DNA lin~ers converting the termini to ~indIII recognitin sites (Emtaoe et al 1983). This fragment was then lioated to ~indIII
di~ested pP~Gl~ DNA to give p?'G427 (Figure 5). p`&427 was more stable than p~iG4~5 and when tranfor~ants were gro~Yn in ruedi~ containing tryptophan ~00 ~ it e~Yhibi~ed a low copy number. '.'1hen such a culture was ~llift~ in~o ~ediu~ lac~ing tryptophan, the copy number increasea rnpidly.
~nis demoilstratcs that ~ II transcription can be controlled by the levels r.~tn~olites or chemicals in the external medium, and that controlled copy ~I~.ber chQn~es can be effected by agents other than temperature.
~LE
.
Construction of Dual Osigin Piasmids with Inducible Copy Number U~der Control of the 'tac' Promoter In this E~nmple a vector ~ns constructed where the ~II promoter was r~placed ~y the tnc promoter tPtac) (Rllssell and Bennct 1982), s-ch that ~py n~ber wns controlled by the addition or removal of lactose or a ~a~tose nnnlo~ue. l~ 121bp Ba~ E,coRl DNA fra~ment carrying the Ptac ~ns purificd fro~ pD~5~0. This fragment was ligated to Bam~I-Eco~l di~ested p;~G40~ D~A> and a~picillin resistant, chloramphe~icol sensitive transformants of ~.coli D~l were isolated. Plasmid DNA fro~ one such ~rnnsformant was isolated, analysed by restriction en~yme digestion and shown to contain the 121bp promoter fragment inserted into the chlor-. ., ampllenicol resistance gene (pl,.G421) (Figure 6). p;;lfi421 ~N-~ was digested ~ith ra,,llI, treated with calf intestine alkalinc phosphatase, and ligated to a l.~'ib Bam~II proL~oterless origin fragt~ent isolated from p-l~411.
~picillin resistant transfor~tants of E.coli D.~l were obtained when the tr~nsEorDation mi~ture was plated at either 30C or ~2C. At 42C, ~he low copy nu1~be. origin is inactive. Two distinct plasmid types were identified fro~t the transformants (p~,G415 and pi-lG416) ~Figure 6). pMG415 car~ied 3 single BamLTI origin fragment in the correct orientation, d~nstr~m of the t~lc, whereas p.~S~416 carried 3 copies oE tke origin ~x~ e~.lt as direct repeats, also orientated for e~pression frott~ Ptac II`C 6).
Plasmids p~ 415 and pli~416 were transformed into E.coli D900 ~I CIsq)~ a strain which overproduces t e lac repressor, the controlling element of the Ptac promoter. Induction of Ptac can be ~ffdct~d by the addition of the lsctose analogue IPT~ (isopropylthiogalac-toside~. p~.~G41~ transformants of D900 grown in L-brot~ had a low copy num~r as judged by agarose gel electrophoresis, but this was not increased hy the addition of IPTG. p~lG416 transformants of D900 grown in L-broth ~lso h~d a low copy number, but this increased quic~-ly on the addition of ~ to the culture, demonstrating controllable copy number induction of p.`~ lu f`ro-~ Ptac.
E~ iPLE 5 The E3pression of the Calf Stomach met Prochymosin Gone Cloned into a Dual-origin Plasmid (~)Construetion of p~lG168 To demonstrate that dual-origin plasmids ,vere useful for the e~pression of clotled heterologous genes in E.coli, a plasmid ~vas constructad carryin~ the calf storiach met-prochymosin gene. pCT70 (~mtage et al 19~3) was digested ivitll llindIII and SalI alld a ~ 2.41cb fragment e~rryin~ the mat~proc3lymosin gene under Ptrp control was isolated. This ~ra~ont ~as li~atA(l to t~vo DN~ fragments isolated from p'liil65. the 5.6kb ~atl-~stI ra~rlellt and the llcb IlindIII-PstI fragment ~Figure 3~.
Tho r~sulting plasmid (p~lG168) isolated from transformants OI E.coli ~i~l, coDprised the cloned gene do~-nstream of .he origin of replication, such th-qt any transcriptional read through f~om Pr. ~vould lead to ~dditional transcriptio~t of the met-procnymosin gene (Figure 3).
(b~pression of ~iet-prochymosin Protein from prlGl6~
p'lCl6~ was trausfori,ied into E,coli E103(S) and met-procnymosin P~prassion nn~lysed by polyarrylaDIide gel electrophoresis. E.coli l'.lG3ts) trnnsfor~iants were grown in L-broth at 30C to an OD600 o-4, ranol s'rift_d to 4 C. Cultures ~vsre tihen incubated at 37C w;tih shil~in~, and samplcs removed at hourly intervals for analysis of eopy number and protoin e~pression. DNA copy number ~Yas l~setermined as deseribed ~or p;~G159 and pi`~!G165. For analysis of protein, each cultu~e sa~iple ~vas eontrifu~ed, the pellet collected and resuspended in stop bllffer (1i~J SDS, lO i~l tris ~ICl p~ 7.5) and an equal volluiie oE sample buffer (0.12 rl tris slCl pll 6.~, 2G5 glycerol, 1,2 ~ mercaptoethanol, 6ri'~ SDS) a~id boile~L for 3 ~i.tut~s.
I'oly~cyla~ 1e ~el electropilorcsis, stainin~ and dcst~ lin~ r,.etnous ~Yerc essentially as described i1~ ~'aui2tis et a1 (l9P2). ';taine(l polyacry-larlidc ~els ~vere sca1lncd usins a Jo~cc 1,oc~1 Chro~oscan at 530 1~m. 130th D~r~ pl~smia copy nu.-lbcr an~ mct-prochy~osin protcin levels werc ~reatly increased following a ~emperature s21ift from 30C to 42C (Table 4, ~igures 4 an~ 7).
Copy nu~ber ir~ere3sed rapidly durin~ a 90 ~1inute post induction period ~t 37~C, ~hilst ~et-prochy~osin acc~:1~latio~ was ~ore gradual. On the basis ~ polyaeryl~ide gel seanning, t~e recor.bin~nt gene pro~uct ~ccumul~ted to 2t least lr~ of total extractable protein by 4-S hours after tine tempera.ure shift. Induced copy number values for p~;~l6P, uere lo~_r thln for the parent dual ori~in plasmid (p~'G165). but still increased fro~
3-~ per c'~rorosome to 120-150 per chror.osome. Cell viability fell fo1lo-~in~ induetion of p';Gl6 bu~ not of p~Gl65. It ~as coneluded that th;s loss in ~iability resulted fro~ the to~ic aeeurlulation of reeo~binant ~ene psoduct.
1`Jt~
Incrc~se in hlot-Prochymosin Gene ETpression Timd after Induction ~ ~otal Protein as at 37C (hrs) met-prochymosin O < 0.5 3 7,08a I 9.09 10.25a a) Tnese measureQents are from ~el scans; t1:e unin~uce(l levels o~ protein are di~ficult to measurc accurntcly 1"~ tllis mct1loa.
I`
- 2~ -~L~ 6 Expression of the Chloramphenicol Asetyl Transferase G~ne Cloned onto a Dual Origin Plasmid (~L~ Construction o phlGl60 Bac~use of the liraitations in the accurate quantitation of stained protein b~nds on polyacrylamide gels, cloned ~ene e~pression on dual origin vncto~s ~s further quantitated by assaying the increased activity of c`nlor~ph2nicol acetyl transferase following copy nur.~iber induction of ~lasl~id p.~ l69. Plas~id p~;G169 was made Erom p~lG16~ in an analagous ~ay to cha e~onstruction of p~lG16. (Example 5), except that the purified ilindIII-1 rar~ent carried the structurial gene Cor chloramphenicol acetyltr:~nsf-ar~se ~nder control of Ptrp (Fi~ure 3).
b)~Y~r~ssion of Cloned Chloramphenicol Acotyl Ts~nserase p~ rl~9 W~S transformed into F.coli ~103(S) and chloranphenicol acctyl transIernse levels Laeasured by polyacrylaL~ide gel electrophoresis an~ zyne assay (Sllaw 1975). E.coli E103(S) ~ransform~nts were grown n~ i~dllced by te~,perature shift to 47.C, followed by continued ine~ubation at 3?C as described for p..lG163 transformants in Fxa~ple 5.
Sa~l~s were removed at hourly intervals and plasmid copy n~aber determined ~ ur~ 4, ~)~ chloraripher.ico'L acetyl trar3ferase specific aci~ivities dat~rL~inad (Sllaw 1975, ~ead and h'orthcote 19~1), (Figure 8, Table ~), and 3~ples run on poly3crylamide gels (Pigure 9). The plas~;d copy number incre~sed with similar kinetics to that of p~i'G168, and with si~ilar absollte v~lues (Figure 4). Chloramphenicol acetyl transferasè iLssays àa~onstrated that the spccific activity of the en~y~ie in crude e~tracts increased appro~ iately 80-fold as a res-llt oE the copy nu~ber induction 2~
~igere ~.). C~lc~la~i~as o~ t~e pLorortio~ oL ~A;tl-3c,~ble ~rotcin preseIIt as chlorallphenicol acetyl transferase were madc from a ~noiwled~e of the specific activitj of the pure protein (Table 5~. Uninduced levels c~pressed from p`;~l69 represented 0.25~ extracted protein, whilst afteI 6 ho~-rs induction tnis had risen to 21.S5.
Increase in Chloramphenicol Acetyl Transferase Specific Ac~ivity Tlm~ ~t~r Induction a) ~ Total Protein as at 37C (hrs) ChloramphenicoI
acetyl transfe~ase 0 ~.25 5.6 2 15.5 _ 18.1 18.5 21.~
6 21.9 7 20.9 e~e vall~es were calculated from the l~own specific activity of pnre c]~lorampllenicol ncetyl transfernse ~195 units pcr mg protein).
-~A~PLE 7 Plasmid St~bility S~udies of Dual Origin Plasmids Plasmids p~iG165 and p~i!G16~ were transformed i~to E.~Q~ P~Y30~.
.nd p~;G163 into E.coli E103(S) to study their stability at 30Cder conditions of chemostat growth without antibio~ic selection. All cxperimen,s were started from a single colony o~ the appropriate E.
. . ~ . .
Goli strain, ta~c~ iro~ an antibiotic-containing a~ar plaLe and inoculated into t00 ~ls of L-broth i~ a 250 ml conical flask with steel spring baffle. The culture W2S incubated in an orbital shaker (37~`, 240 rpm) until stationary phase was reache&. ~ne cells were harvested, resuspended in sterile defined medi~ minus glucose and inoculated into a fer~enter vessel. ~le defined medi-~ was glucose 4 gl~ ~4/2S04 5 g 1~ a2HP04 7 gl-l; ~2P0~ 3 gl-l; proline 200 ~g 1-1;
leucina, 100 mg 1-1; thiamiile 10 mg 1-1; rlgS04.7H20 200 mg 1-C~Cl~,6~0, 5 mg 1-1, ZnS0~.7~0 20 ~g 1-1 1iinSO~.4H20 2 mg ~ m~ ; CuS0~,5~20, 5 mg 1-1, CoC12.6H20, 0.5 mg 1 1; ~cS0,t.7~0 100 m~ ; NaCl 200 mg 1-1; E~TA ~ia2 600 mg 1-1; ~a0H 1~0 ~g 1-1]. Tnis nedium was supplemented with tryptophan 100 ~g 1-1 unless othen~ise stated. Anti-foam (polypropylene-glycol 2000) present in the ~ediu~ at 0.001r,~ v/v, Tlle fermenter cell population wns allo~Yed to grow 2S a closed batch system until the biomass ~as at least 605'~
of the maxi~um supported by the medium. The pump was then turned on and t~a ~ystem run as a cher,~ostat. When the total bio~ass i3 the fermenter was constnnt, it was assu~ed that the initial transient gro~vth phase had ceased ~nd thera~fter the number of generations in the steady state was calculated usin~ tha formula~ n(nur.~ber of generations) = ~tlln2 whe~e ,u = growth rate whic~ is aqual to the dilution rate under steady state conditions, and t =
t.ima, S3m~1es wc~e withdrawn from the continuous cultures, diluted and plated onto L-a~a~. 100 single colonies were picked onto both antibiotic supplc~entcd agar and L-agar as a control. The numoer of colonies resistant to the antibiotic was expressed as a percentage of the number growing on the L-agar plate, and taken as representillg t~e proportion of ~iL2721~3 tha popul2Li~n carryin~ the plasmid. Sa;nples were re~oYcd periodically and plas~lid I,NA prepa~ations ~ade and annlysed for 2ny gross alte}ations.
Cha~ostat analysi3 of stability of p~'G165 and pi;'G16S in E.coli ~V30S havs demonstrated that these plas~ids are completely stable for at leas, 6S generations (Table 6) ~Yith tryptophan in the ~edium. Under these conditions transcription of met-prochy~osin gene on p~G168 was repressed.
Plasmid Stability E.coli Plasmid Stability Numb~r of ~e~erations St~in % ~ Trp - Trp r~v3o~ p~ l65 100 72.5 30 ~V3~ p`;G16S 100 68 ~n ~lOa (~)p~I~168 105 20 Stability analysis is still in progress for grovth in the abse..ce of ~ry~tophanl but a~ter 30 generations no plasmid loss has been detected.
~tability analysis of p~l&l68 in E. coli E103(8) is st~ -cgress but nftar 20 ~en~rations no pl2s~ cs had been obser~ed. The dual-ori~in plasnids appear ~o be s-table ~nder conditions of lo~v copy nu~ber, .?~.~ raplication being directed frorl the par~, pSC101 ori~in.
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Yt~3 - 2~
REFF.RENCES
Enta~e, J.S, Angal, S, Doel, ~I.T, liarris, T.J.P. Jen~ins, B, Lilley, G
and Lo~Ye, P.A. Proc Natl Acad Sci U~A 80 (19S3) 3671-3675.
llashi~oto-Gotoh, TJ Fran1~1in, F.C.~, Nordhei~, A and Tim~is, ~.N. Gene 16 ~19Sl) 227-235.
Ish-~orowitz, D and Bur~e, J.F. Nucl Acids ~es 9 (1981) 2989-2998.
iatis, T, Fritsch, E.F and Sambroo~, J. Molecular Cloning Cold Sprino ~arbor E3boratory (1982).
t~een, C. J ~ol and Applied Genet 2 (1983) 1-10.
r~ad, S.~i alld ~30rthcote, D.~. Analytical Biochem 116 (1981) 53-64.
Russell, ~.~ and Bennett, G.N. Gene 20 (1982) 231-243.
San~r. F, ~ic~len, S. and CoulsPn, A.r~. Proc Natl Acad Sci USA 74 (1977), 5~63-~4S7.
Sha~, '.~.V. ~'etho~s in ~nz~ology 43 (1975) 737-755.
Tatch~ , Nas~yth, ~.A and ~all, B D. Cell 27 (1981) 25-35.
. ~ , .
~ ~2~-~ j _ 29 -List of Escherichia coli strains mentioned in the Examples Strain Genotype Reference or Source DHl F ,rec~l,endAl,gyrA96, Man~atis et al (1982) thi-l,hsdR17(rk,mk) supE44,~
HB101 F ,hsdS20(rB,mB),recA13,Maniatis et al (1982) aral4,proA2,1acYl,galK2, rpsL20,(Smr),xyl-5,mtl-1, supE44,~
RV308 ~ ,F ,Smr,gal305 a) D900 F'i q,~p ,proA B /laci , J.R.Sadler (Denver, laco+,lac~+,lacy ,proB, USA) Sm .
QY7 F ,lacam,trpam, ~bio256- S.Brenner (Cambridge -cI857~ Hl, ~uvrB. UK) El03S L.D.Simon (New Jersey, USA) ~) ATCC is the Rmerican Tylce Culture C~llection desi~nation .
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~7~
The present inventlon makes possible -the crea-tion o~ a new l~ind o~ plasmid in which the copy number can be deliberately controlled by regula-table promote~ such as PL or PR`.
The presence of the XhoI linker 30 base pairs from the 5' end of RNA II allows the replacement of the natural promoter for RNA II by other promoters. Thus, plasmid pMG404 put RNA II under the control of the kanamycin resistance gene promoter on the plasmid 1~ pH~'15 it is believed that both originsin pr~404 func-ti~n, the teMperature sensitive pSC101 origin from ~H~G41~ ~nd the ColEl origin under control of the KmR
promoter. Although the exact point of DNA initiation h~s rot been determined in these plasmids, -the function-ing o~ the hybrid origin is indicated by the highercopy number of pMG404 than pHSG415, and the replication o~ p~IG40~ at 42C. The construction of pMG410, a recircularised BalI fragment carrying the hybrid origin but n~ the pSC101 temperature sensitive originj is ~0 ~dditional evidence that the hybrid origin is functional.
The properties of pMG404 demons-trate tha-t the RNA
II promoter and the first 30 bases of RNA II can be re-placed by another promoter without abolishing the initiation of DNA replication. However the Km ~5 promo-ter is constitutively expressed and it is not there~ore possible to alter the copy number of pMG404.
Several well-defined controllable promoters exist which ~unction in E.coli; the PL promoter from bacteriophage ~
w~s used to construct plasmid pMG411, with a ColEl hybrid origin under direct control. pMG411 retained the origin from pHSG415, and was maintained at a low copy number a-t 30 in a strain carrying a ts ~ repressor gene (QY7).
~lhen the ~ repressor was inactivated at 42, -the copy number increased, indicating that replication of the ColEl origin was being driven by PL. It is therefore clear that the copy number of such plasmids can be deliberately controlled from regulated promoters and this opens the way to constructing plasmids whose copy number can be controlled either by tempera-ture (as wi~h PL in pMG411~ or more importantly by the altera-5 tion of the concentration of metabolites such as trypto-phan or lactose.
Various other solutions have beenproposed to over-5 come the problem of s-table maintenance of high copy number plasmids in host cells. For example, in UK
Patent Specification No:1,557,774 it has been proposed to use mutant plasmids having a temperature-dependent plasmid copy number pattern such that the plasmid shows a controlled constant plasmid copy number when host bacteria carrying the plasmid are cultivated at one temperature, but an altered plasmid copy number pattern, allowing a much higher or to-tally uncontrolled copy number, when the host bacteria carrying the plasmid are .15 ~rown at a different temperature. Thus cells may be propagated to desired production size cul-ture at one temp~erature at which the plasmid replicates at low copy number and at which its gene products do not signifi-cantly-inhibit cell growth. The temperature ~y then ~ be altered, greatly increasing the plasmid copy number and also the corresponding production of gene products.
The introduction of copy number temperature dependence in such mu-tant plasmids, however, may introduce a source of instability into the plasmid, and i-t is likely that ~5 these mutant plasmids may be unstable or subject to loss when cells carrying them are propagated over a prolonged period of time.
The replication of plasmids is controlled by nucleotide sequencescontained within the overall DNA
3~ sequence of the plasmid. These sequences include a sequence defining the origin of replication at which DNA replication is initiated and often also associated sequences which control the :initiation of replication at the origin and the copy number at which the plasmid is maintained. For example, certain plasmids, of which ColEl is a typical example, have plasmid replication systems having a number of features in common, These systems comprise a DNA sequence defining an origin of replication and upstream thereof a DNA sequence coding for transcription, in opposing directions 9 of two RNA
species, RNAII and RNAI. The RNAII species provides an RNA primer which forms a complex a-t or near the origin from which DNA synthesis is initiated; -the ~NAI
species interferes with the formation of this initiation comple~, Transcription of the two RNA species is con-trolled by separate promoter sequences associated with the DNA sequences which code for their transcription.
In ~ddit.ion there is a small polypeptide (the rop protein) which is believed to interact with the promoter for RNAII; this polypeptide is not essential for re-plication and its role is unclear. The origin of replication, the RNA coding sequences and associated promoters together provide an internally self-regulated system,.which controls the replication incompatibility ?O and the copy number of these plasmids. Certain other plasmids, exemplified by RI and some Staphyloccocal plasmids, also control replication initiation at the transcriptional level, but by a messenger RNA species whose product provides an initiation factor, probably ~5 a polypeptide, which is involved in DNA replication.
It is an object of the present invention to provide new DNA vectors which have controllable copy number patterns and therebyovercome problems associated with stable maintenance of vectors which replicate at hi~h copy number only, which new vectors will not be subject to the potential instability of previously described mutant plasmids which have temperature-dependent copy number patterns, and which, furthermore, will have the advantage that their copy number can be controlled by agents other than temperature, e.g.
metabolite concentration.
Accordingly, in a first embodiment the invention provides a DNA vector comprising two replication systems;
a first origin of replication resulting in a low copy number and stable inheritance of the vector and a second, high copy number origin of replication at which replica-tion is directly controllable as the result of replacement or alteration by DNA manipulation of the natural vector s~uence(s) which control replication at said origin.
By means of the invention, when host cells carrying the vector are propagated under a first set of conditions, replication takes place mainly, and preferably exclusively, from the low copy number origin, and when the cells are pro~agated under a different set of conditions, replication takes place at high copy number at the second origin.as 1~ well and the production of large amounts of foreign gene products encoded by the vector is induced.
In a preferred embodiment the invention provides that the second, high copy number, origin of replication comprises an origin of replication and an associated DNA
-~ se~uence encoding an RNA species which provides a primer or i~nitiation factor (e.g. a polypeptide) which initiates DN~ replication by formation of a complex at or near the origin of replication, in which transcription of said RNA
species i~5 directly controllable such that, when host ~ells carrying the vector are propagated under selected conditions, replication takes plase at high copy number ~rom the ori~in and the production of large amounts of Eoreign gene products encoded by the vector is initiated.
~he invention also includes a method for the 3~ preparation of a vector according to the first aspect, ~:72~3 - 5a -comprising including in the DNA sequence coding for the second replication system a DNA sequence which per-mits direct control of replication at the second origin.
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Methods for the preparation of vectors according to the flrst aspect of the invention suitably com-prise ligating a first DNA sequence coding for the re-plication system comprising the first origin wi-th a second DNA sequence coding for the secondary replication system. The DNA sequence which permits direct control of replication at the second origin may be incorporated into the second DNA sequence either before or after ligation with the first DNA sequence. Thus the inven-tion further provides methods for the preparation ofvectors according to the second aspect of the inven-tion 9 comprising including in the second DNA sequence a DNA
s~uence which permits control of replication at the second origin by controlling transcription of the RNA species.
~5 The invention further includes a process for the production of a polypeptide, protein or other gene product which comprises transforming host cells with a vector according to the first aspect of the invention, in which said vector contains a gene sequence coding for ~0 production of said polypeptide, proteinor other gene product, propagating said transformed cells under a first set ofconditions at which replication takes place at low copy number mainly, and preferably exclusively, from the first origin, and then propagating said ~5 transformed cells under a second set of conditions at which replication takes place at high copy number also (or exc-lusively) from the second origin and the expression of said polypeptide, protein or other gene product is induced.
By means of the process of the invention, trans-formed cells are propagated to give the large scale culturesrequired for economic production of polypeptide~
protein or other products under conditions where the vector replicates at low copy number and the instability problems associated with high copy number vectors are avoided, followed by propagation under different condi-tions where the vector replicates at high copy number ..~
~ 2~zle~3 wth concomi-tant high yield of polypeptide, pro-tein or other products.
In particular it has been found, when the ex-pression of the gene product is under the control of a promoter which is regulated by cytoplasmic levels of a repressor, that the increase in vector copy number leads to outstripping of the repressor control and high level expression of the gene product. This is so even in the case when the synthesis of the repressor is autoregulated, e.g. when the promoter/repressor system is that of the tryptophan operon.
The control systemswhich are used to con-trol the copy number of the vectors of the invention may comprise anyof the control systems which are known for controll-ing replication (and/or expression) in recombinant DNAtechnology. In particular, the copy number of the controllable origin of replication may be controlled by temperature or one or more metabolites or metabolite analogues. Examples of metabolite-dependent sys~ems which may be used include: tryptophan, lactose, galactose, arabinose or any other metabolite or meta-bolite analogue, the presence, removal or further metabolism of which can be used to activate transcrip-tion from a given promoter.
~5 The controllable replication system used in the vectors of the invention may be derived from high copy number cloning vectors, such as ColEl-like plasmids, e.g. pAT153, NTPl, CloDF13, RSF1030 or P15A, which ha~Je eopy number control systems which involve transcription 3~ o~ RNAII or a similar RNA species which provides a primer which initiates DNA replication by formation of a complex at or near the origin of replication. In addi-tion other plasmid replication origins whose replication is controlled by an mRNA species and/or its product~s~
may provide the controllable replication systems used in the vectors of -the invention. Such replication origins ~7~L3 - 7a -may be obtained from Gram ve bacterialspecies and are exemplified by Rl, R6, R100, RP4, or Gram +ve bac-terial species, which are exemplifiedbypUB100, pC194 and certain other Staphylococcal plasmids. Furthermore, bacteriophage origins may be used for secondary con-trollable origin, e.g. those from ~, T3, T4, T7, M13,~X174, SPPl, SP02 etc.
In an important embodiment of the invention, the controllable replication systems may be prepared from such high copy number cloning vectors by replacement of 1~ the natural promoter, which pr~motes transcription of the RNA species, by a contr~llable promoter, such as the PL promoter, PR prorroter, Pre pr~moter, P'R prornoter, T71ate pr~moters, t~p prorr~ter, tac proToter, lac promoter, gal pr~rnoter~ara promoter or _ecA promoter 5 (the origin OI replication in such a system is tenned a "hybrid origin"). Altematively the natural promo-ter may be used and trans-cription of the RNA species made controllable by incorpc)rating a regulating f~nction, such as an operator sequence, e.g. the lac operator or OL or OR operators of phage lambda~into the replication systems.
The plasmid pMG9 (containing an Xho I linker DNA
sequence inserted as described by K Tatchell e-t al, Cell, Vol 27, pages 25-35, November 1981 (Part 2)) provides a convenient starting material for preparation 15 Of a controllable replication system based on the ColEl replication origin. This plasmid has a unique Xho I
restriction site, close to the start of the sequence coding for transcription of the RNAII species, which we have found may be used for insertion of operator and 20 controllable promoter sequences, e.g. ;tPL, to give a directly controllable replication system. Plasmid pM&9 was deposited at the National Collection of Type Cultures, Central Public Health Laboratory. Colindale Avenue, London NW9 5HT on 24 ~. rch 1983, under NCTC
No.11539, as a culture of cells of E coliK12, strain DHl, containing the plasmid pMG9.
The replication system comprising the first origin of replication may be obtained from any suitable low copy number plasmid. For example, a replication 30 system comprising the pSC101 origin may be used, and the plasmid pHSG415 (T Hashimoto-Gotoh et al~ &ene, 16 (1981) pages 227-235) provides a convenient source for such a replication system. It will be appreciated that pHSG415 has a temperature sensitive replication 35 origin, but that this origin may be replaced by its wild-type temperature-stable counterpart from pSClO1.
pHSG415, however, provided a convenient, temperature ~ 272~L~3 sensiti~e replication origin for use in the exarnples hereinafter described.
Controllable functions may be incorporated into high copy number replication systems, high copy number and low copy number replication systems may be ligated, and foreign genes may be incorporated into the vectors to produce vectors according to the invention using techniques which are known and understood by workers skilled in the recombinant DNA art. The resultant vectors may then be used to transform suitable host cells usina standard procedures -to produce foreign poly-peptide, protein and other products.
The host cells may comprise eucaryotic cells, includir.g yeast cells e.g. ~.cerevisae, or, more usually, bacterial cells, of species such as B.subtilis or, especially, E.coli.
I
.
Tne invention is furche} ~escribea vy way of illustration only in the following E.samples, Examples 1-7. These Exan~ples relate to the construction of spacific dual origil~ plasmids according to the invention, and to studies of copy number control, heterologous ~ene e~pression and stability of tllese plasmids. It will be appreciated that the invention is not limited to the specific plasmids and methods described.
Thesa E~amples refer to tne accompanying diaDrams in which:
~ iguro 1 shows plas~id restriction maps and indicates the DNA
~aniplllations which were used to prepare a dual origin plasmid according to th-e il~vention, p2lG411;
Finure 2 is an agarose gel oL DNA isolated fro~. c~ltures of a p~7G411 trans~ormaint of E.coli QY7 ta~en at hourly intervals following a tenperatLre shift from 30C to 42C (lanes 1-7, hours C-6);
Figuro 3 snows plasmid restriction maps and indicates the DNA
~anipulations used to prepare two furtller dual origin plasmids according to ehe invention, p~iGl~9 and p~lG16~;
~ i~ur3 ~l is a graph of copy nu~ber induction following a te~perature shi~t (30C to 42S with subsequent incubation at 37C) of trans-~ormants of ~.coli carrying pr.l&lS9, pMGlbS, pb!G168 and p~ l69;
~ igure 5 shows plasmid restriction maps and i~.dicates tl~e DNA~anipulations used to prepare a metabolite controllable dual origin ~ector according to the present invention, pPIG427;
~72~3 Figure S sho-Ys plasmid restriction maps and indicates the DNi~
~anipulations used to prepare two f~rther metabolite controllable dual origin vectors according to the invention, p~lG415 and p.~.iG416;
Figure 7 is a lG.~o polyacrylamide gel of protein ~ro~ total cell e~tracts prepared from E.coli E103(S) transforrJed by p~lG169 after tenperature shift treat~ent, samples being taken at hourly intervals ~lanes 1-8, hours 0-7);
~ igure 3 is a graph showing incrcase in plas~id copy nu~ber and ~hlorn~pllenicol acetyltransferase activity for cultures of E.coli El03(S) transforrled by pi'>lG169 after teDperature shock treat~ent, and Figure 9 is a 10~ polyacrylamide gel of protein from total cell e~tracts of cultures of p~lG169 transformed E.coli ElU3(S) at hourly intervals over a perio~ of S hours after te~perature snoc~ treatment (lanes 6-l, hours 0-5).
ElV~lPLE 1 Constr~ction of ~ Dual-origin Plasmid ~ith Copy Number under Pl Control (a) Construction of a Dual-origin Plasmid pMG4U4 Plasmid p~Gl5 (Figure l) is a derivative of ~Sa-12 (pBR322 replicon) which has constitutively high copy nunber (approxirnately 300 copies per chromosor.e) due to the insertion of a XhoI linker (CCTCGA~G) into the ori~in region (Table l) (Tatchell et al, 1981)o During construction of ~S~ the ro~ gene was lost by spontaneous deletion extending beyond the ~S~1I site in the tet gene, bllt reLnining the Bami1I site. Tne position of the hoI linker ~vas determined by inserting the l.lkb ~hnI-P~m~1I DNA fragment of p~-!Gl5 into bacteriophage ~.113mp~ and DN~
sequencin~ from the XhoI site by the method of Sanger et al, (1977).
Ins_rtion of the linker was at a point 30 bases do-N~s~raam of the tra1lscriptional start of ~AII (Table l). ~lis ~hoI linker provided a ~iquc clcavn~e site close to the S' end of the ~lAII transcript, such that th~ l.lkb ~hoI-Xa~-lI fragment isolated from p~lGl5 carried a promoter-lcss P'~AII sequence (Figure l). To determine if this seq-~ence could fl~ction as n primer of DNA replication when coupled to another promoter, the l.lkb fra~ment was inserted downsteam of the ~m resistance gene (~rl~) nromoter of pl~s~id pT1S~4lS (Tlashimoto-Gotoh ~ al, 1901) ~Fi:~urc 1). p~SG415 is a stable low copy number Apr~ ~m~ Cn1~ plasmid whoso replicQtion origin is derived from pSClOl; this plasmid replicates at 30~C b~lt not ~t 42C.
Site of Insertion of XhoI Linker near the Origin WILD TYPE S~nUE~'CE
~h'AII
~' CTTGC~-~ACA~A~ CCACCGCTACCM CGGTGGTTT
~555 p`~lG15 SEQUENCE
P~NAII
5' CTTGC~U~C.~A~ ACCACCGCTACC~C~GTCCTCGAGG&&TT
I XhoI Linkei -~55 Conditions for restriction enzyme digestions, DNA ligations, and sg3rose gel cl3ctrophoresis were as described in ;~;ianiatis et al (1982).
Insertion of the XhoI-~amEI frag~.ent fro~ pilG15, coupling the pIomoterless ~'~AII sequenca to the ~mP~ promoter, gave tne dual origin plas~id pr~lG404 (Figur~ l~. Analysis OI the plasmid pilG404 by restriction en7~e dig~stion verifie~ its structure (results not shown), and plasmid r~ yields demonstrated that its copy number was considerably higher than thnt o~ p~iSG415, In addition, pdlG404 was maintained in bac,eria at 4~C
wll~r3ns p}iSG4l5 was lost due to its in3bility to replicate at that ~p3ratur-e, It was conclllded tnat the hybrid 'ColEl origin' w~s functioning in prllG404, da~onstrating that the ~ gene pro~oter could substitute for tne ~NAII promoter, Confirmation of the functioning of the hybrid origin was obtained by digesting p~.G404 with BalI and recircularising the larger fragment .. . . . ..
~2~
co~ltaining onl~ t~e hy~rid ori~in. This ~eneraced a plasr,~id ~L~ which ~vas capablc of antono~ous replication at both 30~ and ~2C, clemonstrating thæt tne hybrid origin was functional.
(b)Construction of Dual-origin p~G411 Plasmid p~lG404 has two origins of replication: the lo~v copy number origin of pSC101 and the 'Col~l' hybrid origin. The pSC101 origin should ensura stable replication of the plasmid at 30C (~shi~oto-Go.oh et ll, l9~1), allosYing the construction of derivati~es with the 'ColEl' hybrid Ol`i~iU driven by a contro]lable pror,~oter. A 0.5~b SalI-~coRl `ra~ment carryin~ .he Pl promoter OI bac~eriopnage ~ was isolated from the plasmid pCT10~0 (Figure 1) and ligated to the 5.5kb EcoRl-XhoI
fr~gment from p~lG404. The ligation mixture wæs used to transform E
coli ~Y7 (l cI~57 defective lysogen) and D~ll at JO~, and the Apr~
.ransfor~ants screened for CmS clones. One of these, prlG411 was chosen ~or further st~dy (Figure 1). Plasmid phG411 transformants of Dl~l and QY7 ~rew well under selective conditions and copy n~mber determinations were m~de ~t a variety of gro~vth temperatures.
~c)Copy Number ~leasurements of phlG411 Plasmid copy num3er ~vas determinecl by tl,vo mothods. One aepended upon tho separation of chromosomal and supercoiled DNA by eaesiu~ chloride eentri~ug~tion~ Tho second method depended upon the separation of Ghromosomal and plasmid DNA by agarose gel electrophoresis. Cells were grown overnight in L-broth containing ampicillin, and diluted one hundred fold into minimal ?,'9 salts medium containing a~picillin. At an OV~60 of 0.~, 2-deo:cyadenosine (200 ~g/ml) thymidine (1 ~g/ml1 and ~3il]-thymidine .
IZ7~ 3 ~ ~g Ci/ml) wera a~ded, and incu~ation continued for 2 hours. Cells wcrc centrifllg-d, ~Yashed and resuspended in an equal volume of 50 ~F,J tris L~Cl 7.~ sucrose (-~/v) and lysozyme (200 ~g/ml). After incubation for minutes at 0C, EDTA y1as added to a concentration of 10 ~I and incubation contined for 10 minu~es, and finally sarkosyl ~0.4~ w/v) ~as sdded. Chromosomal DNA in the lysate was sheared by 6 passages through a 1~ ga~ge needle, and eell debris removed by centriEugation. ~NA
preparations were futher purified by a single phenol and chloroform e~traction and ethanol precipitation. This labelled DNA prepaIation was th~n u~ed for eopy number determination by either method.
For the ngarose gel method, samples were electrophoresed on a 0.7$
a~arose gel (in 0.04 ~i tris acetate, 0.001 ~I EDTA, plI 7.9) for 12 hours at ~0 V. DNA bands were visualised by stai~ins in ethidiu~ b~omide. Tke chromosomal~ and plas~id DNA bands were cut from the gel, dissolved in saturated sodiu~ iodide and the DNA precipitated by the addition of 10~
(wlv) triehloroacetic acid (TCA). Precipitates were collected on GF/C
lihat.~lan filters, ~ashed in 1~ (w/v) TCA, ethanol and then air dried.
~adioactivity was determined by counting in a liquid scintillation counter, ~ or eopy nl~er determinations by caesium chloride ce~trirugation DNA
s~rl~lcS ~vere eentrif~lged to equilibrium in caesius chloride-ethidiu~
bro~ a in a r.ec~man TI50 rotor at 4S,000 rpm for 24 hours. The gradients ~ere fraetionated, and the fractions precipitated witn TCA onto GF/C
~`~hatman filters, washed and processed as described above.
~ 3 PlasmiG GOpy l~r~nbers were determined fro~. pIIGl5, pAT153 and p~iIC411 at 30C, 37C and ~C and the results obtained are given in Table 2.
. . .
Variation of Plasmid Copy N~bar with T~mperat~re PlasmidBacterial Strain Percenta~c of Copies pRr (growth temperature) total DNAChromosome n~;Gl~~101 (37C) 33 309 p~l`l5~ ~E101 (37C) 5.~ 59 ~:G~llQ~'7 t3noc) 0.7 4 ~t~741.' ~Y7 (37C) 13.~ 7 p~lG411 QY7 (42C) 24.~ 143 I~Ieasurenents of p~lG411 copy nt~ber in strain QY7 ~l lyso~en) at various te~ceratures de~onstrated that tne A repressor controls pi~G411 cop~ number.
After Drowtlt at 30C, copy n~ber was estimated at 4 per c~ro~.osome, ~hilst at 37C it had increased to 78 and at 42C it had increased to 1~.3 ~er chror~.oso~e (Table 2).
To de~onstrate that the hybrid 'ColEl' origin in p~'G411 could be s~vitched OII by inactivatino the ~ repressor, a transform~tnt of QY7 was ~ro~vn at 30C~ and then the tempe~ature raised to 42C durin~
o~ponantial ~rowth. The plasmid copy number increase ~as followed by agaroso gcl analysis of D~IA isolated from 1 ml cultures ~Figure 2).
Samplas wera taken at 1 hour intervals over a period of 7 hoursO As prodictod, an increase in pbG~ll copy number was observea over the 7 hour period.
EX~IPLE 2 Constrllction of Dual Origin Plasmids with Ind~cible Copy Number under Pr Control ~)Construction of Dual Origin Plasmids pblG159 and pMG165 Plasmid p~lG411 has two origins of replication, the low copy n~ber origin of pSC101, and the Pl-driven ColEl origin. Since the repressor gene, cIgs7 is resuired for copy number control~ this limits the use of p`l.'G411 to lysogenic bacterial strains~ To overcome this constraint, a du~l-ori~,in plasmid carrying the cIgs7 gene was constructed.
~ irstly, to facilitate subsequent cloning of foreign genes an oligo-nucleotide comprisin~ a SalI and nindIII restriction site was inserted into the a~I site immediately downstream of the hybrid origin in p~ ll; this gave p~rsl53 (Figure 3). The 5.9'~b EcoRl-Ea~II fragnent of p~ 71:;3 w~s purified and ligated to a l.lkb ~col~-Ea~lI fragnent of p~V2 (Queen, 198~), to give pi.lG159. The EcoRl-Za~I fragment of pCQV2 cnrries both Pr and the cIs~7 gene, and on insertion into p~C153, r~places the Pl containing fragment, fusing the ColEl origin RNAIT Seqnence to Pr. Note that the repressor gene is transcribed away fron tne origin IFi~ure 3). To increase plas~id stability (see bclow) when the dual-origin v~ctcrs are maintained at 30C, the pS~101 2.85kb ~incII fragnent ~nrryin~ the ori~in of replication, replication protein and par sequence, ~las introduced into p.,lG159, by replacine the 3.25kb EcoRl-BalI fragment w~ith the pSC101 l~incII fragment with an EcoBl linker at one end. This ~ave ph'&l65 ~Figure 3).
(b)Copy Number Control of Pr Driven Dual ~rigin Plasmids To demonstrnte t~at the 'Col~l' hybrid origin driven 'Dy Pr was controlled by tl1e ter.~per~ture sensitive repressor expressed fro~ ~he cloned cIs~7 gene, p~'GlS9 and plUGl65 were transfo~med into E.coli El03(S).
Copy number determinations at 30C were made as previoasly described from 31~-thymidine labelled cells (Table 3). Copy number induction after temperature sihift to 42C was followed by agarose gel electrophoresis.
To quantitate the copy n~ber change and kinetics of induction in L-brot~, a 'spot' hybridisation method was used.
T.~BLE 3 Variation o~ Plas~id Copy Nu~ber with T3mperaturs Pl~smidB~cterinl Strain Copies p~r Chromosome (g.owth temperature) p~ 9~103(S) (30C) 3-4 p~ 103tS) (30C) 3-4 ~Gl5gElO3(3) (~2C~ ~ 320 p'~ l5~El03(S) (4~C) ~ 248 ~ or the 'spot' hybridisation method 32P-labelled probe DMA was pre~a~ed by n-cl~ translation (-`~aaidtiS et al, l~?). D~A samples were orepared by tne al~aline lysis method (Ish-~orowitz and Rurke, lgSl) from l ml of cu1ture takel1 at various times after the temperature shift. Samples Or thc D~A preparations were treated with P~!ase at 37C for 5 minutes, follo~Yed by incuDation with restriction enzy~e ~a~I for 30 ~inutes, and héat denaturation at lO0C for 3 minutes. Varying amounts of the digested ~IA preparations were spotted onto nitrocellulose filters, which were then dried at 70C, ~.nd subseq~ently hybridised ovcrnight (15 ho~rs) with t'ne den~tured probe Di`~A at 37C in 2 x SSC/50~ for~amide. ~ilters ~vere thell washed twice in 2 x SSCtS0~ formamide and once in 2 x SSC, and then air dried, ~adioactivity was determined by co~ttin~ in a liquid scintillation counter. The values obtained were corrected for cell growth durin~ induction, and the magnitude of the eopy nu~ber change over the uni~dueed value was determined.
~ otll p~i~l5~ and pi.G165 sho~led a rapid copy number induction ~ollowing a t~p~ratuxe shift to 42C and continued incubation at 37C. Uninduced valuas of 3-~ copies per chromosome rising to 90-100 copies after 2 hours inlluetion, and up to 300-400 eopies after 4-5 hours induction were obtained (Fi~u~e 4), clearly de~onstrating the eontrol of copy n~ber e~erted by the eloned Pr promoter and cIgs7 gene.
EXA~LE 3 Construction of Dual Origin Plas~ids with Inducible Copy N~nber under Ptrp Co~trol The t~o ~bove Esatnples of dual origin plas~nids with eontrollable eopy nl~ber, emI)loyed the use of temperature as the indueing agent, this ~sample desGri'~es 'clle use of a ,metaboii~e to control copy nu~b~r. '~he 6.2~b llindIII-E~lII fra&ment of p~iG411 was ligated to the 0~65kb ~indfII-~a~I fra&~ent o~ pCT54 (Figure 5) using standard burfers and techniques ('~!nniatis et al, 1982) to give pMG426. This plas~id was unstable even under a~pieillin seleetion and witll tryptophan present in the medium (whicn should repress transeripcion fro~ Ptrp). It ~as concluded that the instability was due to the inability to repress completely transcriptin ~IOm Ptrp, tnereby giving a high copy nl~mber. To decrease transcription tllrough the origin, a transcriptional ,erminator ~as inserted behYeen the Ptrp nlld the 'ColE1' origin sequence. Such terminators reuuce levels o.
transcripLion appro~i~ately 10-fold. ~ l~Obp AluI fragment from bncteriopll3~e T7 DNA, carr-~ing the early transcription terminator was lignted to DNA lin~ers converting the termini to ~indIII recognitin sites (Emtaoe et al 1983). This fragment was then lioated to ~indIII
di~ested pP~Gl~ DNA to give p?'G427 (Figure 5). p`&427 was more stable than p~iG4~5 and when tranfor~ants were gro~Yn in ruedi~ containing tryptophan ~00 ~ it e~Yhibi~ed a low copy number. '.'1hen such a culture was ~llift~ in~o ~ediu~ lac~ing tryptophan, the copy number increasea rnpidly.
~nis demoilstratcs that ~ II transcription can be controlled by the levels r.~tn~olites or chemicals in the external medium, and that controlled copy ~I~.ber chQn~es can be effected by agents other than temperature.
~LE
.
Construction of Dual Osigin Piasmids with Inducible Copy Number U~der Control of the 'tac' Promoter In this E~nmple a vector ~ns constructed where the ~II promoter was r~placed ~y the tnc promoter tPtac) (Rllssell and Bennct 1982), s-ch that ~py n~ber wns controlled by the addition or removal of lactose or a ~a~tose nnnlo~ue. l~ 121bp Ba~ E,coRl DNA fra~ment carrying the Ptac ~ns purificd fro~ pD~5~0. This fragment was ligated to Bam~I-Eco~l di~ested p;~G40~ D~A> and a~picillin resistant, chloramphe~icol sensitive transformants of ~.coli D~l were isolated. Plasmid DNA fro~ one such ~rnnsformant was isolated, analysed by restriction en~yme digestion and shown to contain the 121bp promoter fragment inserted into the chlor-. ., ampllenicol resistance gene (pl,.G421) (Figure 6). p;;lfi421 ~N-~ was digested ~ith ra,,llI, treated with calf intestine alkalinc phosphatase, and ligated to a l.~'ib Bam~II proL~oterless origin fragt~ent isolated from p-l~411.
~picillin resistant transfor~tants of E.coli D.~l were obtained when the tr~nsEorDation mi~ture was plated at either 30C or ~2C. At 42C, ~he low copy nu1~be. origin is inactive. Two distinct plasmid types were identified fro~t the transformants (p~,G415 and pi-lG416) ~Figure 6). pMG415 car~ied 3 single BamLTI origin fragment in the correct orientation, d~nstr~m of the t~lc, whereas p.~S~416 carried 3 copies oE tke origin ~x~ e~.lt as direct repeats, also orientated for e~pression frott~ Ptac II`C 6).
Plasmids p~ 415 and pli~416 were transformed into E.coli D900 ~I CIsq)~ a strain which overproduces t e lac repressor, the controlling element of the Ptac promoter. Induction of Ptac can be ~ffdct~d by the addition of the lsctose analogue IPT~ (isopropylthiogalac-toside~. p~.~G41~ transformants of D900 grown in L-brot~ had a low copy num~r as judged by agarose gel electrophoresis, but this was not increased hy the addition of IPTG. p~lG416 transformants of D900 grown in L-broth ~lso h~d a low copy number, but this increased quic~-ly on the addition of ~ to the culture, demonstrating controllable copy number induction of p.`~ lu f`ro-~ Ptac.
E~ iPLE 5 The E3pression of the Calf Stomach met Prochymosin Gone Cloned into a Dual-origin Plasmid (~)Construetion of p~lG168 To demonstrate that dual-origin plasmids ,vere useful for the e~pression of clotled heterologous genes in E.coli, a plasmid ~vas constructad carryin~ the calf storiach met-prochymosin gene. pCT70 (~mtage et al 19~3) was digested ivitll llindIII and SalI alld a ~ 2.41cb fragment e~rryin~ the mat~proc3lymosin gene under Ptrp control was isolated. This ~ra~ont ~as li~atA(l to t~vo DN~ fragments isolated from p'liil65. the 5.6kb ~atl-~stI ra~rlellt and the llcb IlindIII-PstI fragment ~Figure 3~.
Tho r~sulting plasmid (p~lG168) isolated from transformants OI E.coli ~i~l, coDprised the cloned gene do~-nstream of .he origin of replication, such th-qt any transcriptional read through f~om Pr. ~vould lead to ~dditional transcriptio~t of the met-procnymosin gene (Figure 3).
(b~pression of ~iet-prochymosin Protein from prlGl6~
p'lCl6~ was trausfori,ied into E,coli E103(S) and met-procnymosin P~prassion nn~lysed by polyarrylaDIide gel electrophoresis. E.coli l'.lG3ts) trnnsfor~iants were grown in L-broth at 30C to an OD600 o-4, ranol s'rift_d to 4 C. Cultures ~vsre tihen incubated at 37C w;tih shil~in~, and samplcs removed at hourly intervals for analysis of eopy number and protoin e~pression. DNA copy number ~Yas l~setermined as deseribed ~or p;~G159 and pi`~!G165. For analysis of protein, each cultu~e sa~iple ~vas eontrifu~ed, the pellet collected and resuspended in stop bllffer (1i~J SDS, lO i~l tris ~ICl p~ 7.5) and an equal volluiie oE sample buffer (0.12 rl tris slCl pll 6.~, 2G5 glycerol, 1,2 ~ mercaptoethanol, 6ri'~ SDS) a~id boile~L for 3 ~i.tut~s.
I'oly~cyla~ 1e ~el electropilorcsis, stainin~ and dcst~ lin~ r,.etnous ~Yerc essentially as described i1~ ~'aui2tis et a1 (l9P2). ';taine(l polyacry-larlidc ~els ~vere sca1lncd usins a Jo~cc 1,oc~1 Chro~oscan at 530 1~m. 130th D~r~ pl~smia copy nu.-lbcr an~ mct-prochy~osin protcin levels werc ~reatly increased following a ~emperature s21ift from 30C to 42C (Table 4, ~igures 4 an~ 7).
Copy nu~ber ir~ere3sed rapidly durin~ a 90 ~1inute post induction period ~t 37~C, ~hilst ~et-prochy~osin acc~:1~latio~ was ~ore gradual. On the basis ~ polyaeryl~ide gel seanning, t~e recor.bin~nt gene pro~uct ~ccumul~ted to 2t least lr~ of total extractable protein by 4-S hours after tine tempera.ure shift. Induced copy number values for p~;~l6P, uere lo~_r thln for the parent dual ori~in plasmid (p~'G165). but still increased fro~
3-~ per c'~rorosome to 120-150 per chror.osome. Cell viability fell fo1lo-~in~ induetion of p';Gl6 bu~ not of p~Gl65. It ~as coneluded that th;s loss in ~iability resulted fro~ the to~ic aeeurlulation of reeo~binant ~ene psoduct.
1`Jt~
Incrc~se in hlot-Prochymosin Gene ETpression Timd after Induction ~ ~otal Protein as at 37C (hrs) met-prochymosin O < 0.5 3 7,08a I 9.09 10.25a a) Tnese measureQents are from ~el scans; t1:e unin~uce(l levels o~ protein are di~ficult to measurc accurntcly 1"~ tllis mct1loa.
I`
- 2~ -~L~ 6 Expression of the Chloramphenicol Asetyl Transferase G~ne Cloned onto a Dual Origin Plasmid (~L~ Construction o phlGl60 Bac~use of the liraitations in the accurate quantitation of stained protein b~nds on polyacrylamide gels, cloned ~ene e~pression on dual origin vncto~s ~s further quantitated by assaying the increased activity of c`nlor~ph2nicol acetyl transferase following copy nur.~iber induction of ~lasl~id p.~ l69. Plas~id p~;G169 was made Erom p~lG16~ in an analagous ~ay to cha e~onstruction of p~lG16. (Example 5), except that the purified ilindIII-1 rar~ent carried the structurial gene Cor chloramphenicol acetyltr:~nsf-ar~se ~nder control of Ptrp (Fi~ure 3).
b)~Y~r~ssion of Cloned Chloramphenicol Acotyl Ts~nserase p~ rl~9 W~S transformed into F.coli ~103(S) and chloranphenicol acctyl transIernse levels Laeasured by polyacrylaL~ide gel electrophoresis an~ zyne assay (Sllaw 1975). E.coli E103(S) ~ransform~nts were grown n~ i~dllced by te~,perature shift to 47.C, followed by continued ine~ubation at 3?C as described for p..lG163 transformants in Fxa~ple 5.
Sa~l~s were removed at hourly intervals and plasmid copy n~aber determined ~ ur~ 4, ~)~ chloraripher.ico'L acetyl trar3ferase specific aci~ivities dat~rL~inad (Sllaw 1975, ~ead and h'orthcote 19~1), (Figure 8, Table ~), and 3~ples run on poly3crylamide gels (Pigure 9). The plas~;d copy number incre~sed with similar kinetics to that of p~i'G168, and with si~ilar absollte v~lues (Figure 4). Chloramphenicol acetyl transferasè iLssays àa~onstrated that the spccific activity of the en~y~ie in crude e~tracts increased appro~ iately 80-fold as a res-llt oE the copy nu~ber induction 2~
~igere ~.). C~lc~la~i~as o~ t~e pLorortio~ oL ~A;tl-3c,~ble ~rotcin preseIIt as chlorallphenicol acetyl transferase were madc from a ~noiwled~e of the specific activitj of the pure protein (Table 5~. Uninduced levels c~pressed from p`;~l69 represented 0.25~ extracted protein, whilst afteI 6 ho~-rs induction tnis had risen to 21.S5.
Increase in Chloramphenicol Acetyl Transferase Specific Ac~ivity Tlm~ ~t~r Induction a) ~ Total Protein as at 37C (hrs) ChloramphenicoI
acetyl transfe~ase 0 ~.25 5.6 2 15.5 _ 18.1 18.5 21.~
6 21.9 7 20.9 e~e vall~es were calculated from the l~own specific activity of pnre c]~lorampllenicol ncetyl transfernse ~195 units pcr mg protein).
-~A~PLE 7 Plasmid St~bility S~udies of Dual Origin Plasmids Plasmids p~iG165 and p~i!G16~ were transformed i~to E.~Q~ P~Y30~.
.nd p~;G163 into E.coli E103(S) to study their stability at 30Cder conditions of chemostat growth without antibio~ic selection. All cxperimen,s were started from a single colony o~ the appropriate E.
. . ~ . .
Goli strain, ta~c~ iro~ an antibiotic-containing a~ar plaLe and inoculated into t00 ~ls of L-broth i~ a 250 ml conical flask with steel spring baffle. The culture W2S incubated in an orbital shaker (37~`, 240 rpm) until stationary phase was reache&. ~ne cells were harvested, resuspended in sterile defined medi~ minus glucose and inoculated into a fer~enter vessel. ~le defined medi-~ was glucose 4 gl~ ~4/2S04 5 g 1~ a2HP04 7 gl-l; ~2P0~ 3 gl-l; proline 200 ~g 1-1;
leucina, 100 mg 1-1; thiamiile 10 mg 1-1; rlgS04.7H20 200 mg 1-C~Cl~,6~0, 5 mg 1-1, ZnS0~.7~0 20 ~g 1-1 1iinSO~.4H20 2 mg ~ m~ ; CuS0~,5~20, 5 mg 1-1, CoC12.6H20, 0.5 mg 1 1; ~cS0,t.7~0 100 m~ ; NaCl 200 mg 1-1; E~TA ~ia2 600 mg 1-1; ~a0H 1~0 ~g 1-1]. Tnis nedium was supplemented with tryptophan 100 ~g 1-1 unless othen~ise stated. Anti-foam (polypropylene-glycol 2000) present in the ~ediu~ at 0.001r,~ v/v, Tlle fermenter cell population wns allo~Yed to grow 2S a closed batch system until the biomass ~as at least 605'~
of the maxi~um supported by the medium. The pump was then turned on and t~a ~ystem run as a cher,~ostat. When the total bio~ass i3 the fermenter was constnnt, it was assu~ed that the initial transient gro~vth phase had ceased ~nd thera~fter the number of generations in the steady state was calculated usin~ tha formula~ n(nur.~ber of generations) = ~tlln2 whe~e ,u = growth rate whic~ is aqual to the dilution rate under steady state conditions, and t =
t.ima, S3m~1es wc~e withdrawn from the continuous cultures, diluted and plated onto L-a~a~. 100 single colonies were picked onto both antibiotic supplc~entcd agar and L-agar as a control. The numoer of colonies resistant to the antibiotic was expressed as a percentage of the number growing on the L-agar plate, and taken as representillg t~e proportion of ~iL2721~3 tha popul2Li~n carryin~ the plasmid. Sa;nples were re~oYcd periodically and plas~lid I,NA prepa~ations ~ade and annlysed for 2ny gross alte}ations.
Cha~ostat analysi3 of stability of p~'G165 and pi;'G16S in E.coli ~V30S havs demonstrated that these plas~ids are completely stable for at leas, 6S generations (Table 6) ~Yith tryptophan in the ~edium. Under these conditions transcription of met-prochy~osin gene on p~G168 was repressed.
Plasmid Stability E.coli Plasmid Stability Numb~r of ~e~erations St~in % ~ Trp - Trp r~v3o~ p~ l65 100 72.5 30 ~V3~ p`;G16S 100 68 ~n ~lOa (~)p~I~168 105 20 Stability analysis is still in progress for grovth in the abse..ce of ~ry~tophanl but a~ter 30 generations no plasmid loss has been detected.
~tability analysis of p~l&l68 in E. coli E103(8) is st~ -cgress but nftar 20 ~en~rations no pl2s~ cs had been obser~ed. The dual-ori~in plasnids appear ~o be s-table ~nder conditions of lo~v copy nu~ber, .?~.~ raplication being directed frorl the par~, pSC101 ori~in.
.. .. ... .. .. ..
Yt~3 - 2~
REFF.RENCES
Enta~e, J.S, Angal, S, Doel, ~I.T, liarris, T.J.P. Jen~ins, B, Lilley, G
and Lo~Ye, P.A. Proc Natl Acad Sci U~A 80 (19S3) 3671-3675.
llashi~oto-Gotoh, TJ Fran1~1in, F.C.~, Nordhei~, A and Tim~is, ~.N. Gene 16 ~19Sl) 227-235.
Ish-~orowitz, D and Bur~e, J.F. Nucl Acids ~es 9 (1981) 2989-2998.
iatis, T, Fritsch, E.F and Sambroo~, J. Molecular Cloning Cold Sprino ~arbor E3boratory (1982).
t~een, C. J ~ol and Applied Genet 2 (1983) 1-10.
r~ad, S.~i alld ~30rthcote, D.~. Analytical Biochem 116 (1981) 53-64.
Russell, ~.~ and Bennett, G.N. Gene 20 (1982) 231-243.
San~r. F, ~ic~len, S. and CoulsPn, A.r~. Proc Natl Acad Sci USA 74 (1977), 5~63-~4S7.
Sha~, '.~.V. ~'etho~s in ~nz~ology 43 (1975) 737-755.
Tatch~ , Nas~yth, ~.A and ~all, B D. Cell 27 (1981) 25-35.
. ~ , .
~ ~2~-~ j _ 29 -List of Escherichia coli strains mentioned in the Examples Strain Genotype Reference or Source DHl F ,rec~l,endAl,gyrA96, Man~atis et al (1982) thi-l,hsdR17(rk,mk) supE44,~
HB101 F ,hsdS20(rB,mB),recA13,Maniatis et al (1982) aral4,proA2,1acYl,galK2, rpsL20,(Smr),xyl-5,mtl-1, supE44,~
RV308 ~ ,F ,Smr,gal305 a) D900 F'i q,~p ,proA B /laci , J.R.Sadler (Denver, laco+,lac~+,lacy ,proB, USA) Sm .
QY7 F ,lacam,trpam, ~bio256- S.Brenner (Cambridge -cI857~ Hl, ~uvrB. UK) El03S L.D.Simon (New Jersey, USA) ~) ATCC is the Rmerican Tylce Culture C~llection desi~nation .
. . . ... . . . ..... ... . .
~7~
The present inventlon makes possible -the crea-tion o~ a new l~ind o~ plasmid in which the copy number can be deliberately controlled by regula-table promote~ such as PL or PR`.
The presence of the XhoI linker 30 base pairs from the 5' end of RNA II allows the replacement of the natural promoter for RNA II by other promoters. Thus, plasmid pMG404 put RNA II under the control of the kanamycin resistance gene promoter on the plasmid 1~ pH~'15 it is believed that both originsin pr~404 func-ti~n, the teMperature sensitive pSC101 origin from ~H~G41~ ~nd the ColEl origin under control of the KmR
promoter. Although the exact point of DNA initiation h~s rot been determined in these plasmids, -the function-ing o~ the hybrid origin is indicated by the highercopy number of pMG404 than pHSG415, and the replication o~ p~IG40~ at 42C. The construction of pMG410, a recircularised BalI fragment carrying the hybrid origin but n~ the pSC101 temperature sensitive originj is ~0 ~dditional evidence that the hybrid origin is functional.
The properties of pMG404 demons-trate tha-t the RNA
II promoter and the first 30 bases of RNA II can be re-placed by another promoter without abolishing the initiation of DNA replication. However the Km ~5 promo-ter is constitutively expressed and it is not there~ore possible to alter the copy number of pMG404.
Several well-defined controllable promoters exist which ~unction in E.coli; the PL promoter from bacteriophage ~
w~s used to construct plasmid pMG411, with a ColEl hybrid origin under direct control. pMG411 retained the origin from pHSG415, and was maintained at a low copy number a-t 30 in a strain carrying a ts ~ repressor gene (QY7).
~lhen the ~ repressor was inactivated at 42, -the copy number increased, indicating that replication of the ColEl origin was being driven by PL. It is therefore clear that the copy number of such plasmids can be deliberately controlled from regulated promoters and this opens the way to constructing plasmids whose copy number can be controlled either by tempera-ture (as wi~h PL in pMG411~ or more importantly by the altera-5 tion of the concentration of metabolites such as trypto-phan or lactose.
Claims (20)
1. A DNA vector for use in bacterial host cells comprising two replication systems; a first origin of replication resulting in a low copy number and stable inheritance of the vector, and a second, high copy number, origin of replication at which replication is directly controllable as the result of replacement or alteration by DNA manipulation of the natural vector sequence(s) which control replication at said second origin.
2. A vector according to claim 1 wherein the second, high copy number, origin of replication comprises an origin of replication and an associated DNA sequence encoding an RNA species which provides a primer or initiation factor which initiates DNA replication by formation of a complex at or near the origin of replication, in which transcription of said RNA species is directly controllable such that, when host cells carrying the vector are propagated under selected conditions, replication takes place at a high copy number from the origin.
3. A vector according to claim 1 or 2, wherein the copy number of the controllable origin or replication is controlled by temperature or one or more metabolites or matabolite analogues.
4. A vector according to claim 1 or 2, wherein the controllable origin of replication is derived from a naturally occurring high copy number plasmid having a copy number control system involving transcriptional control by RNAII or a similar RNA species.
5. A vector according to claim 2, wherein the natural promoter which promotes transcription of the RNA species is replaced by a controllable promoter.
6. A vector according to claim 5 wherein the controllable promoter is the PL promoter, PR
promoter, Pre promoter, Prm promoter, P'R
promoter, T7 late promoters, trP promoter, tac promoter, lac promoter, qal promoter, ara promoter or recA promoter.
promoter, Pre promoter, Prm promoter, P'R
promoter, T7 late promoters, trP promoter, tac promoter, lac promoter, qal promoter, ara promoter or recA promoter.
7. A vector according to claim 2 wherein transcription of the RNA species is controlled by incorporating a regulating function into the replication system.
8. A vector according to claim 7 wherein said regulating function comprises the lac operator or the OL or OR operator of phage lambda.
9. A method for producing a vector according to claim 1, comprising ligating a first DNA sequence coding for the first origin of replication with a second DNA
sequence coding for the second origin of replication.
sequence coding for the second origin of replication.
10. A method for producing a vector as defined in claim 2 comprising ligating a first DNA sequence coding for the first origin of replication with a second DNA
sequence coding from the second origin or replication, the second DNA sequence including a DNA sequence which permits direct control of replication of the second origin by controlling transcription of the said RNA
species.
sequence coding from the second origin or replication, the second DNA sequence including a DNA sequence which permits direct control of replication of the second origin by controlling transcription of the said RNA
species.
11. A process for the production of a gene product com-prising transforming bacterial host cells with a DNA vector according to claim 1, said DNA vector containing a gene sequence coding for production of said gene product, propagating said transformed cells under a first set of conditions in which replication takes place at low copy number mainly (or exclusively) from the first origin of replication, and then propagating said transformed cells under a second set of conditions in which replication take place at high copy number also (or exclusively) from the second origin of replication and the expression of said gene product is induced.
12. A process according to claim 11 for the production of preprochymosin, met-prochymosin or met-chymosin.
13. A process according to claim 11 in which the expression of the gene product is under the control of a promoter which is regulated by cytoplasmic levels of a repressor and in which the increase in vector copy number on propagating the transformed cells under the second set of conditions leads to outstripping of the repressor control and high level expression of the gene product.
14. A process according to claim 13 wherein the synthesis of the repressor is autoregulated.
15. A process according to claim 14 wherein the promoter/repressor system is that of the tryptophan operon.
16. A process for the production of a gene product comprising propagating bacterial host cells transformed with a DNA vector according to claim 1, said DNA vector containing a gene sequence coding for production of said gene product under a first set of conditions in which replication takes place at low copy number mainly (or exclusively) from the first origin of replication, and then propagating said transformed cells under a second set of conditions in which replication takes place at high copy number also (or exclusively) from the second origin or replication and the expression of said gene product is induced.
17. A process according to claim 16 for the production of preprochymosin, met-prochymosin or met-chymosin.
18. A process according to claim 16 in which the expression of the gene product is under the control of a promoter which is regulated by cytoplasmic levels of a repressor and in which the increase in vector copy number on propagating the transformed cells under the second set of conditions leads to outstripping of the repressor control and high level expression of the gene product.
19. A process according to claim 18 wherein the synthesis of the repressor is autoregulated.
20. A process according to claim 19 wherein the promoter/repressor system is that of the trypophan operon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000450724A CA1272143A (en) | 1984-03-28 | 1984-03-28 | Dna vector comprising a low copy number and a high copy number origin of replication and their use in recombinant dna technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000450724A CA1272143A (en) | 1984-03-28 | 1984-03-28 | Dna vector comprising a low copy number and a high copy number origin of replication and their use in recombinant dna technology |
Publications (1)
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|---|---|---|---|
| CA000450724A Expired CA1272143A (en) | 1984-03-28 | 1984-03-28 | Dna vector comprising a low copy number and a high copy number origin of replication and their use in recombinant dna technology |
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1984
- 1984-03-28 CA CA000450724A patent/CA1272143A/en not_active Expired
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