IE914045A1 - A NOVEL PHASMID VECTOR IN E.coli - Google Patents
A NOVEL PHASMID VECTOR IN E.coliInfo
- Publication number
- IE914045A1 IE914045A1 IE404591A IE404591A IE914045A1 IE 914045 A1 IE914045 A1 IE 914045A1 IE 404591 A IE404591 A IE 404591A IE 404591 A IE404591 A IE 404591A IE 914045 A1 IE914045 A1 IE 914045A1
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- vector
- parc035
- parc032
- phasmids
- phasmid
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- 239000013598 vector Substances 0.000 title claims abstract description 57
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 31
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 45
- 108020004414 DNA Proteins 0.000 claims abstract description 37
- 238000010367 cloning Methods 0.000 claims abstract description 36
- 230000001580 bacterial effect Effects 0.000 claims abstract description 11
- 238000000338 in vitro Methods 0.000 claims abstract description 9
- 229930027917 kanamycin Natural products 0.000 claims abstract description 9
- 229960000318 kanamycin Drugs 0.000 claims abstract description 9
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims abstract description 9
- 229930182823 kanamycin A Natural products 0.000 claims abstract description 9
- 241000724791 Filamentous phage Species 0.000 claims abstract description 6
- 229960000723 ampicillin Drugs 0.000 claims abstract description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims abstract description 4
- 238000002741 site-directed mutagenesis Methods 0.000 claims abstract description 3
- 108020005091 Replication Origin Proteins 0.000 claims abstract 4
- 230000001002 morphogenetic effect Effects 0.000 claims abstract 3
- 239000012634 fragment Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 238000010276 construction Methods 0.000 claims description 11
- 206010042566 Superinfection Diseases 0.000 claims description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 102000053602 DNA Human genes 0.000 claims description 4
- 101710137500 T7 RNA polymerase Proteins 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 241000701832 Enterobacteria phage T3 Species 0.000 claims description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 3
- 230000037431 insertion Effects 0.000 claims description 3
- 108091034117 Oligonucleotide Proteins 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 230000002285 radioactive effect Effects 0.000 claims description 2
- 238000010561 standard procedure Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000013518 transcription Methods 0.000 claims description 2
- 230000035897 transcription Effects 0.000 claims description 2
- 238000001727 in vivo Methods 0.000 claims 1
- 241001515965 unidentified phage Species 0.000 claims 1
- 239000013612 plasmid Substances 0.000 abstract description 38
- 230000010076 replication Effects 0.000 abstract description 9
- 239000003550 marker Substances 0.000 abstract description 6
- 239000002245 particle Substances 0.000 abstract description 4
- 230000003612 virological effect Effects 0.000 abstract description 3
- 238000001712 DNA sequencing Methods 0.000 abstract description 2
- 239000003242 anti bacterial agent Substances 0.000 abstract description 2
- 230000003115 biocidal effect Effects 0.000 abstract description 2
- 239000013604 expression vector Substances 0.000 abstract description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 abstract 1
- 230000009466 transformation Effects 0.000 abstract 1
- 238000011144 upstream manufacturing Methods 0.000 abstract 1
- 101150077543 st gene Proteins 0.000 description 7
- 238000010369 molecular cloning Methods 0.000 description 6
- 238000002703 mutagenesis Methods 0.000 description 6
- 231100000350 mutagenesis Toxicity 0.000 description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 108091029795 Intergenic region Proteins 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 101100459256 Cyprinus carpio myca gene Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 241000701533 Escherichia virus T4 Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 101150023479 hsdS gene Proteins 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 101150059159 proA2 gene Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Cloning and expression vectors comprise (i) a replication origin and morphogenetic signal sequence derived from filamentous bacteriophage of E.coli having F pili (f1, M13, fd) and (ii) aT7 (T7 O 10) phage promoter upstream of a multiple cloning site. The vectors carry an antibiotic resistance marker gene ampicillin (pARC032 and pARC035) or Kanamycin (pARC036) and a bacterial origin of replication pARC032 and pARC035 are identically constructed except the f1 replication origin sequence lies in opposite orientation. Upon transformation of E.coli male specific strains with phasmid containing a recombinant insert, is plasmid DNA packaged as a viral particle can be produced when the transformants are superinfected with a helper phage. This ss phasmid DNA can also be used as template for ss DNA sequencing and in vitro site directed mutagenesis. Genes inserted at the multiple cloning site of the phasmids can be expressed in appropriate host strains.
Description
A NOVEL PHASMID VECTOR IN E.COLI FIELD OF THE INVENTION In this patent application we have disclosed the construction of a novel phasmid vector of E.coli which can be used for cloning gene(s) of interest and for hyperexpression of gene products in suitable host bacteria. This vector when incorporated into a ’’male specific" E.coli strain, upon superinfection with helper phage, can give rise to single stranded plasmid DNA which can be sequenced directly by dideoxy sequencing methods and which also can be used as a template for site directed in vitro mutagenesis.
BACKGROUND OF THE INVENTION Development of a large number of recombinant DNA molecules have been due to the concurrent development in the construction of tools ’’vectors" for cloning and manipulation of DNA. These vectors are generally characterised by the presence of a genetic determinant, the phenotype of which is altered by insertional inactivation. In addition, these vectors contain multiple restriction enzyme cleavage sites allowing the direct cloning of a variety of restriction fragments.
The first cloning vector constructed was pBR322 (A survey of Molecular Cloning Vectors and their uses, 1988, Biotechnology Series, Boskin, Butterworth, Rodrigues, M. & Denhardt D. (eds)). This vector could be used for only cloning purposes and not for manipulating the expression of inserted gene. For such purposes, a number of inducible expression system was developed. An example of —2— such an expression system is the T7 system developed by Studier & Moffat (1986, J.Mol.Biol. 189, 113). This system makes use of the high specificity of the T7 RNA polymerase to recognise and transcribe any gene downstream of the T7 0 10 promoter. In the study of gene structure and function, the technique of DNA analysis such as DNA sequencing and site directed in vitro mutagenesis are efficiently carried out on single stranded (ss) DNA templates. In 1981, Dotto et al., (Virology 114. 463) showed that a plasmid carrying the intergenic region (IG) of filamentous phage fl could be packaged as ss DNA into a viral particle by a helper phage. On the basis of these observations, a number of cloning vectors have been developed which contain the IG region of a filamentous phage, such as pEMBL (Dente et al., 1983, Nucl.Acid Res. 11. 1645), Blue scribe (Strategene, San Diego, CA, USA), pGEM (Promega, Madison, WI, USA), etc.
Utilizing the same logic, a second generation of pUC vectors such as pUC118 and pUC119 which can give rise to ss plasmid DNA upon superinfection with helper phage have also been developed (Viera & Messing, 1987, Methods Enzymol. 153. 3). In a similar fashion, a series of cloning vectors with the property of releasing ss plasmid upon superinfection with helper phage have been developed by Konings et al. (pKUN series) where the specific DNA sequences of the filamentous phage Ff and the Ike are utilized (Konings et al., 1987, Methods enzymol. 153. 12; Peeters, B.P.H., Konings, R.N.H. & Schoenmakers, J.G.G. 1987). All these plasmid vectors which have the capability of producing ss plasmid DNA packaged inside a viral coat, upon superinfection with another helper phage are commonly referred to as phasmid vectors. However all —3— these vectors that have been mentioned above are suitable only for general cloning purposes. Number of phasmid vectors which can be utilized not only for general cloning but also for expression are very few at present.
One of them pFSE4 employs a λ-phage promotor (PL) including the ell ribosome binding site (RBS). Hypothetically under appropriate conditions insertion can be expressed in this vector, though this vector is not considered as a hyperexpression vector (Cloning vectors.
A laboratory manual, Pouwels et al. (eds.), 1986). The other phasmid expression vector belongs to the pSP6 family where pSP6 the promoter of a Salmonella phage gene is utilized for the expression of the cloned gene (Cloning vectors. A laboratory manual, Pouwels et al. (eds), 1986). We wanted to incorporate all the above individual features of the known vectors and develop the current vectors which can take advantage of the properties of the above vectors. Hence, in this patent application, the inventors have taken advantage of the phage fl origin of replication and constructed a novel phasmid vector which can be used for general cloning, production of single stranded plasmid DNA and for hyperexpression of the cloned gene under appropriate host condition. In other words, the novel phasmid vector is a genetic engineering tool, which is used for cloning, sequencing and hyperexpression of a gene. The only limitation of the invention could be the cloning of the same fragment in both the vectors to be able to manipulate both the strands of the DNA fragment.
DETAILED DESCRIPTION OF THE INVENTION To develop a phasmid vector with the ability to hyperexpress any cloned gene and to produce ss DNA of —4— said gene the following features were included in the plasmid: a. Introduction of the origin of replication of the single stranded DNA phage fl which will enable the ss plasmid DNA to be packaged into the viral particle. b. Introduction of a strong promoter preceding the cloning site(s) for expression of the cloned gene. c. Introduction of an antibiotic resistance marker for proper selection and maintenance of the plasmid in E.coli host. d. Introduction of an E.coli origin of replication for propagation of the plasmid in E.coli.
To achieve the desired vector construction E.coli pET7 plasmid (Fig. 1; Studier and Moffat, 1986, J.Mol.Biol. 189. 113) was used as a source of the T7 0 10 promoter of Bacteriophage T7 which is specifically recognized by T7 RNA polymerase. For the construction of the plasmid described in this invention pET7 was digested with PvuII and Nrul and the large fragment was purified. In the next step of the construction the origin of the replication of E.coli fl was introduced into this plasmid. The fragment containing the fl origin of replication was excised out of pGEM7Zf(+) (Promega Corporation Catalogue; YanishPerron et al. (1985) Gene 33. 103-109) plasmid by digestion using the restriction enzyme Sau3AI. Subsequently the restriction fragments were filled-in using the Klenow DNA polymerase. A 788 bp fragment separated on agarose gel contained the fl origin of replication. This fragment was ligated to the PvuII and Nrul digested large fragment of pET7 to generate pARC032 and pARC035 (Figs. 2 and 3). —5— Special features of the phasmid vector 1. pARC032 and pARC035 in E.coli male specific hosts upon superinfection with helper phage R408 (Russel et al., 1986, Gene 45. 333) can package alternative strands of the plasmids as ss DNA into the virus particle. 2. Both the plasmids pARC032 and pARC035 can be used as cloning vectors. 3. The gene of interest can be cloned into any of the unique multicloning cites (e.g. BamHI, EcoRV, Hindlll, Clal and EcoRI) available following the T7 0 10 promotor element. 4. The cloned gene can be overexpressed in the presence of a functional T7 RNA polymerase using suitable host systems.
. Upon introduction of these plasmids into E.coli JM101, the transformed strains can be superinfected with the helper phage and ss DNA can be isolated for sequencing purposes and/or for other manipulations. 6. Phasmid vector with Kanamycin marker (eg. pARC036) can be used to derive ss plasmid DNA which can be used as a template for in vitro mutagenesis.
EXAMPLE 1 Construction of PARC036 In order to be able to utilize the ss plasmid DNA which can be generated from these plasmids as a template for in vitro mutagenesis the Ampicillin resistance marker of pARC032 was replaced with the Kanamycin resistance cartridge, since the E.coli CJ236 used in the preparation of the template for in vitro mutagenesis sometimes become spontaneously ampicillin resistant (Unpublished observations - Dwarakanath, P. & Goutam Das). A plasmid containing the kanamycin resistance gene was digested —6— with Pstl and the 1.3 Kb fragment containing the KanR gene was purified. The fragment was blunt ended with bacteriophage T4 DNA polymerase. This fragment was ligated to the large fragment of EcoRI and Pstl digested pARC032 following T4 DNA polymerase treatment. The ligated product was used to transform E.coli HB101 and kanamycin resistant colonies were selected and the plasmid was characterized. This plasmid pARC036 (Fig. 4) has the T7 0 10 promoter, fl origin of replication, as well as the Kanr marker. Detailed diagrammatic depiction of the construction of pARC032 and pARC035 is shown in Fig. 5 and that of pARC036 is shown in Fig. 6.
EXAMPLE 2 Cloning of E.coli st gene in PARC036 The utility of the plasmid pARC036 was checked by inserting an E.coli plasmid gene at the BamHI - Hindlll site of pARC036. The gene cloned was E.coli st gene as a BamHI - Hindlll fragment (Dwarakanath et al., 1989, Gene 81. 219). The cloning strategy is shown in Fig. 7. The final construct is shown in Fig. 8. Advantage of this cloning strategy was that if the Hindlll sites of the vector pARC036 were used, upon successful cloning in a 3way ligation process the Kanr property should be restored. The resulting plasmid containing the st gene insert is designated as pARC044 (Fig. 8). The plasmid pARC044 was used to transform E.coli JM101, BL21 (DE3) and CJ236. The transformed strains maintained the plasmid stably. Upon superinfection with helper phage R408, the JM101 (pARC044) produced ss plasmid DNA containing the st gene insert which can be sequenced following the isolation of the ss plasmid DNA according to the known standard procedure (Molecular Cloning, Maniatis et al. (eds) CSH Publ. 1989). —7— EXAMPLE 3 Hyperexpression of st gene cloned in the novel phasmid vector E.coli hyperexpression strain BL21 (DE3) which has the T7 5 RNA polymerase gene cloned under the lac UV5 promoter at the chromosomal locus (Studier and Moffat, 1986, J.Mol.Biol. 189. 113) was transformed with pARC 044. The transformed clones were characterized and further grown in M9 media (Dwarakanath et al., 1989, Gene 81. 219) and were induced to hyperexpress ST as a secreted product according to the procedure previously described by Dwarakanath et al. (1989, Gene 81. 219). The level of secreted ST under induced condition was comparable to the wild-type level as determined by a previously standardized ELISA method (Dwarakanath et al., 1989, Gene 81, 219).
EXAMPLE 4 ss phasmid DNA as a template for site directed in vitro mutagenesis E.coli CJ236 (dut, ung) was transformed with pARC044 and the transformants were characterized. One of the transformants was grown in liquid medium and was superinfected with helper phage. After overnight incubation at 37°C with vigorous shaking, the culture supernatant containing the phasmid, virions were separated from the bacterial pellet, and the ss plasmid DNA was isolated according to Maniatis. (Molecular Cloning, Maniatis et al., (eds) CSH Publ., 1989). As shown in Fig. 9, this phasmid DNA was used as a template to introduce a mutation in the st gene. —8— The present invention is summarized in the following clauses: 1. A phasmid construct containing a DNA phage fl origin of replication, a T7 phage promoter followed by a multiple cloning site and a kanamycin resistance selection marker. 2. The design of a phasmid as described in Fig. 4 containing features as in paragraph 1 or part thereof. 3. A phasmid to which a gene or a gene segment can be introduced at the multiple cloning site described therein. 4. A phasmid which can replicate in any bacterial host.
. A phasmid which can produce single stranded DNA of the gene inserted at the multiple cloning site in any host cell upon superinfection by a helper phage. 6. A phasmid wherein a synthetic oligonucleotide containing a mismatch flanked by complementary sequences to the gene inserted at the multiple cloning site, which produces a variant ss DNA mutagenized at a preselected spot. 7. A phasmid capable of producing ss DNA under appropriate culture conditions which can be labelled with radioactive elements such as 32P or other chemical markers suitable for determining the nucleotide sequence of the ss DNA. 8. A phasmid which upon insertion into a suitable host cell, can be selected by growing the host cell in kanamycin containing medium. 9. A phasmid which contains a bacteriophage T7 promoter for transcription.
. Any bacterial cell transformed by the phasmid which produces a protein or a peptide encoded by the gene inserted at the multiple cloning site. —9— 11. Any bacterial cell transformed by the phasmid which produces a mutagenized protein or peptide encoded by the mutagenized ss DNA.
Materials and Methods The bacterial strains used were: E.coli HB101 (F", hsd S20(rB,mB"), supE44, recA13, aral4, proA2, rpsL20(Str), xyl-l,mlt-l,); E.coli JM101 (thi, (lac-proAB), [F', traD36, proAB, lacIqZM15]); E.coli CJ236 (dutl, ungl, thi-1, relAl/pCJ105 (cam1 F')); E.coli BL21(DE3) (hsdS, gal (clts857 indl Sam7 nin5 lacUV5-T7genel).
Helper phage R408.
Plasmids pET7 (a kind gift from Dr. Studier) and pGEM7Zf(+) from Promega.
The restriction enzymes BcoRI, BamHI, ZfindIII,PstI, Sau3AI were from Boehringer Mannheim. T4 DNA ligase, T4 DNA polymerase, Klenow fragment were from Boehringer Mannheim.
All DNA manipulations were done by essentially following protocols described in ’’Molecular cloning - A Laboratory Manual", Maniatis et al. (eds) CSH Publ., 1989.
-IOREFERENCES 1. Rodriguez, R.L., and Denhardt, D.T. (eds) Vectors: A Survey of Molecular Cloning Vectors and their uses 1988, Biotechnology Series, Boston, Butterworth. 2. Studier & Moffat, 1986, J.Mol.Biol. 189, 113. 3. Dotto et al. 1981, Virology, 114. p. 463. 4. Dotto et al. 1983, Nucl.Acid.Res. 11. p. 1645.
. Peeters, B.P.K., Konings et al., European Patent Application No.209 204-A1. 6. Viera & Messing, 1987, Methods Enzymol. 153. p.3. 7. Konings et al., 1987, Methods Enzymol. 153. p. 12. 8. Pouwels et al., (eds) Cloning Vectors. A laboratory Manual, 1986. 9. Yanish-Perron et al. 1985, Gene 33., p. 103.
Maniatis et al., Molecular Cloning - A laboratory Manual, CSH Publ., 1989. 11. Dwarakanath et al., 1989, Gene 81, p. 219 —11— FIGURE LEGENDS E.coli pET7 plasmid 5 Eigr-i E.coli pARCO32 plasmid Fig·—J.
E.coli pARC035 plasmid Fig. 4 E.coli pARC036 plasmid Fig. 5 Construction of pARC032 and pARC035 Fig^6 Construction of pARC036 Fiq.,7 Cloning of E.coli st gene in pARC036 Fig. 8 E.coli pARC044 plasmid Fig,.9 Strategy for site directed mutagenesis using ss phasmid DNA as a template
Claims (21)
1. Vector phasmids pARC032, pARC035 constructed from a starting vector incorporating 5 (a) in opposite orientation, relative to the T7 0 io promoter, the replication origin and a morphogenetic signal, or an active mutant thereof, of a filamentous bacteriophage of E.coli bacteria having F pili; and 10 (b) T7 phage promoter followed by a multiple cloning site.
2. Vector phasmids pARC032, pARC035 which contain the replication origin and a morphogenetic signal, or an 15 active mutant thereof, of the filamentous bacteriophage Ff (M13, fd and fl) or a mutant of said bacteriophage.
3. Vector phasmids pARC032 and pARC035 in which the 20 ampicillin resistance phenotype has been replaced by kanamycin resistance phenotype as exemplified by the construction of pARC036 from pARC035.
4. Vector phasmid pARC 032 having the structure shown 25 in Figure 2.
5. Vector phasmid pARC 035 having the structure shown in Figure 3. 30
6. Vector phasmids pARC032_. and pARC035 into which a fragment of double-stranded DNA has been inserted at the multiple cloning site. —13—
7. Vector phasmids pARC032 and pARC035 which can replicate in a bacterial host.
8. Vector phasmids pARC032 and pARC035 which can 5 produce single stranded DNA of a gene inserted at the multiple cloning site in an appropriate host cell upon superinfection by a helper phage.
9. Vector phasmids pARC032 and pARC035 wherein a 10. Synthetic oligonucleotide containing a mismatch flanked by complementary sequences to the gene is inserted at the multiple cloning site.
10. Vector phasmids pARC032 and pARC035 capable of 15 producing ss DNA under standard culture conditions which can be labelled with radioactive elements such as 32 P or other chemical markers suitable for determining the nucleotide sequence of the ss DNA. 20
11. Vector phasmids pARC032 and pARC035 as claimed in. claim 1 which,. U pon insertion in a suitable host cell, can be selected by growing the host cell in kanamycin containing medium. 25
12. Vector phasmids pARC032 and pARC035 wherein the new restriction sites could be introduced into the multiple cloning site by standard method of in vitro site directed mutagenesis.
13. Vector phasmids pARC032 and pARC035 which contain a bacteriophage T7 promoter for transcription and which is transcribed in vivo or in vitro by a T7 RNA polymerase. —14—
14. A bacterial cell, transformed by the vector phasmid pARC032 or pARC035, which produces a protein or a peptide encoded by a gene inserted at the multiple cloning site.
15. A bacterial cell, transformed by the vector phasmid pARC032 or pARC035, which produces a mutagenized protein or peptide encoded by the mutagenized ss DNA.
16. A process for the production of a protein or a peptide, encoded by the gene inserted at the multiple cloning site, by using a bacterial cell transformed by the vector phasmid pARC032 or pARC035.
17. A process for the production of a mutagenized protein or peptide, encoded by the mutagenized ss DNA, by using a bacterial cell transformed by the vector phasmid pARC032 or pARC035.
18. Vector phasmids pARC032 and pARC035 obtained by a method for their construction substantially as hereinbefore described.
19. Vector phasmid pARC036 having the structure shown in Figure 4.
20. A process for the production of a protein or peptide according to claim 16 or 17 substantially as hereinbefore described.
21. Protein or peptide prepared by the process claimed in claim 16, 17 or 20.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN933MA1990 | 1990-11-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| IE914045A1 true IE914045A1 (en) | 1992-06-03 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IE404591A IE914045A1 (en) | 1990-11-21 | 1991-11-21 | A NOVEL PHASMID VECTOR IN E.coli |
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| Country | Link |
|---|---|
| GB (1) | GB2250290A (en) |
| IE (1) | IE914045A1 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL8502061A (en) * | 1985-07-17 | 1987-02-16 | Stichting Katholieke Univ | NEW VECTOR PLASMIDS, THEIR CONSTRUCTION AND APPLICATION, AND MICRO-ORGANISMS INCLUDING THEM. |
| EP0354727A3 (en) * | 1988-08-08 | 1990-11-22 | Eli Lilly And Company | Novel single stranded phasmid vectors and methods for transforming streptomyces and other actinomycetes |
-
1991
- 1991-11-20 GB GB9124556A patent/GB2250290A/en not_active Withdrawn
- 1991-11-21 IE IE404591A patent/IE914045A1/en unknown
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| Publication number | Publication date |
|---|---|
| GB9124556D0 (en) | 1992-01-08 |
| GB2250290A (en) | 1992-06-03 |
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