GB2087896A - Plasmid pUC2 and its microbiological preparation - Google Patents
Plasmid pUC2 and its microbiological preparation Download PDFInfo
- Publication number
- GB2087896A GB2087896A GB8110672A GB8110672A GB2087896A GB 2087896 A GB2087896 A GB 2087896A GB 8110672 A GB8110672 A GB 8110672A GB 8110672 A GB8110672 A GB 8110672A GB 2087896 A GB2087896 A GB 2087896A
- Authority
- GB
- United Kingdom
- Prior art keywords
- plasmid
- puc2
- dna
- nrrl
- streptomyces
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000013612 plasmid Substances 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title description 4
- 230000002906 microbiologic effect Effects 0.000 title description 2
- 241000187438 Streptomyces fradiae Species 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 12
- 108091008146 restriction endonucleases Proteins 0.000 claims description 12
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 238000003776 cleavage reaction Methods 0.000 claims description 6
- 239000006166 lysate Substances 0.000 claims description 6
- 230000007017 scission Effects 0.000 claims description 6
- 239000004471 Glycine Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 235000015097 nutrients Nutrition 0.000 claims description 4
- 230000002934 lysing effect Effects 0.000 claims description 3
- 238000003306 harvesting Methods 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 13
- 238000010367 cloning Methods 0.000 abstract description 5
- 108020004511 Recombinant DNA Proteins 0.000 abstract description 3
- 108090001061 Insulin Proteins 0.000 abstract 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 abstract 2
- 102000004877 Insulin Human genes 0.000 abstract 1
- 229940125396 insulin Drugs 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 30
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 15
- 241000187747 Streptomyces Species 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- 230000002068 genetic effect Effects 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 4
- 241001655322 Streptomycetales Species 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 229960005542 ethidium bromide Drugs 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 108020004638 Circular DNA Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000013600 plasmid vector Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 238000009631 Broth culture Methods 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- JOCBASBOOFNAJA-UHFFFAOYSA-N N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid Chemical compound OCC(CO)(CO)NCCS(O)(=O)=O JOCBASBOOFNAJA-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007994 TES buffer Substances 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- FLJPGEWQYJVDPF-UHFFFAOYSA-L caesium sulfate Chemical compound [Cs+].[Cs+].[O-]S([O-])(=O)=O FLJPGEWQYJVDPF-UHFFFAOYSA-L 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 101100148606 Caenorhabditis elegans pst-1 gene Proteins 0.000 description 1
- 108010073254 Colicins Proteins 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 102100033195 DNA ligase 4 Human genes 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 241000617590 Escherichia coli K1 Species 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 241000606768 Haemophilus influenzae Species 0.000 description 1
- 241000606766 Haemophilus parainfluenzae Species 0.000 description 1
- 101000927810 Homo sapiens DNA ligase 4 Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 101150093335 KIN1 gene Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000364057 Peoria Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 108010059712 Pronase Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000005157 Somatostatin Human genes 0.000 description 1
- 108010056088 Somatostatin Proteins 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000187419 Streptomyces rimosus Species 0.000 description 1
- 241000531819 Streptomyces venezuelae Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 108010049705 endonuclease R Proteins 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- -1 for example Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 229940047650 haemophilus influenzae Drugs 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229940016590 sarkosyl Drugs 0.000 description 1
- 108700004121 sarkosyl Proteins 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000011877 solvent mixture Substances 0.000 description 1
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 1
- 229960000553 somatostatin Drugs 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The plasmid pUC 2 can be obtained from the microorganism Streptomyces fradiae, NRRL 11444. The plasmid can be used as a cloning vehicle in recombinant DNA work. By way of example, the insulin gene can be inserted into the plasmid and then a suitable host containing the resulting plasmid can be used to produce insulin.
Description
SPECIFICATION
A novel plasmid and its microbiological preparation
The present invention relates to a novel plasmid which can have utility as a cloning vehicle in recombinant DNA work.
British Patent Publication No. 2045252A discloses and claims a plasmid which is given the code name pUC2. It is stated that pUC2 can be prepared by incubating fragmented mycelia obtained by growing Streptomyces fradiae having the deposit number NRRL 11444. In fact, pUC2 cannot be prepared from the microorganism of this deposit.
Accordingly, British Patent Publication No. 2045252A does not make the plasmid publicly available.
The development of plasmid vectors useful for recombinant DNA genetics among microorganisms is well known. The editorial in Science, Vol. 196, April, 1977, gives a good summary of
DNA research. This editorial is accompanied by a number of supporting papers in the same issue of Science.
Similar DNA work is currently being done on industrially important microorganisms of the genus Streptomyces. [Bibb, M.J., Ward, J.M., and Hopwood, D.A. 1978. "Transformation of plasmid DNA into Streptomyces at high frequency". Nature 274, 398-400]. Though plasmid
DNA's have been detected in several Streptomycetes [Huber, M.L.B. and Godfrey, 0. 1 978. "A general method for lysis of Streptomyces species". Can. J. Microbiol. 24, 631-632.] [Schrempf, H., Bujard, H., Hopwood, D.A. and Goebel, W. 1975. "Isolation of covalently closed circular deoxyribonucleic acid from Streptomyces coelicolorA3(2)". J. Bacteriol. 121, 416-421.] [Umezawa, H. 1977. "Microbial secondary metabolities with potential use in cancer treatment (Plasmid involvement in biosynthesis and compounds)".Biomedicine 26, 236-249.], [Malik, V.S. 1 977. Preparative Method for the isolation of super-coiled DNA from a chloramphenicol producing Streptomycete. J. Antibiotics 30, 897-899.], only one Streptomycete plasmid has been physically isolated and extensively characterized in the literature [Schrempf, supra]. The existence of other plasmids in the genus Streptomyces has been inferred from reported genetic data as follows:
(1) Akagawa, H., Okanishi, M. and Umezawa, H. 1975. "A plasmid involved in chloramphenicol production in Streptomyces venezuelae: Evidence from genetic mapping". J. Gen.
Microbiol. 90, 336-346.
(2) Freeman, R.F. and Hopwood, D.A. 1 978. "Unstable naturally occurring resistance to antibiotics in Streptomyces". J. Gen. Microbiol. 106, 377-381.
(3) Friend, E.J., Warren, M. and Hopwood, D.A. 1978. "Genetic evidence for a plasmid controlling fertility in an industrial strain of Streptomyces rimosus". J. Gen. Microbiol. 106, 201-206.
(4) Hopwood, D.A. and Wright, H.M. 1973. "A plasmid of Streptomyces coelicolorcarrying a chromosomal locus and its inter-specific transfer". J. Gen. Microbiol. 79, 331-342.
(5) Hotta, K., Okami, Y. and Umezawa, H. 1 977. "Elimination of the ability of a kanamycinproducing strain to biosynthesize deoxystreptamine moiety by acriflavine". J. Antibiotics 30, 1146-1149.
(6) Kirby, R., Wright, L.F. and Hopwood, D.A. 1 975. "Plasmid-determined antibiotic synthesis and resistance in Streptomyces coelicolor". Nature 254, 265-267.
(7) Kirby, R. and Hopwood, D.A. 1 977. "Genetic determination of methylenomycin synthesis by the SCPI plasmid of Streptomyces coelicolorA3(2)". J. Gen. Microbiol. 98, 239-252.
(8) Okanishi, M., Ohta, T. and Umezawa, H. 1969. "Possible control of formation of aerial mycelium and antibiotic production in streptomyces by episomic factors". J. Antibiotics 33, 45-47.
Plasmid pUC2 is obtainable from the microorganism Streptomyces fradiae, NRRL 12323.
This plasmid can be obtained from NRRL 1 2323 by growing the culture on a suitable medium, fragmenting the mycelia, incubating the fragmented mycelia, harvesting the culture after a suitable time, and then lysing the mycelia. From this lysate it is possible to isolate essentially pure pUC2. Plasm it pUC2 sensitivities to a variety of restriction endonucleases should allow its ready modification and adaptation to a number of host vector systems.
pUC2 is characterized by standard characterization tests which include its molecular weight, approximately 29.8 megadaltons, a restriction map as shown in the drawing, and presence at 1-2 copies per S. fradiae NRRL 12323 cell.
pUC2 is useful as a cloning vector in DNA work wherein desired genes are incorporated into the plasmid; the plasmid may then be transformed into a suitable host.
The accompanying drawing depicts the restriction endonuclease cleavage map for pUC2. The map is constructed on the basis of plasmid pUC2 having a molecular weight of ca. 29.8 + 0.7 megadaltons or a molecular length of ca. 45.0 kilobases. The map positions of the various restriction sites are given as kilobase coordinates relative to the Xba I restriction site at 0.0/45.0 kilobases. The restriction endonuclease abbreviations are as follows: (1) Bg1 II is an enzyme from Bacillus globigii; (2) Hind ill is an enzyme from Haemophilus influenzae; and (3)
Xba I is an enzyme from Xanthomanas badrii.
pUC2 may be obtained from Streptomyces fradiae NRRL 12323. This biologically pure culture is available from the permanent collection of the Northern Regional Research Laboratory,
U.S. Department of Agriculture, Peoria, Illinois, U.S.A. It was deposited on 14th November 1980.
Characteristics of pUC2
Molecular weight: ca. 29.8 megadaltons.
Copies Per Cell: 1-2
Restriction Endonuclease Sensitivities:
pUC2 has the following sensitivities to restriction endonucleases. Please refer to the drawing for the restriction endonucleases cleavage map for pUC2.
Plasmid Sensitivities To Restriction En don ucleases # Cleavage Sites # Cleavage Sites
Enzyme pUC2 Enzyme pUC2
BamH1 10 Hind ílí 2
EcoRI O Kin 1 10
Pst 1 > 11 Who 1 9 Xba I 2 Smal 12 Bgl II 4 Bcl I 5
These results were obtained by digestion of pUC2 DNA in the presence of an excess of restriction endonuclease. The number of restriction sites were determined from the number of resolvable fragments in either 0.7 or 1.0% agarose gels.
pUC2 can be used to create recombinant plasmids which can be incorporated into host bacteria transformation. The process of creating recombinant plasmids is well known in the art.
Such a process comprises cleaving the isolated vector plasmid, e.g., pUC2, at a specific site(s) by means of a restriction endonuclease, for example, Hind Ill, Xba I, and the like. The plasmid, which is a circular DNA molecule, is thus converted into a linear DNA molecule by the enzyme which cuts the two DNA strands at a specific site. Other non-vector DNA is similarly cleaved with the same enzyme. Upon mixing the linear vector or portions thereof and non-vector DNAs, their single-stranded or blunt ends can pair with each other and in the presence of a second enzyme known as polynucleotide ligase can be covalently joined to form a single circle of DNA.
The above procedure also can be used to insert a length of DNA from a higher animal into pUC2. For example, the DNA which codes for ribosomal RNA in the frog can be mixed with pUC2 DNA that has been cleaved. The resulting circular DNA molecules consist of plasmid pUC2 with an inserted length of frog rDNA.
The recombinant plasmids containing a desired genetic element, prepared by using pUC2, can be introduced into a host organism for expression. Examples of valuable genes which can be inseted into host organisms by the above described process are genes coding for somatostatin, rat proinsulin, and proteases.
The usefulness of plasmid pUC2 is derived from its capacity to function as a plasmid vector in industrially important microorganisms, e.g. Streptomyces. Hence, cloning of genetic information from Streptomyces into pUC2 provides a means of increasing the production of commercially important products from these organisms, e.g. antibiotics.
This approach is compared to the concept of cloning genes for antibiotic production into the well characterized Escherichia coli K-1 2 host-vector system. The E. coli system has the disadvantage that it has been found that genes from Gram-positive organisms, e.g. Bacillus, do not express well in the Gram-negative E. coli host. Likewise, plasmids from Gram-negative are not maintained in Gram-positive hosts, and Gram-negative genetic information is either expressed poorly or not at all in Gram-positive hosts. This clearly argues for the advantage of a
Gram-positive host-vector system and argues the usefulness of plasmid pUC2 in such a system.
In general, the use of a host-vector system to produce a product foreign to that host requires the introduction of the genes for the entire biosynthetic pathway of the product to the new host.
As discussed above, this may lead to problems of genetic expression, but may also generate
new and/or increased problems in the fermentation of the microorganisms and in the extraction and purification of the product. A perhaps more useful approach is to introduce a plasmid vector, e.g. pUC2, into a host which normally produces the product and clone onto that plasmid the genes for boisynthesis of the product. At the very least, problems of fermentation and product extraction and purification should be minimized. Additionally, in this cloning system it may not be necessary to clone and amplify all the genes of the biosynthetic pathway, but rather it may be necessary only to clone regulatory genes or genes coding for the enzymes that are rate limiting in product biosynthesis.Since pUC2 is a streptomycete plasm id, it is ideally suited for these purposes in the genus Streptomyces. Furthermore, since pUC2 is also a plasmid from a
Gram-positive organism, it may serve as a vector in a number of other microorganisms, e.g.
Bacillus, Arthrobacter, etc.
Streptomyces fradiae, NRRL 12323, can be grown in an aqueous nutrient medium under submerged aerobic conditions. The organism can be grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps, and the like.
Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.
The inoculated medium can be incubated at any temerature conductive to satisfactory growth of the microorganism, for example, between about 18 and 50 C., and preferably between about 20 and 37 C. Ordinarily, optimum growth of the microorganism is obtained in about 3 to 1 5 days. The medium normally remains acidic during the growth cycle. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.
When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the growth of the microorganism and the attendant inefficient utilization of the equipment.
Accordingly, it is desirable to produce a vegetative inoculm in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil liquid N2 agar plug, or a slant culture.
When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the growth of the microorganism so long as a good growth of the microorganism is obtained.
The following examples are illustrative of the process and products of the subject invention but are not to be construced as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Example 1-Isolation Of Plasmid pUC2 From A Biologically
Pure Culture Of Streptomyces fradiae
NRRL 12323
The spores from a biologically pure culture of Streptomyces fradiae, NRRL 12323 are inoculated into 10 ml of the following medium which contains 1 % glucose; 0.4% peptone; 0.4% yeast extract; 0.05% MgSO47H2O; 0.2% KH2PO4; and 0.4% K2HPO4.
The medium has previously been sterilized in a 50 ml Erlenmeyer flask. After inoculation, the flask is incubated at 32 C. for about 24 to 36 hours on a Gump of New Brunswick rotary shaker operating at 100-250 rpm. Upon completion of the incubation, 0.5 ml of the culture is tranferred into 10 ml of the above medium containing 0.5 to 2.0% (w/v) glycine in a 50 ml Erlenmeyerflask.
The addition of glycine facilitates the subsequent lysing of the cells. The amount of glycine in the medium can be varied by routine adjustements with the goal being to facilitate the subsequent lysing of the celis. The flask is than incubated further for another 24 to 36 hours at 32 C. on a Gump rotary shaker, as above. After this incubation, the mycelia are separated from the broth by low speed centrifugation, for example, at 6000 X g for 1 5 minutes at 4" C. and decantation of the supernatant from the mycelial pellet.
The supernatant is discarded and the pellet is resuspended in 1.5 ml of an isotonic buffer, e.g.
TES buffer [0.03 M tris(hydroxymethyl)aminomethane (Tris), 0.005 M EDTA and 0.05 M NaCL; pH = 8.0] containing 20% (w/v) sucrose. Next, 0.3 ml of a 5 mg/ml lysozyme and 0.15 ml of a 1 mg/ml RNase in the same buffer are added and the mixture is incubated at 37"C. for 30 minutes with occasional mixing. Then, 0.6 ml of 0.25 M EDTA (pH = 8.0) is added and this is incubated 15 minutes at 37 C. Then 0.3 ml of 5 mg/ml pronase is added and the material is incubated 10 minutes at 37 C. Subsequently, the cell suspension is lysed by the addition of 3.0 ml of a 2% sarkosyl in TES buffer and incubation of this mixture at 37 C. for 20-30 minutes. The lysate is then sheared by passing it 5-10 times through a 50 ml disposable syringe without a needle.
This crude lysate material is then mixed with a salt, for example, cesium chloride (preferred), and cesium sulfate, and the intercalating dye ethidium bromide to give a solution of density = 1.550.
This solution is centifuged to equilibrium at 145,000 X g (isopycnic density gradient centrifugation). The covalently closed circular plasmid DNA is then visible in the centrifuge tube under long wave ultraviolet (320 nm) illumination as a faint fluorescent band below the intensely flourescent band of linear chromosomal and plasmid DNAs.
Covalently closed circular plasmid DNA is prepared for characterization by removing it from the isopycnic gradients, extracting the ethidium bromide by two treatments with one third volume of isopropyl alcohol and then dialyzing the aqueouss phase against an appropriate buffer, e.g.
0.1 x SSC buffer (0.015 M NaCL, 0.0015 M Na3QH5O72H20; pH = 7.4 to yield essentially pure pUC2.
Procedures For Characterizing pUC2
The size of pUC2 was determined by sedinentation in neutral and alkaline sucrose gradients using an internal marker plasmid DNA having a molecular weight of approximately 28.0 megadaltons and a corresponding sedimentation value of approximately 58S. From the neutral sucrose gradients the sedimentation value of pUC2 was determined to be 64S. The molecular weight for pUC2 was calculated from the equations by Hudson et al. [Hudson, B., Clayton, D.A. and Vinograd, J. 1 968.
"Complex mitochondrial DNA". Cold Spring Harbor Symp. Quant. Biol. 33, 435-442]. This molecular weight is in good agreement with that estimated from the alkaline sucrose gradients.
An estimate of pUC2 molecular weight was also obtained by electron microscopy of individual
DNA molecules [Kleinschmidt, A.K. (1968). Monolayer techniques in electron microscopy of nucleic acid molecules. In "Method in Enzymology" (S.P. Colowick and N.O. Kaplan, eds.) Vol. 1 2B, pages 361-377. Academic Press, New York]. Plasmid pUC1 was found to have an average contour length of 1 5.2 + 0.6 m and a corresponding molecular weight of 29.8 + 0.7 megadaltons.
The percent plasmid DNA in Streptomyces fradiae NRRL 12323 was determined by labeling the culture with [methyl-3H] thymidine, preparing crude lysates, and centrifuging samples of the lysates in cesium chloride ethidium bromide density gradients. The gradients are fractionated, the isotopic counting performed, and the percent radioactivity in the plasmid band used to quantitate the plasmid
DNA and calculate the plasmid copy number [Radloff, R., Bauer, W. and Vinograd, J. 1967. "A dye-buoyant density method for detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells". Proc. Nat. Acad. Sci. USA 57, 1514-1520].
Restriction En don uclease Digestion And Agarose Gel Electrophoresis
Restriction endonucleases were obtained as commercial preparations from Miles Laboratories and
New England Biolabs. Enzyme digestions were prepared in accordance with the conditions specified by the suppliers using at least a two-fold excess of endonuclease.
In some experiments plasmid DNA was digested with more than one endonuclease. Two methods were used in these experiments. In the first method, the plasmid DNA was digested first with the enzyme having the lower ionic strength requirements, and then digested with the enzyme having higher ionic strength requirments after the addition of an equal volume of 2X buffer of the second enzyme. In the second method, restriction fragments of one enzyme digest were isolated from a preparative agarose gel as described by Tanaka and Weisblum [Tanka, T., and Weisblum, B. 1 975.
Construction of a colicin El-R factor composite plasmid in vitro: Means for amplification of deoxyribonucleic acid. J. Bacteriol. 121, 354-362]. The isolated restriction fragments were concentrated by ethanol precipitation and then with other restriction enzymes. Double digest experiments were compared with single digest experiments to ensure that no abnormal restriction
patterns were obtained, i.e. no non-specific cleavage of DNA by an enzyme occurs after altering the
ionic strength of the digestion mixture.
The digested samples were applied to 0.7-1% agarose gels and were electrophoresed for 2 hours at a constant applied voltage of 10-15 v/cm of gel height. [Sharp, P.A., Sugden, J. and Sambrook, J. 1 973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using
analytical agarose-ethidium bromide electrophoresis. Biochemistry 12, 3055-3063]. The molecular weights of restriction fragments were determined relative to the standard migration patterns of
bacteriophage lambda DNA digested with enzyme EcoRI [Helling, R.B., Goodman, H.M. and Boyer,
H.W. 1974. Analysis of endonuclease R EcoRI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virology 14, 1 235-1 244].
The work described herein was all done in conformity with physical and biological containment
requirments specified in the NIH Guidelines.
Claims (5)
1. Essentially pure plasmid pUC2 which is characterized by a molecular weight of approximately
29.8 megadaltons, and a restrictionendonuclease cleavage map as shown in the drawing.
2. A process for isolating essentially pure pUC2 from Streptomyces fradiae, NRRL 12323, which comprises:
(a) growing S. fradiae, NRRL 1 2323 on a suitable S. fradiae growth medium until sufficient
mycelial growth is obtained;
(b) fragmenting said mycelia;
(c) incubating said fragmented mycelia in a suiable growth medium, as above;
(d) harvesting the culture after a suitable time;
(e) lysing the harvested mycelia; and
(f) isolating essentially pure pUC2 from the lysate.
3. A process, according to claim 2, which comprises cultivating Streptomyces fradiae, NRRL 12323, in a nutrient medium at a temperature of about 32 C. for about 24 to 36 hours.
4. A process according to claim 2 or claim 3 in which the fragmented mycelia are incubated in a growth medium containing glycine.
5. A process according to claim 2 substantially as described in the Example.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US20886180A | 1980-11-21 | 1980-11-21 |
Publications (2)
Publication Number | Publication Date |
---|---|
GB2087896A true GB2087896A (en) | 1982-06-03 |
GB2087896B GB2087896B (en) | 1984-05-10 |
Family
ID=22776332
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB8110672A Expired GB2087896B (en) | 1980-11-21 | 1981-04-06 | Plasmid p uc2 and its microbiological preparation |
Country Status (6)
Country | Link |
---|---|
JP (1) | JPS56135498A (en) |
DE (1) | DE3105757A1 (en) |
FR (1) | FR2494718A1 (en) |
GB (1) | GB2087896B (en) |
IT (1) | IT1144690B (en) |
NL (1) | NL8100853A (en) |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2045252A (en) * | 1979-03-29 | 1980-10-29 | Upjohn Co | A plasmid and its microbiological preparation |
-
1981
- 1981-02-17 DE DE19813105757 patent/DE3105757A1/en not_active Withdrawn
- 1981-02-19 JP JP2232381A patent/JPS56135498A/en active Pending
- 1981-02-20 NL NL8100853A patent/NL8100853A/en not_active Application Discontinuation
- 1981-03-12 FR FR8104977A patent/FR2494718A1/fr not_active Withdrawn
- 1981-03-19 IT IT20592/81A patent/IT1144690B/en active
- 1981-04-06 GB GB8110672A patent/GB2087896B/en not_active Expired
Also Published As
Publication number | Publication date |
---|---|
GB2087896B (en) | 1984-05-10 |
FR2494718A1 (en) | 1982-05-28 |
DE3105757A1 (en) | 1982-07-01 |
NL8100853A (en) | 1982-06-16 |
IT1144690B (en) | 1986-10-29 |
JPS56135498A (en) | 1981-10-22 |
IT8120592A0 (en) | 1981-03-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US4273875A (en) | Plasmid and process of isolating same | |
US4332900A (en) | Construction of co-integrate plasmids from plasmids of Streptomyces and Escherichia | |
US4338400A (en) | Co-integrate plasmids and their construction from plasmids of Escherichia and Streptomyces | |
US4340674A (en) | Cointegrate plasmids and their construction from plasmids of Escherichia and Streptomyces | |
US4332898A (en) | Hybrid plasmid and process of making same | |
US4343906A (en) | Hybrid plasmid of pBR322 and Streptomyces plasmid and E. coli containing same | |
HU197354B (en) | Process for selecting streptomyces containing recombinant dna | |
EP0213898B1 (en) | A host-vector system | |
US4393137A (en) | Cloning plasmid for streptomyces | |
GB2045253A (en) | A plasmid and its microbiological preparation | |
US4362816A (en) | Hybrid plasmid and process of making same | |
GB2046272A (en) | A novel plasmid and its microbiological preparation | |
GB2045252A (en) | A plasmid and its microbiological preparation | |
EP0038156A2 (en) | A plasmid and its microbiological preparation | |
EP0035914A2 (en) | Plasmid vectors, plasmids and their preparation, and cloning processes using them | |
US4401761A (en) | Process for stabilizing plasmids by deletion of DNA | |
GB2045251A (en) | A plasmid and its microbiological preparation | |
GB2087896A (en) | Plasmid pUC2 and its microbiological preparation | |
GB2045254A (en) | A plasmid and its microbiological preparation | |
US4518698A (en) | Plasmid and production thereof | |
MacNeil | A flexible boiling procedure for isolating plasmid DNA from gram-positive microorganisms | |
GB2044773A (en) | A plasmid and its microbiological preparation | |
US4460691A (en) | Streptomyces plasmid prophage pUC13 | |
US4478937A (en) | Plasmid and production thereof | |
GB2121051A (en) | A plasmid cloning vector for bacillus subtilis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PCNP | Patent ceased through non-payment of renewal fee |