CN113025641B - 一种将dna片段随机插入枯草芽孢杆菌染色体的方法及其应用 - Google Patents
一种将dna片段随机插入枯草芽孢杆菌染色体的方法及其应用 Download PDFInfo
- Publication number
- CN113025641B CN113025641B CN202110355936.6A CN202110355936A CN113025641B CN 113025641 B CN113025641 B CN 113025641B CN 202110355936 A CN202110355936 A CN 202110355936A CN 113025641 B CN113025641 B CN 113025641B
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- plasmid
- chromosome
- pusigh
- fragment
- 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.)
- Active
Links
- 239000012634 fragment Substances 0.000 title claims abstract description 47
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 39
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 39
- 210000000349 chromosome Anatomy 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000013612 plasmid Substances 0.000 claims abstract description 36
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims abstract description 15
- 239000000203 mixture Substances 0.000 claims abstract description 13
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 9
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 8
- 238000013518 transcription Methods 0.000 claims description 33
- 229960005091 chloramphenicol Drugs 0.000 claims description 13
- 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 claims description 13
- 238000012216 screening Methods 0.000 claims description 10
- 229960000268 spectinomycin Drugs 0.000 claims description 7
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 claims description 7
- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 claims description 5
- 230000004927 fusion Effects 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 108010084455 Zeocin Proteins 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 claims description 4
- 230000006698 induction Effects 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 2
- 108010020764 Transposases Proteins 0.000 abstract description 8
- 230000006801 homologous recombination Effects 0.000 abstract description 6
- 238000002744 homologous recombination Methods 0.000 abstract description 6
- 102000008579 Transposases Human genes 0.000 abstract description 4
- 230000001939 inductive effect Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 27
- 238000003780 insertion Methods 0.000 description 11
- 230000037431 insertion Effects 0.000 description 11
- 230000010354 integration Effects 0.000 description 6
- 230000002759 chromosomal effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000007857 nested PCR Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000005944 Chlorpyrifos Substances 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 101150117564 ITR2 gene Proteins 0.000 description 2
- 101710198366 Parathion hydrolase Proteins 0.000 description 2
- 108020005091 Replication Origin Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- SBPBAQFWLVIOKP-UHFFFAOYSA-N chlorpyrifos Chemical compound CCOP(=S)(OCC)OC1=NC(Cl)=C(Cl)C=C1Cl SBPBAQFWLVIOKP-UHFFFAOYSA-N 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000009191 jumping Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- RLBIQVVOMOPOHC-UHFFFAOYSA-N parathion-methyl Chemical compound COP(=S)(OC)OC1=CC=C([N+]([O-])=O)C=C1 RLBIQVVOMOPOHC-UHFFFAOYSA-N 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000017105 transposition Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101100267391 Escherichia coli (strain K12) yjbH gene Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101150053510 ITR1 gene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101150024831 ahpC gene Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000012966 insertion method Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 101150004591 spxH gene Proteins 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 101150038987 xylR gene Proteins 0.000 description 1
Images
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
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- 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
- C12N15/67—General methods for enhancing the expression
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/08—Phosphoric triester hydrolases (3.1.8)
- C12Y301/08001—Aryldialkylphosphatase (3.1.8.1), i.e. paraoxonase
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/90—Vectors containing a transposable element
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种将DNA片段随机插入枯草芽孢杆菌染色体的方法及其应用。一种组合物，由选自转座子元件或含有该转座子元件的质粒和质粒pUSIGH组成。该组合物在把目标DNA片段随机插入枯草芽孢杆菌染色体中的应用。将组合物共同转化到枯草芽孢杆菌中后，转座子元件或含有该转座子元件的质粒会通过同源重组将转座子元件整合到宿主染色体中，pUSIGH可在宿主内正常独立复制。用木糖诱导pUSIGH上的转座酶基因表达，在转座酶的作用下转座子随机插入到染色体上；进而可筛选出目标蛋白表达水平较高的克隆。
Description
技术领域
本发明属于生物高技术领域，公开了一种将DNA片段随机插入枯草芽孢杆菌染色体的方法及其应用。
背景技术
枯草芽孢杆菌(Bacillus subtilis)是重要的重组蛋白表达宿主，其具有安全好、发酵成本低、分泌蛋白能力强及生长迅速等优点。在构建表达菌株时，可利用多拷贝质粒携带目标基因，或者将目标基因整合于宿主染色体上。这两种方式分别被称为质粒型表达和染色体整合型表达。质粒型表达的缺点是稳定性差且通常携带抗性基因，优点是基因拷贝数高。染色体整合型表达稳定性好且无抗性基因问题，但这种方式的缺点是基因拷贝数较少、目标蛋白产量较低，需要通过各种手段优化表达水平。
整合型表达方式中，提高蛋白产量的方法之一是改变目标基因在染色体的位置(Englaender et al..ACS Synth Biol.6:710-20.Doi:10.1021/acssynbio.6b00350.Sauer et al..2016.ACS Synth Biol.9:942-46.Doi:10.1021/acssynbio.6b00065)。依据是位置效应，即基因的表达水平受整合位置影响。位置效应的原因主要有三个。一是剂量效应，位于复制起始位点oriC附近的基因在染色体复制回合中具有更多的拷贝数，从而具有相对较高的表达水平(Block et al..2012.Nucleic AcidsRes.18:8979-92.Doi:10.1093/nar/gks694)。二是大多数基因的转录方向与DNA复制的方向是相同，从而与DNA复制的冲突(Bryant et al..Nucleic Acids Res.18:11383-92.Doi:10.1093/nar/gku828)。三是上游启动子的转录加强效应。总之，在采用染色体整合型表达方式时要为目标基因挑选合适的整合位置。
挑选位置的常用策略是将目标基因随机插入到染色体不同位置，然后筛选表达水平高的克隆。该策略可通过转座元件实现，即将目标基因置于转座子中，利用转座子随机插入的特性将目标基因随机插入染色体不同位置。在枯草芽孢杆菌中，常用的转座子系统是pMarA(Breton et al..2006.Appl Environ Microbiol.72:327-333.Doi:10.1128/AEM.72.1.327-33.)。然而，该系统的不足之处是在50℃条件下进行筛选且存在多拷贝插入现象；50℃可能会失活温度敏感性蛋白，多拷贝插入则导致假阳性。因此，需要为枯草芽孢杆菌建立更高效的染色体随机插入的方法。
发明内容
针对枯草芽孢杆菌中现有的染色体随机插入方法存在筛选温度较高和假阳性问题，本发明建立一种新的将DNA片段随机插入枯草芽孢杆菌染色体的方法，实现在常温条件下将DNA片段以单拷贝形式高效随机插入枯草芽孢杆菌染色体，并证明该方法可用于优化重组蛋白在枯草芽孢杆菌中的表达水平。
本发明的目的可通过以下技术方案实现：
一种用于把目标DNA片段随机插入枯草芽孢杆菌染色体的组合物，由以下两组分组成：
组分A：质粒pUSIGH，质粒pUSIGH全序列如SEQ ID NO.2所示；质粒pUSIGH用于控制转座酶的表达，从而控制转座子的随机插入。
组分B：选自转座子元件或含有该转座子元件的质粒；所述的转座子元件为LFITR1-Transcription terminator-Cm-目标DNA-ITR2-RF融合片段，其中LFITR1-Transcription terminator-Cm片段的核苷酸序列如SEQ ID NO.3所示，ITR2-RF片段的核苷酸序列如SEQ ID NO.4所示；质粒转座子元件或含有该转座子元件的质粒用于将目标DNA置于转座子中。将上述两种组分共同转化到枯草芽孢杆菌中后，转座子元件或含有该转座子元件的质粒会通过同源重组将转座子元件整合到宿主染色体中，pUSIGH可在宿主内正常独立复制。用木糖诱导pUSIGH上的转座酶基因表达，在转座酶的作用下转座子随机插入到染色体上；进而可筛选出目标蛋白表达水平较高的克隆。
本发明所述的组合物在把目标DNA片段随机插入枯草芽孢杆菌染色体中的应用。
本发明所述的组合物在构建目的基因被随机插入染色体的枯草芽孢杆菌基因工程菌中的应用。
一种枯草芽孢杆菌基因工程菌PDZC(pUSIGH)，将本发明所述的组合物共同转化枯草芽孢杆菌168，经氯霉素和壮观霉素筛选得到。
本发明所述的质粒组合物在优化目标蛋白在枯草芽孢杆菌中表达水平中的应用。
一种把目标DNA片段随机插入枯草芽孢杆菌染色体的方法，包括：将本发明所述的组合物共同转化到枯草芽孢杆菌菌株168中，通过氯霉素和壮观霉素抗性筛选得到菌株PDZCM(pUSIGH)，经过添加木糖诱导菌株PDZCM(pUSIGH)后涂布于含有氯霉素和零霉素的LB平板，获得目标DNA片段随机插入枯草芽孢杆菌染色体的克隆。
作为本发明的一种优选，所述的转座子元件LFITR1-Transcription terminator-Cm-目标DNA-ITR2-RF通过化学合成制备。
作为本发明的一种优选，所述的通过同源重组的方式将目标DNA片段插入质粒pTMCZ的转座子ITR1-Transcription terminator-Cm-ITR2中的方法为：PCR扩增或化学合成LF-ITR1-Transcription terminator和ITR2-RF片段,LF-ITR1-Transcriptionterminator序列如SEQ ID NO.7所示，ITR2-RF序列如SEQ ID NO.8所示；通过重叠延伸PCR将LF-ITR1-Transcription terminator、目标DNA片段和ITR2-RF三个片段连接，生成LFITR1-Transcription terminator-Cm-目标DNA-ITR2-RF融合片段。
作为本发明的一种优选，以本发明所述的菌株PDZC(pUSIGH)总DNA为模板，分别使用引物对P20/P21和P22/P23扩增LF-ITR1-Transcription terminator和ITR2-RF；所述的引物对P20/P21序列如SEQ ID NO.5/SEQ ID NO.6所示，所述的引物对P22/P23序列如SEQID NO.7/SEQ ID NO.8所示，所述的LF-ITR1-Transcription terminator序列如SEQ IDNO.3，所述的ITR2-RF序列如SEQ ID NO.4。
有益效果：
利用本发明的方法可实现在常温条件下将DNA片段以单拷贝形式高效随机插入枯草芽孢杆菌染色体，可用于优化重组蛋白在枯草芽孢杆菌中的表达水平。
附图说明
图1本发明的主要技术原理，染色体转座示意图
图2目标蛋白在染色体中随机插入示意图
图3甲基对硫磷水解酶MPH突变体文库的筛选
具体实施方式
实施例1质粒pTMCZ和pUSIGH的构建过程
大肠杆菌和枯草芽孢杆菌用LB培养基(蛋白胨1％，酵母粉0.5％，氯化钠1％，pH7.0)在37℃培养。转座子的左侧重复序列ITR1(Breton et al..2006.Appl EnvironMicrobiol.72:327-333.Doi:10.1128/AEM.72.1.327-33.)和转录终止子B0015(http://parts.igem.org/Part:BBa_B0015)委托生物公司合成，然后经引物P1/P2扩增后得到片段ITR1-Transcription terminator。以质粒pNW33N(GenBank Number AY237122)为模板，用引物P3/P4扩增氯霉素抗性基因。以质粒pMarA(Breton et al..2006.Appl EnvironMicrobiol.72:327-333.Doi:10.1128/AEM.72.1.327-33.)为模板，使用引物P5/P6扩增ITR2。最终，使用引物P1/P6，通过重叠延伸PCR将ITR1-Transcription terminator、氯霉素抗性基因和ITR2三片段连接，生成片段ITR1-Transcription terminator-Cm-ITR2。为了将片段ITR1-Transcription terminator-Cm-ITR2置于零霉素抗性基因(zeo)(Yan etal..2008.Appl Environ Microbiol.74:5556-62.Doi:10.1128/AEM.01156-08.)的启动子和核糖体结合位点之间，用引物P7/P6扩增ITR1-Transcription terminator-Cm-ITR2获得片段Pzeo-ITR1-Transcription terminator-Cm-ITR2，用引物P8/P9扩增质粒p7Z6(Yan etal..2008.Appl Environ Microbiol.74:5556-62.Doi:10.1128/AEM.01156-08.)，然后用引物P7/P9通过重叠延伸PCR将这两个片段融合成片段Pzeo-ITR1-Transcriptionterminator-Cm-ITR2-Zeo。以枯草芽孢杆菌168(ATCC 23857)基因组为模板，分别使用引物P10/P11和P12/P13扩增上游同源臂(LF)和下游同源臂(RF)。然后使用引物P10/P13通过重叠延伸PCR将LF,Pzeo-ITR1-Transcription terminator-Cm-ITR2-Zeo和RF进行三片段融合生成片段LF-Pzeo-ITR1-Transcription terminator-Cm-ITR2-Zeo-RF，接着将该片段连接至pUC19(GenBank Number M77789.2)的EcoR V位点，转化到大肠杆菌DH5α后经20ppm氯霉素筛选后得到质粒pTMCZ。
以质粒pAX01(
et al..2001.J Bacteriol.183:2696-99.Doi:10.1128/JB.183.14.4393-4393.2001.)为模板，分别使用引物P14/P15扩增xylR片段和启动子Pxyl。以质粒pMarA(Breton et al..2006.Appl Environ Microbiol.72:327-333.Doi:10.1128/AEM.72.1.327-33.)为模板，使用引物P16/P17扩增转座酶基因Himar1。然后使用引物P14/P17将上述两片段通过重叠延伸PCR融合，得到片段xylR-Pxyl-Himar1。以质粒pUS20(Yeal..2018.J Biotechnol.284:57-62.Doi:10.1016/j.jbiotec.2018.08.001)为模板，使用引物P18/P19扩增整个质粒片段后，通过同源重组的方式将xylR-Pxyl-Himar1片段插入到质粒pUS20中，转化到大肠杆菌DH5α后经100ppm壮观霉素筛选后得到质粒pUSIGH。
实施例2将转座子随机插入枯草芽孢杆菌染色体
如图1所示，将质粒pTMCZ和pUSIGH共同转化到枯草芽孢杆菌168中。枯草芽孢杆菌转化参考Anagnostopoulos et al.(1961.J Bacteriol.81:741-746.)建立的方法进行。质粒pTMCZ进入菌株168后，片段Pzeo-ITR1-Transcription terminator-Cm-ITR2-Zeo将通过同源重组的方式插入到宿主染色体的amyE位点，而质粒pUSIGH进入菌株168后可独立复制。通过抗性筛选(10ppm氯霉素和100ppm壮观霉素)和PCR验证后得到菌株PDZC(pUSIGH)。将菌株PDZC(pUSIGH)培养至OD600＝1.0后添加1％木糖诱导质粒pUSIGH上的转座酶基因Himar1的表达(诱导3小时)，在转座酶的作用下转座子ITR1-Transcription terminator-Cm-ITR2会从amyE位置跳出，并随机插入到染色体其他位置。转座子ITR1-Transcriptionterminator-Cm-ITR2从amyE位置的跳出会导致启动子Pzeo启动零霉素抗性基因zeo基因的表达，使宿主获得零霉素抗性。用含有10ppm氯霉素和20ppm零霉素的LB平板筛选，即可获得发生转座子跳跃的克隆。转座效率达10-5，即每108个细胞中有1000个发生转座。
实施例3将目标DNA随机插入枯草芽孢杆菌染色体
为了以转座子ITR1-Transcription terminator-Cm-ITR2为载体，将目标DNA随机插入枯草芽孢杆菌染色体，先将目标DNA(如图2中EC)通过同源重组插入到该转座子中，然后用木糖诱导转座子跳跃(同实施例2)。为了验证可行性，将甲基对硫磷水解酶(MPH)的表达盒PNBP3510-mpd(Zhou et al..2019.Microb Cell Fact.18(1):111.Doi:10.1186/s12934-019-1159-0)插入到转座子ITR1-Transcription terminator-Cm-ITR2，并产生菌株PDZCM(pUSIGH)。具体步骤为：以菌株PDZC(pUSIGH)总DNA为模板，分别使用引物对P20/P21和P22/P23扩增LF-ITR1-Transcription terminator和ITR2-RF；以菌株PD8NM(Zhou etal..2019.Microb Cell Fact.18(1):111.Doi:10.1186/s12934-019-1159-0)总DNA为模板，使用引物P24/P25扩增表达盒PNBP3510-mpd；使用引物P20/P23通过重叠延伸PCR将LF-ITR1-Transcription terminator、ITR2-RF和PNBP3510-mpd三个片段连接，生成片段LFITR1-Transcription terminator-Cm-PNBP3510-mpd-ITR2-RF；然后将融合后片段转化与质粒pUSIGH共同转化到菌株168中，通过抗性筛选(10ppm氯霉素和100ppm壮观霉素)筛选得到菌株PDZCM(pUSIGH)。经过添加木糖诱导后涂布于含有氯霉素和零霉素的LB平板，获得发生转座子跳跃的克隆(具体条件同实施例2)。将获得的克隆点在含有50mg/L毒死蜱的LB平板上生长，当毒死蜱被MPH水解后，在菌落周围会形成透明圈；透明圈大小和MPH活力高低成正比。如图3A所示，从约15000个菌落中筛选出5个突变株T1,T2,T3,T4和T5,其水解圈明显大于WT菌株(表达盒PNBP3510-mpd整合在枯草杆菌168淀粉酶基因处)。通过Arbitrary PCR(Black et al..1993.Insect Mol Biol.2:1-6.Doi:10.1111/j.1365-2583.1993.tb00118.x.)鉴定，在5个突变株中PNBP3510-mpd插入位置分别为ahpC,veg,crdA,qoxB和yjbH。相对WT菌株，突变体菌株的酶活分别提高了12％,18％,25％,37％和50％(图3B和3C)。对5株突变株的染色体位置进行比对，其中MT1,MT2,MT3和MT4靠近复制起始位点oriC(图3D)。MT1和MT2中表达盒PNBP3510-mpd的转录方向同插入基因转录方向一致。
表1本发明所用引物
序列表
<110> 南京农业大学
<120> 一种将DNA 片段随机插入枯草芽孢杆菌染色体的方法及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6891
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gcgttctgtt tctgcttcgg tatgtgattg tgaagctggc ttacagaaga gcggtaaaag 60
aagaaataaa aaagaaatca tcttttttgt ttggaaagcg agggaagcgt tcacagtttc 120
gggcagcttt ttttatagga acattgattt gtattcactc tgccaagttg ttttgataga 180
gtgattgtga taattttaaa tgtaagcgtt aacaaaattc tccagtcttc acatcggttt 240
gaaaggagga agcggaagaa tgaagtaaga gggatttttg actccgaagt aagtcttcaa 300
aaaatcaaat aaggagtgtc aagaatgttt gcaaaacgat tcaaaacctc tttactgccg 360
ttattcgctg gatttttatt gctgtttcat ttggttctgg caggaccggc ggctgcgagt 420
gctgaaacgg cgaacaaatc gaatgagctt acagcaccgt cgatcaaaag cggaaccatt 480
cttcatgcat ggaattggtc gttcaatacg ttaaaacaca atatgaagga tattcatgat 540
gcaggatata cagccattca gacatctccg attaaccaag taaaggaagg gaatcaagga 600
gataaaagca tgtcgaactg gtactggctg tatcagccga catcgtatca aattggcaac 660
cgttacttag gtactgaaca agaatttaaa gaaatgtgtg cagccgctga agaatatggc 720
ataaaggtca ttgttgacgc ggtcatcaat cataccacca gtgattatgc cgcgatttcc 780
aatgaggtta agagtattcc aaactggaca catggaaaca cacaaattaa aaactggtct 840
gatcgatggg atgtcacgca gaattcattg ctcgggctgt atgactggaa tacacaaaat 900
acacaagtac agtcctatct gaaacggttc ttagacaggg cattgaatga cggggcagac 960
ggttttcgat ttgatgccgc caaacatata gagcttccag atgatggcag ttacggcagt 1020
caattttggc cgaatatcac aaatacatct gcagagttcc aatacggaga aatcctgcag 1080
gatagtgcct ccagagatgc tgcatatgcg aattatatgg atgtgacagc gtctaactat 1140
gggcattcca taaggtccgc tttaaagaat cgtaatctgc ttgatatggc tttttatatg 1200
tgttactcta catacagata acaggttggc tgataagtcc ccggtctgac aactttatct 1260
gagaatagtc aatcttcgga aatcccaggt ggcatgctaa aagtctcgta aagcgttcta 1320
tcaataaccc gttggtgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 1380
ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc 1440
ttcgggtggg cctttctgcg tttataccgt ctcagaatcg gccgttataa aagccagtca 1500
ttaggcctat ctgacaattc ctgaatagag ttcataaaca atcctgcatg ataaccatca 1560
caaacagaat gatgtacctg taaagatagc ggtaaatata ttgaattacc tttattaatg 1620
aattttcctg ctgtaataat gggtagaagg taattactat tattattgat atttaagtta 1680
aacccagtaa atgaagtcca tggaataata gaaagagaaa aagcattttc aggtataggt 1740
gttttgggaa acaatttccc cgaaccatta tatttctcta catcagaaag gtataaatca 1800
taaaactctt tgaagtcatt ctttacagga gtccaaatac cagagaatgt tttagataca 1860
ccatcaaaaa ttgtataaag tggctctaac ttatcccaat aacctaactc tccgtcgcta 1920
ttgtaaccag ttctaaaagc tgtatttgag tttatcaccc ttgtcactaa gaaaataaat 1980
gcagggtaaa atttatatcc ttcttgtttt atgtttcggt ataaaacact aatatcaatt 2040
tctgtggtta tactaaaagt cgtttgttgg ttcaaataat gattaaatat ctcttttctc 2100
ttccaattgt ctaaatcaat tttattaaag ttcatattta gttcctcctt tctgtatgta 2160
gagtaacaca tataaaaagc catatcaaga agctttttag acatctaatc ttttctgaag 2220
tacatccgca actgtccata ctctgatgtt ttatatcttt tctaaaagtt cgctagatag 2280
gggtcccgag cgcctacgag gaatttgtat cggggtatag atctaaaacg aattttaaca 2340
aaaaaatgtg tttttctttg ttagaccggg gacttatcag ccaacctgtt aaaggaggaa 2400
ctaaatatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga 2460
gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc 2520
gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg 2580
ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg 2640
tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc 2700
gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc 2760
gtggccgagg agcaggactg atatctccca ctatgcatct gatgtgtctg cggacaagct 2820
agtgacatgg gtagagtcgc atgatacgta tgccaatgat gatgaagagt cgacatggat 2880
gagcgatgat gatatccgtt taggctgggc ggtgatagct tctcgttcac ggcagtacgc 2940
ctcttttctt ttccagacct gagggaggcg gaaatggtgt gaggttcccg gggaaaagcc 3000
aaataggcga tcgcgggagt gctttatttg aagatcaggc tatcactgcg gtcaatagat 3060
ttcacaatgt gatggctgga cagcctgagg aactctcgaa cccgaatgga aacaaccaga 3120
tatttatgaa tcagcgcggc tcacatggcg ttgtgctggc aaatgcaggt tcatcctctg 3180
tctctatcaa tacggcaaca aaattgcctg atggcaggta tgacaataaa gctggagcgg 3240
gttcatttca agtgaacgat ggtaaactga caggcacgat caatgccagg tctgtagctg 3300
tgctttatcc tgatgatatt gcaaaagcgc ctcatgtttt ccttgagaat tacaaaacag 3360
gtgtaacaca ttctttcaat gatcaactga cgattacctt gcgtgcagat gcgaatacaa 3420
caaaagccgt ttatcaaatc aataatggac cagagacggc gtttaaggat ggagatcaat 3480
tcacaatcgg aaaaggagat ccatttggca aaacatacac catcatgtta aaaggaacga 3540
acagtgatgg tgtaacgagg accgagaaat acagttttgt taaaagagat ccagcgtcgg 3600
ccaaaaccat cggctatcaa aatccgaatc attggagcca ggtaaatgct tatatctata 3660
aacatgatgg gagccgagta attgaattga ccggatcttg gcctggaaaa ccaatgacta 3720
aaaatgcaga cggaatttac acgctgacgc tgcctgcgga cacggataca accaacgcaa 3780
aagtgatttt taataatggc agcgcccaag tgcccggtca gaatcagcct ggctttgatt 3840
acgtgctaaa tggtttatat aatgactcgg gcttaagcgg ttctcttccc cattgagggc 3900
aaggctagac gggacttacc gaaagaaacc atcaatgatg gtttcttttt tgttcataaa 3960
tcagacaaaa cttttctctt gcaaaagttt gtgaagtgtt gcacaatata aatgtgaaat 4020
acttcacaaa caaaaagaca tcaaagagaa acataccctg gaaggatgat taatgatgaa 4080
caaacatgta aataaagtag ctttaatcgg agcgggtttt gttggaagca gttatgcatt 4140
tgcgttaatt aaccaaggaa tcacagatga gcttgtggtc attgatgtaa ataaagaaaa 4200
agcaatgggc gatgtgagtc gacctgcagg catgcaagct tggcactggc cgtcgtttta 4260
caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 4320
cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 4380
cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt 4440
atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 4500
cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca 4560
tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg 4620
tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat 4680
gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga 4740
acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 4800
ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 4860
gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 4920
ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 4980
gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 5040
agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag 5100
caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 5160
gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 5220
agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 5280
gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 5340
aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 5400
ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 5460
tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 5520
tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 5580
gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 5640
atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 5700
ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 5760
aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 5820
ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 5880
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 5940
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 6000
cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 6060
gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 6120
gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 6180
tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 6240
ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 6300
gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 6360
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 6420
tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 6480
ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 6540
gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 6600
acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg 6660
cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg 6720
aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag 6780
gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt 6840
cacacaggaa acagctatga ccatgattac gaattcgagc tcggtacccg g 6891
<210> 2
<211> 7386
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggatccttat aattttttta atctgttatt taaatagttt atagttaaat ttacattttc 60
attagtccat tcaatattct ctccaagata actacgaact gctaacaaaa ttctctccct 120
atgttctaat ggagaagatt cagccactgc atttcccgca atatcttttg gtatgatttt 180
acccgtgtcc atagttaaaa tcatacggca taaagttaat atagagttgg tttcatcatc 240
ctgataatta tctattaatt cctctgacga atccataatg gctcttctca catcagaaaa 300
tggaatatca ggtagtaatt cctctaagtc ataatttccg tatattcttt tattttttcg 360
ttttgcttgg taaagcatta tggttaaatc tgaatttaat tccttctgag gaatgtatcc 420
ttgttcataa agctcttgta accattctcc ataaataaat tcttgtttgg gaggatgatt 480
ccacggtacc atttcttgct gaataataat tgttaattca atatatcgta agttgctttt 540
atctcctatt ttttttgaaa taggtctaat tttttgtata agtatttctt tactttgatc 600
tgtcaatggt tcagatacga cgactaaaaa gtcaagatca ctatttggtt ttagtccact 660
ctcaactcct gatccaaaca tgtaagtacc aataaggtta ttttttaaat gtttccgaag 720
tatttttttc actttattaa tttgttcgta tgtattcaaa tatatcctcc tcactatttt 780
gattagtacc tattttatat ccatagttgt taattaaata aacttaattt agtttattta 840
tagatttcat tggcttctaa attttttatc tagataataa ttattttagt taattttatt 900
ctagattata tatgatatga tctttcattt ccataaaact aaagtaagtg taaacctatt 960
cattgtttta aaaatatctc ttgccagtca cgttacgtta ttagttatag ttattataac 1020
atgtattcac gaacgaaaat cggatcctct agagattgct agcaagcatg tactgatatc 1080
atactaaacg gctcaaacac aatctataca agtttttaag ataggccaaa gggaataaca 1140
gtatacgtta gtgaaatccc acaggaaaaa tatattaaaa tactaatgtt ctatcaaacg 1200
aacaaccctt aaaaaggaac ttaaaacctc tgggttttaa ggaaattcgc attttattta 1260
gtgtttttct cttgactttg agaacttgaa actagcagaa tagctgactg ttctaggaaa 1320
cagggcgaat ttcgattgcc tatgtctgtc gcgcaaaaaa taaaaacgga cagacatagg 1380
caatcgatca ggatttgaaa ctagcgtcat agagacgtct gaggtttcca gctctgcctt 1440
gctatcgcca ggctttcgcc tgccatgacc tttttacata caatgcttgt cctgtatgca 1500
acttctatgg ggtttgtctc gtgttctctc acacggtcac actcaattgt gtgccgctgc 1560
atagaagctt ggccatagtt gcccgcaccg tagtgcgcca agcaacctag tggtttaggc 1620
acattctccg gaccgttaat ggccgtcctc gccattcacc acaagcgcag caaggaacgc 1680
ttattgtggt atatccccgg gtttgcggtg gacggggcaa ctcctgacgt cagtttattt 1740
tacacccctt aacggcagct gggtgacaaa caaaaaacga cagaaaacca cggtttgata 1800
ccctccaaac agtggttttc tgtcgtccaa aaatagccga aaagtgttga cgtatacact 1860
tgttttcggt aaaatgaaga cataacttaa acattgtaag tgagggctta caaaccaagt 1920
gttcgatgct gcaacatcgg acacttttta tttgtcattc tttatttgta ttcaattttg 1980
caaatagctc gcaaacaaaa tatgtatcat caaatctatt aaccttgttg tctgcaaaca 2040
acagggtttt ttgttgttta tttagaataa ctagaaccag aattcaatgc caaaactttc 2100
acattgactt aacttgactt tatcttacac gatttttttt ttgacgtaaa gccccgggcc 2160
tggtaccagt cgacctgcag gcatgcttca gaacgctcgg ttgccgccgg gcgtttttta 2220
tgcagcaatg gcaagaacgt cccggggagc tcctaactta taggggtaac acttaaaaaa 2280
gaatcaataa cgatagaaac cgctcctaaa gcaggtgcat tttttcctaa cgaagaaggc 2340
aatagttcac atttattgtc taaatgagaa tggactctag aagaaacttc gtttttaatc 2400
gtatttaaaa caatgggatg agattcaatt atatgatttc tcaagataac agcttctata 2460
tcaaatgtat taaggatatt ggttaatcca attccgatat aaaagccaaa gttttgaagt 2520
gcatttaaca tttctacatc atttttattt gcgcgttcca caatctcttt tcgagaaata 2580
ttcttttctt ctttagagag cgaagccagt aacgcttttt cagaagcata taattcccaa 2640
cagcctcgat ttccacagct gcatttgggt ccattaaaat ctatcgtcat atgacccatt 2700
tccccagaaa aaccctgaac acctttatac aattcgttgt taataacaag tccagttcca 2760
attccgatat taatactgat gtaaacgatg ttttcatagt tttttgtcat accaaatact 2820
ttttcaccgt atgctcctgc attagcttca ttttcaacaa aaaccggaac attaaactca 2880
ctctcaatta aaaactgcaa atctttgata ttccaattta agttaggcat gaaaataatt 2940
tgctgatgac gatctacaag gcctggaaca caaattccta ttccgactag accataaggg 3000
gactcaggca tatgggttac aaaaccatga ataagtgcaa ataaaatctc ttttacttca 3060
ctagcggaag aactagacaa gtcagaagtc ttctcgagaa taatatttcc ttctaagtcg 3120
gttagaattc cgttaagata gtcgactcct atatcaatac caatcgagta gcctgcattc 3180
ttattaaaaa caagcattac aggtcttctg ccgcctctag attgccctgc cccaatttca 3240
aaaataaaat ctttttcaag cagtgtattt acttgagagg agacagtaga cttgtttaat 3300
cctgtaatct cagagagagt tgccctggag acaggggagt tcttcaaaat ttcatctaat 3360
attaattttt gattcatttt ttttactaaa gcttgatctg caatttgaat aataaccact 3420
cctttgttta tccaccgaac taagttggtg ttttttgaag cttgaattag atatttaaaa 3480
gtatcatatc taatattata actaaatttt ctaaaaaaaa cattgaaata aacatttatt 3540
ttgtatatga tgagataaag ttagtttatt ggataaacaa actaactcaa ttaagatagt 3600
tgatggataa acttgttcac ttaaatcaaa gggggaaatg acaaatggtc caaactagtg 3660
agtcgactaa ggaggtgaga gcaatggaaa aaaaggaatt tcgtgttttg ataaaatact 3720
gttttctgaa gggaaaaaat acagtggaag caaaaacttg gcttgataat gagtttccgg 3780
actctgcccc agggaaatca acaataattg attggtatgc aaaattcaag cgtggtgaaa 3840
tgagcacgga ggacggtgaa cgcagtggac gcccgaaaga ggtggttacc gacgaaaaca 3900
tcaaaaaaat ccacaaaatg attttgaatg accgtaaaat gaagttgatc gagatagcag 3960
aggccttaaa gatatcaaag gaacgtgttg gtcatatcat tcatcaatat ttggatatgc 4020
ggaagctctg tgcgaaatgg gtgccgcgcg agctcacatt tgaccaaaaa caacaacgtg 4080
ttgatgattc tgagcggtgt ttgcagctgt taactcgtaa tacacccgag tttttccgtc 4140
gatatgtgac aatggatgaa acatggctcc atcactacac tcctgagtcc aatcgacagt 4200
cggctgagtg gacagcgacc ggtgaaccgt ctccgaagcg tggaaagact caaaagtccg 4260
ctggcaaagt aatggcctct gttttttggg atgcgcatgg aataattttt atcgattatc 4320
ttgagaaggg aaaaaccatc aacagtgact attatatggc gttattggag cgtttgaagg 4380
tcgaaatcgc ggcaaaacgg ccccacatga agaagaaaaa agtgttgttc caccaagaca 4440
acgcaccgtg ccacaagtca ttgagaacga tggcaaaaat tcatgaattg ggcttcgaat 4500
tgcttcccca cccgccgtat tctccagatc tggcccccag cgactttttc ttgttctcag 4560
acctcaaaag gatgctcgca gggaaaaaat ttggctgcaa tgaagaggtg atcgccgaaa 4620
ctgaggccta ttttgaggca aaaccgaagg agtactacca aaatggtatc aaaaaattgg 4680
aaggtcgtta taatcgttgt atcgctcttg aagggaacta tgttgaataa aagcttggca 4740
ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 4800
cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 4860
ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt 4920
acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat 4980
gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 5040
tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 5100
cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 5160
tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 5220
ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 5280
ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 5340
attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 5400
gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 5460
ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 5520
cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 5580
gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 5640
tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 5700
gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 5760
ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 5820
tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 5880
gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 5940
caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 6000
cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 6060
atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 6120
gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 6180
attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 6240
cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 6300
atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6360
tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6420
ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6480
ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 6540
cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6600
gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6660
gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6720
acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6780
gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6840
agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6900
tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6960
agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 7020
cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 7080
gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 7140
ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac 7200
aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact 7260
cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg 7320
agcggataac aatttcacac aggaaacagc tatgaccatg attacgaatt cgagctcggt 7380
acccgg 7386
<210> 3
<211> 1484
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gcgttctgtt tctgcttcgg tatgtgattg tgaagctggc ttacagaaga gcggtaaaag 60
aagaaataaa aaagaaatca tcttttttgt ttggaaagcg agggaagcgt tcacagtttc 120
gggcagcttt ttttatagga acattgattt gtattcactc tgccaagttg ttttgataga 180
gtgattgtga taattttaaa tgtaagcgtt aacaaaattc tccagtcttc acatcggttt 240
gaaaggagga agcggaagaa tgaagtaaga gggatttttg actccgaagt aagtcttcaa 300
aaaatcaaat aaggagtgtc aagaatgttt gcaaaacgat tcaaaacctc tttactgccg 360
ttattcgctg gatttttatt gctgtttcat ttggttctgg caggaccggc ggctgcgagt 420
gctgaaacgg cgaacaaatc gaatgagctt acagcaccgt cgatcaaaag cggaaccatt 480
cttcatgcat ggaattggtc gttcaatacg ttaaaacaca atatgaagga tattcatgat 540
gcaggatata cagccattca gacatctccg attaaccaag taaaggaagg gaatcaagga 600
gataaaagca tgtcgaactg gtactggctg tatcagccga catcgtatca aattggcaac 660
cgttacttag gtactgaaca agaatttaaa gaaatgtgtg cagccgctga agaatatggc 720
ataaaggtca ttgttgacgc ggtcatcaat cataccacca gtgattatgc cgcgatttcc 780
aatgaggtta agagtattcc aaactggaca catggaaaca cacaaattaa aaactggtct 840
gatcgatggg atgtcacgca gaattcattg ctcgggctgt atgactggaa tacacaaaat 900
acacaagtac agtcctatct gaaacggttc ttagacaggg cattgaatga cggggcagac 960
ggttttcgat ttgatgccgc caaacatata gagcttccag atgatggcag ttacggcagt 1020
caattttggc cgaatatcac aaatacatct gcagagttcc aatacggaga aatcctgcag 1080
gatagtgcct ccagagatgc tgcatatgcg aattatatgg atgtgacagc gtctaactat 1140
gggcattcca taaggtccgc tttaaagaat cgtaatctgc ttgatatggc tttttatatg 1200
tgttactcta catacagata acaggttggc tgataagtcc ccggtctgac aactttatct 1260
gagaatagtc aatcttcgga aatcccaggt ggcatgctaa aagtctcgta aagcgttcta 1320
tcaataaccc gttggtgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc 1380
ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc 1440
ttcgggtggg cctttctgcg tttataccgt ctcagaatcg gccg 1484
<210> 4
<211> 2027
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aagcttttta gacatctaat cttttctgaa gtacatccgc aactgtccat actctgatgt 60
tttatatctt ttctaaaagt tcgctagata ggggtcccga gcgcctacga ggaatttgta 120
tcggggtata gatctaaaac gaattttaac aaaaaaatgt gtttttcttt gttagaccgg 180
ggacttatca gccaacctgt taaaggagga actaaatatg gccaagttga ccagtgccgt 240
tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg accggctcgg 300
gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg acgtgaccct 360
gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct gggtgtgggt 420
gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga acttccggga 480
cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg agttcgccct 540
gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact gatatctccc 600
actatgcatc tgatgtgtct gcggacaagc tagtgacatg ggtagagtcg catgatacgt 660
atgccaatga tgatgaagag tcgacatgga tgagcgatga tgatatccgt ttaggctggg 720
cggtgatagc ttctcgttca cggcagtacg cctcttttct tttccagacc tgagggaggc 780
ggaaatggtg tgaggttccc ggggaaaagc caaataggcg atcgcgggag tgctttattt 840
gaagatcagg ctatcactgc ggtcaataga tttcacaatg tgatggctgg acagcctgag 900
gaactctcga acccgaatgg aaacaaccag atatttatga atcagcgcgg ctcacatggc 960
gttgtgctgg caaatgcagg ttcatcctct gtctctatca atacggcaac aaaattgcct 1020
gatggcaggt atgacaataa agctggagcg ggttcatttc aagtgaacga tggtaaactg 1080
acaggcacga tcaatgccag gtctgtagct gtgctttatc ctgatgatat tgcaaaagcg 1140
cctcatgttt tccttgagaa ttacaaaaca ggtgtaacac attctttcaa tgatcaactg 1200
acgattacct tgcgtgcaga tgcgaataca acaaaagccg tttatcaaat caataatgga 1260
ccagagacgg cgtttaagga tggagatcaa ttcacaatcg gaaaaggaga tccatttggc 1320
aaaacataca ccatcatgtt aaaaggaacg aacagtgatg gtgtaacgag gaccgagaaa 1380
tacagttttg ttaaaagaga tccagcgtcg gccaaaacca tcggctatca aaatccgaat 1440
cattggagcc aggtaaatgc ttatatctat aaacatgatg ggagccgagt aattgaattg 1500
accggatctt ggcctggaaa accaatgact aaaaatgcag acggaattta cacgctgacg 1560
ctgcctgcgg acacggatac aaccaacgca aaagtgattt ttaataatgg cagcgcccaa 1620
gtgcccggtc agaatcagcc tggctttgat tacgtgctaa atggtttata taatgactcg 1680
ggcttaagcg gttctcttcc ccattgaggg caaggctaga cgggacttac cgaaagaaac 1740
catcaatgat ggtttctttt ttgttcataa atcagacaaa acttttctct tgcaaaagtt 1800
tgtgaagtgt tgcacaatat aaatgtgaaa tacttcacaa acaaaaagac atcaaagaga 1860
aacataccct ggaaggatga ttaatgatga acaaacatgt aaataaagta gctttaatcg 1920
gagcgggttt tgttggaagc agttatgcat ttgcgttaat taaccaagga atcacagatg 1980
agcttgtggt cattgatgta aataaagaaa aagcaatggg cgatgtg 2027
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctttgcggta gtggtgctt 19
<210> 6
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtgccggtac cccaagtgat gatgacttga tatggctttt tatatgtgtt ac 52
<210> 7
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggacagcata aaaaataccc tgtctgaaag ctttttagac atctaatctt ttctg 55
<210> 8
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctcatctgtg attccttgg 19
Claims (7)
1.一种用于把目标DNA片段随机插入枯草芽孢杆菌染色体的组合物，其特征在于，由以下两组分组成：
组分A：质粒pUSIGH，其序列如SEQ ID NO.2所示；
组分B：质粒pTMCZ，其序列如SEQ ID NO.1所示。
2.权利要求1所述的组合物在把目标DNA片段随机插入枯草芽孢杆菌染色体中的应用。
3.权利要求1所述的组合物在构建目的基因被随机插入染色体的枯草芽孢杆菌基因工程菌中的应用。
4.一种枯草芽孢杆菌基因工程菌，其特征在于将权利要求1所述的组合物共同转化枯草芽孢杆菌168，经氯霉素和壮观霉素筛选得到。
5.权利要求1所述的组合物在优化目标蛋白在枯草芽孢杆菌中表达水平中的应用。
6.一种把目标DNA片段随机插入枯草芽孢杆菌染色体的方法，其特征在于，包括：将表达目标DNA的表达盒与权利要求1中所述的质粒pTMCZ的扩增片段进行融合，制备转座子元件LF-Pzeo-ITR1-Transcription terminator-Cm-目标DNA-ITR2-Zeo-RF融合片段，其中LF-Pzeo -ITR1-Transcription terminator-Cm片段的核苷酸序列如SEQ ID NO.3所示，ITR2-Zeo-RF片段的核苷酸序列如SEQ ID NO.4所示，将该转座子元件或含有该转座子的质粒与权利要求1中的质粒pUSIGH共同转化到枯草芽孢杆菌菌株168中，通过氯霉素和壮观霉素抗性筛选得到菌株PDZCM-pUSIGH，经过添加木糖诱导菌株PDZCM-pUSIGH后涂布于含有氯霉素和零霉素的LB平板，获得目标DNA片段随机插入枯草芽孢杆菌染色体的克隆。
7.根据权利要求6所述的方法，其特征在于所述的转座子元件通过化学合成或融合PCR制备。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110355936.6A CN113025641B (zh) | 2021-04-01 | 2021-04-01 | 一种将dna片段随机插入枯草芽孢杆菌染色体的方法及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110355936.6A CN113025641B (zh) | 2021-04-01 | 2021-04-01 | 一种将dna片段随机插入枯草芽孢杆菌染色体的方法及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113025641A CN113025641A (zh) | 2021-06-25 |
| CN113025641B true CN113025641B (zh) | 2023-05-23 |
Family
ID=76454419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110355936.6A Active CN113025641B (zh) | 2021-04-01 | 2021-04-01 | 一种将dna片段随机插入枯草芽孢杆菌染色体的方法及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113025641B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115181714B (zh) * | 2022-05-31 | 2023-08-11 | 南京农业大学 | 一种植物免疫诱抗菌及其构建方法和应用 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063078A (zh) * | 2015-08-03 | 2015-11-18 | 南宁市新科健生物技术有限责任公司 | 一种利用Tn7转座元件整合表达外源蛋白的重组枯草芽孢杆菌构建方法 |
-
2021
- 2021-04-01 CN CN202110355936.6A patent/CN113025641B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063078A (zh) * | 2015-08-03 | 2015-11-18 | 南宁市新科健生物技术有限责任公司 | 一种利用Tn7转座元件整合表达外源蛋白的重组枯草芽孢杆菌构建方法 |
Non-Patent Citations (5)
| Title |
|---|
| A transposon system for random insertion of a gene expression cassette into the chromosome of Bacillus subtilis;Bin Ye，等;J Biotechnol;第10卷;66-73 * |
| In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon;Yoann Le Breton ,等;APPLIED AND ENVIRONMENTAL MICROBIOLOGY;第72卷(第1期);327–333 * |
| Modified mariner Transposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis;Eric R. Pozsgai，等;Applied and Environmental Microbiology;第78卷(第3期);778–785 * |
| 枯草芽孢杆菌OKB015菌株突变体文库的构建及促生相关基因的克隆和功能研究;马欣;中国优秀硕士学士学位论文全文数据库(第7期);全文 * |
| 转座子随机突变芽孢杆菌的研究进展;马欣 等;中国生物防治学报(第3期);394-403 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113025641A (zh) | 2021-06-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2020202823B2 (en) | Engineering plant genomes using CRISPR/Cas systems | |
| KR102264290B1 (ko) | 세포 면역요법을 위한 방법 및 조성물 | |
| CN111349596B (zh) | 产褪黑素的重组大肠杆菌及其构建方法和应用 | |
| CN112703250A (zh) | CRISPRi在高通量代谢工程中的应用 | |
| KR20110076868A (ko) | 분리 조건 및/또는 안전한 작업 범위 하에서의 ｃ5 탄화수소가 없는 이소프렌의 제조 방법 및 조성물 | |
| CN109451729B (zh) | 工程化病毒载体减少了炎症和免疫反应的诱导 | |
| TW201217532A (en) | Nucleic acid construct, recombinant vector and method for producing a target protein | |
| KR20100118973A (ko) | 이소프렌을 생성하기 위한 조성물 및 방법 | |
| CN112725282A (zh) | 携带正交tRNA/氨酰tRNA合成酶的稳定细胞系的构建 | |
| KR20190116282A (ko) | 복수의 숙주를 위한 다중 dna 구조체의 조립 및 편집을 위한 모듈식 범용 플라스미드 디자인 전략 | |
| CN113025641B (zh) | 一种将dna片段随机插入枯草芽孢杆菌染色体的方法及其应用 | |
| CN113564206B (zh) | 一种目的基因多拷贝整合到酿酒酵母染色体rDNA的方法 | |
| CN107429222B (zh) | 在体外培养分节丝状菌的方法 | |
| CN113862166B (zh) | 一种产柚皮素的酿酒酵母菌 | |
| DK2258843T3 (en) | Expression vector with mass production of foreign gene-derived protein by animal cells and use thereof | |
| CN112961832A (zh) | 一种细胞株及其制备方法和用途 | |
| CN109295100A (zh) | 携带正交tRNA/氨酰tRNA合成酶的稳定细胞系的构建 | |
| CN112680450A (zh) | 一种基于CRISPR-Cas系统的全基因组随机突变方法及其应用 | |
| CN101238214A (zh) | 使用改进的调节表达系统治疗疾病 | |
| CN109161545B (zh) | 抑制鸡Sirt1基因表达的microRNA及其重组过表质粒以及LMH细胞系 | |
| CN111534578A (zh) | 一种高通量筛选真核生物细胞与农药互作的靶点基因的方法 | |
| CN106566832A (zh) | 针对nfat3基因靶点的短发卡rna、重组载体及应用 | |
| CN111534544A (zh) | 一种高通量筛选真核生物细胞与病毒互作靶点基因的方法 | |
| CN111388658B (zh) | 基于重组减毒李斯特菌的kras高表达癌症疫苗及其制备方法及其应用方法 | |
| CN112195190B (zh) | 来源于贝莱斯芽孢杆菌质粒的复制元件及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 210043 Jiangsu Nanjing Qixia District Bagua Zhou street Jiangsu Qixia modern agriculture industrial park Nanjing Agricultural University modern horticulture industry science and Technology Innovation Center Patentee after: NANJING AGRICULTURAL University Address before: 211225 Jiangsu Nanjing Lishui District Baima Town National Agricultural Science and Technology Park Nanjing Agricultural University base Patentee before: NANJING AGRICULTURAL University |