CN113667687A - 基于ai-2分子响应的启动元件及其构建的大肠杆菌动态调控系统和方法 - Google Patents

基于ai-2分子响应的启动元件及其构建的大肠杆菌动态调控系统和方法 Download PDF

Info

Publication number
CN113667687A
CN113667687A CN202110968581.8A CN202110968581A CN113667687A CN 113667687 A CN113667687 A CN 113667687A CN 202110968581 A CN202110968581 A CN 202110968581A CN 113667687 A CN113667687 A CN 113667687A
Authority
CN
China
Prior art keywords
dcpf1
lsrr
lsra
pacyduet
gfp
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
Application number
CN202110968581.8A
Other languages
English (en)
Other versions
CN113667687B (zh
Inventor
刘龙
陈坚
刘振民
吕雪芹
堵国成
李江华
苏米亚
林璐
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bright Dairy and Food Co Ltd
Original Assignee
Bright Dairy and Food Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Bright Dairy and Food Co Ltd filed Critical Bright Dairy and Food Co Ltd
Priority to CN202110968581.8A priority Critical patent/CN113667687B/zh
Publication of CN113667687A publication Critical patent/CN113667687A/zh
Priority to US17/972,463 priority patent/US20230167435A1/en
Application granted granted Critical
Publication of CN113667687B publication Critical patent/CN113667687B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • 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
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • 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
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • 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
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • 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)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了一种基于AI‑2分子响应的启动元件及其构建的大肠杆菌动态调控系统和方法，构建了一种基于AI‑2分子响应的细胞密度依赖型启动元件PJ23119‑LsrR‑PlsrA，利用该元件可以自诱导dCpf1的表达，进一步通过组装不同靶基因的crRNA，可以将该自诱导元件用于dCpf1‑CRP对合成途径上的基因进行动态调控。本发明通过构建载体pACYDuet‑PJ23119‑LsrR‑PlsrA‑dCpf1‑CRP、pRSFDuet‑GFP‑mCherry和pETDuet‑crRNA，可以用于同时对不同基因进行转录激活和抑制。本发明重组大肠杆菌的构建方法简单，具有很好的应用前景。

Description

基于AI-2分子响应的启动元件及其构建的大肠杆菌动态调控 系统和方法
技术领域
本发明属于基因工程技术领域，特别涉及一种基于AI-2分子响应的启动元件及其构建的大肠杆菌动态调控系统和方法。
背景技术
大肠杆菌(Escherichia coli)长期以来被广泛用作分子生物学的实验室菌株，促进了DNA复制、转录、翻译和表达中许多重要机制的发现。此外，大肠杆菌因其生长发育快速，培养条件简单，代谢可塑性强，生化和生理功能丰富，是实验室和工业应用的首选原核生物。大肠杆菌较为完善的生物化学、生理学和遗传学性质使大肠杆菌在代谢工程和合成生物学方面的研究取得了迅速进展，已被广泛用作工业宿主代谢合成如赖氨酸、1,3-丙二醇和1,4-丁二醇。大肠杆菌菌株通常被认为是无害的，如实验室菌株E.coli MG1655，被认为是缺乏与人类疾病相关的毒力因子，已被广泛应用于生产各种生物化学品、食品和生物燃料。
可编程的位点特异性基因调控元件在全基因组层次的基因功能研究、代谢通路调控以及人工基因线路设计中发挥重要作用。基于间隔短回文重复序列(Clusteredregularly interspaced shortpalindromic repeat sequences,CRISPR)的基因调控工具是目前最受欢迎的一种基因调控技术，利用CRISPR技术可以对微生物进行多基因的调控。CRISPR基因调控系统是通过引导RNA(如sgRNA、crRNA等)引导缺陷型核酸酶(如dCas9、dCpf1等)定位到靶基因位点时，并不切割DNA，而是与转录阻遏域融合，形成的RNA-蛋白质复合物被引导到靶基因的转录起始位点以抑制其转录；而当dCas蛋白C端融合转录激活因子时，dCas蛋白与转录激活因子可在转录起始位点激活靶基因的转录。近年来，随着CRISPR-dCpf1系统的可编程转录因子的出现，基因表达的合成控制变得更加简单。相比CRISPR-dCas9系统，CRISPR-dCpf1优势明显，具有更低的脱靶效率。此外，dCpf1只需要单个RNA引导，而dCas9的引导需要sgRNA和tracrRNA两个RNA的共同作用。因此，CRISPR-dCpf1系统更适合多个基因位点的调控。
基因表达的动态调控已成为一种普遍的调控策略，微生物能够根据微生物群落或细胞代谢状态自主调节其代谢通量。其中，群体感应(Quorum sensing，QS)是细菌通过分泌信号分子来监测群体密度并协调微生物功能的信息交流机制，已被广泛应用于自动调控代谢合成途径中关键基因的表达，或用于自诱导重组蛋白的表达。而基于AI-2(Autoinducer-2,AI-2)信号分子响应的群体响应系统(AI-2QS)是大肠杆菌MG1655中的一种内源性群体响应系统。在AI-2QS系统中，当细胞密度较低时，转录因子LsrR结合于PlsrA启动子上的lsrAbox序列，从而抑制PlsrA启动子的转录。随着菌体细胞密度增加，胞内合成的信号分子AI-2被不断通过TqsA蛋白转运出胞外，胞外AI-2信号分子不断累积达到一定阈值时，AI-2分子又被LsrABCD转运系统转入进胞内，并在LsrK蛋白作用下形成磷酸化AI-2分子，磷酸化的AI-2分子与PlsrA启动子上的lsrAbox竞争性结合LsrR分子，从而解除LsrR对PlsrA的转录抑制作用，从而激活启动子PlsrA的表达(图1)。因此，可以利用大肠杆菌自身的群体响应系统，开发细胞密度依赖型的启动子PlsrA自动开启CRISPR-dCpf1调控系统，进一步通过组装合成途径中不同基因的crRNA，可以用于动态调控代谢合成途径上的基因。
CRISPR-dCpf1系统是目前较为受欢迎的一种基因编辑和调控系统，相比CRISPR-dCas9系统，引导RNA(crRNA)序列较短，通过构建不同crRNA的阵列，便可用于多基因的调控中。苗成思等人研究了CRISPR-dCpf1系统中目标序列位置、crRNA中重复序列及间隔序列长度、PAM序列等因素对CRISPR-dCpf1系统在大肠杆菌中调控性能的影响，得到了基于异丙基硫代半乳糖苷(Isopropyl-β-D-Thiogalactoside,IPTG)诱导高效CRISPR-dCpf1基因调控工具。武耀康等人也通过设计crRNA阵列在枯草芽孢杆菌中构建了一种基于木糖诱导的CRISPR/Cpf1的多基因编辑和转录调控系统(CAMERS-B)，不仅可以实现B.subtilis中双基因敲除、多点突变或单基因插入，还可以同时对多个基因进行转录抑制和激活。进一步利用CAMERS-B系统抑制乙偶姻分解代谢途径的基因(bdhA和acoA)以及合成副产物乳酸和乙酸的基因(ldh和pta)，同时激活乙偶姻合成途径中的途径基因alsSD及其转录激活因子的基因alsR。通过构建针对bdhA，acoA，ldh，pta和alsR的crRNA阵列，实现了CAMERS-B系统在乙偶姻合成相关途径中的转录抑制和激活，从而使乙偶姻的产量提高了44.8％，达到25.8g/L。
尽管目前已开发了基于IPTG诱导诱导的CRISPR-dCpf1调控系统。但利用IPTG诱导dCpf1的过度表达可能会对细胞造成代谢负担，从而对代谢合成产生负面影响。因此，如果采用自诱导型的信号分子自动触发CRISPR-dCpf1调控系统，可以避免重组蛋白对细胞造成的代谢负担，以及IPTG对细胞的潜在毒害作用。通常在引入一些外源代谢合成途径时，由于响应中间代谢物的阈值有一定浓度范围，或由于缺乏响应中间代谢产物调控机制方面的研究，很难开发出可以响应特定信号分子的自诱导元件用于动态调控合成途径上关键基因的表达。而基于细胞群体响应的动态调控是目前代谢合成中较有潜力的一种调控系统，无需响应特定中间代谢物，利用细胞群体密度的变化，便可分泌相关信号分子，激活或抑制相关基因的表达。因此，利用大肠杆菌内源性细胞密度依赖的AI-2信号分子可以开发出自诱导dCpf1的表达元件，进一步通过构建特定基因的crRNA可以实现动态激活或抑制代谢合成途径中相关基因的表达。
发明内容
为解决上述基于IPTG诱导的CRISPR-dCpf1调控系统可能对细胞产生的潜在毒害作用，本发明构建了基于AI-2信号分子的细胞密度依赖型响应元件PJ23119-LsrR-PlsrA(SEQID NO.1)，利用该元件可以自诱导dCpf1的表达，进一步通过构建特定基因的crRNA可以动态激活或抑制代谢合成途径相关基因的表达，从而避免IPTG等诱导剂对细胞的潜在毒害作用。本发明主要是通过以下技术方案解决上述技术问题。
本发明提供了一种基于AI-2分子响应的启动元件，该启动元件为细胞密度依赖型启动元件PJ23119-LsrR-PlsrA；所述细胞密度依赖型启动元件PJ23119-LsrR-PlsrA是将基于AI-2信号分子响应的野生型启动元件基因片段PlsrR-LsrR-PlsrA中的天然细胞密度依赖型启动子PlsrA进行优化，利用组成型启动子PJ23119表达转录调控因子LsrR，构建基于AI-2分子响应的细胞密度依赖型启动元件PJ23119-LsrR-PlsrA；优选的，所述元件PJ23119-LsrR-PlsrA的核苷酸序列如SEQ ID NO.1所示，所述所述野生型启动元件基因片段PlsrR-LsrR-PlsrA的核苷酸序列如SEQ ID NO.2所示。
本发明还提供了一种基于AI-2分子响应的细胞密度依赖型启动元件PJ23119-LsrR-PlsrA的应用，用于自动触发CRISPR-dCpf1调控系统。
本发明还提供了一种基于AI-2分子响应的大肠杆菌CRISPR-dCpf1动态调控系统，包括dCpf1蛋白表达载体pACYDuet-dCpf1、报告荧光蛋白GFP和mCherry表达载体pRSFDuet-GFP-mCherry和报告蛋白的crRNA表达载体pETDuet-crRNA；优选的，所述pACYDuet-dCpf1表达载体包括细胞密度依赖型启动元件PJ23119-LsrR-PlsrA。
优选的，选用大肠杆菌中转录激活因子CRP融合在dCpf1蛋白的C端。
本发明还提供了一种基于AI-2分子响应的大肠杆菌CRISPR-dCpf1动态调控系统的构建方法，包括以下步骤：
(1)选择质粒pACYDuet、pRSFDuet和pETDuet作为出发载体，将FndCpf1和PJ23119-LsrR-PlsrA基因片段与pACYDuet进行重组连接，构建pACYDuet-PJ23119-LsrR-PlsrA-dCpf1载体；将GFP基因和线性化载体pRSFDuet进行重组连接，构建pRSFDuet-GFP载体；将crRNA片段和线性化载体pETDuet进行重组连接，构建crRNA基因的pETDuet-crRNA载体；
(2)以pACYDuet-PJ23119-LsrR-PlsrA-dCpf1为模板，将转录激活因子CRP融合在dCpf1蛋白的C端，构建dCpf1激活质粒pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP；
(3)以步骤(1)中的pRSFDuet-GFP载体为模板，将mCherry的基因片段与pRSFDuet-GFP进行重组连接，构建荧光激活和抑制的报告载体pRSFDuet-GFP-mCherry；
(4)将pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP质粒与pRSFDuet-GFP-mCherry和pETDuet-crRNA载体共转化到E.coliMG1655感受态中。
本发明还提供了一种基于AI-2分子响应的大肠杆菌CRISPR-dCpf1动态调控系统在大肠杆菌合成途径基因的表达调控中的应用。
与现有技术相比，本发明的积极进步效果在于：
本发明构建了一种基于AI-2分子响应的细胞密度依赖型启动元件PJ23119-LsrR-PlsrA，采用强启动子PJ23119表达转录因子LsrR，可以有效增强报告蛋白的荧光强度。通过组装不同靶基因的crRNA，可以将该自诱导元件用于dCpf1-CRP对合成途径上的基因进行动态调控。本发明通过构建载体pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP、pRSFDuet-GFP-mCherry和pETDuet-crRNA，可以用于同时对不同基因进行转录激活和抑制。本发明重组大肠杆菌的构建方法简单，具有很好的应用前景。
附图说明
图1为基于AI-2信号分子响应的大肠杆菌群体响应系统；
图2为CRISPR-dCpf1调控系统的构建示意图；
图3为转录因子CRP和RpoZ对绿色荧光蛋白的激活强度；
图4为基于阿拉伯糖诱导的CRISPR-dCpf1基因激活和抑制的调控效果图；
图5为基于AI-2信号分子的细胞密度依赖启动子PlsrA的荧光强度；
图6为基于细胞密度依赖的CRISPR-dCpf1基因激活和抑制的调控效果图。
具体实施方式
本发明提供了一种基于AI-2信号分子响应的细胞密度依赖型元件PJ23119-LsrR-PlsrA，利用该元件可以自动触发CRISPR-dCpf1调控系统，为动态调控代谢合成途径中的基因提供了一种工具。本发明中，所述元件PJ23119-LsrR-PlsrA的核苷酸序列如SEQ ID NO.1所示。
本发明中，要实现构建的CRISPR-dCpf1系统同时具有激活和抑制的调控作用，首先构建了3种兼容质粒，包括dCpf1蛋白表达载体pACYDuet-dCpf1、报告荧光蛋白GFP和mCherry表达载体pRSFDuet-GFP-mCherry和报告蛋白的crRNA表达载体pETDuet-crRNA。其次，选用大肠杆菌中转录激活因子(CRP和RpoZ)分别融合在dCpf1蛋白的C端后，检测不同转录因子的激活强度。
本发明中，由于基于AI-2信号分子响应的天然细胞密度依赖型启动子PlsrA的荧光强度较低，需要对该启动子进行优化，包括启动子核心区的替换，RBS序列随机突变，转录因子LsrR的表达。。随后将细胞密度依赖型启动子PlsrA替换CRISPR-dCpf1系统中利用阿拉伯糖诱导的启动子ParaB，构建了同时具有激活和抑制作用的CRISPR-dCpf1动态调控系统。采用本发明构建的基于AI-2分子响应的CRISPR-dCpf1动态调控系统，只需设计将代谢合成途径相关基因的crRNA进行组装后，便可应用于动态调控大肠杆菌中合成途径基因的表达。
本发明还提供了一种基于AI-2信号分子响应的大肠杆菌CRISPR-dCpf1动态调控系统的构建方法，该构建方法包括以下步骤：
(1)CRISPR-dCpf1激活系统的构建
CRISPR-dCpf1调控系统的构建如图2所示，首先选用大肠杆菌常用的质粒pACYDuet、pRSFDuet和pETDuet作为出发载体，使用无缝克隆试剂盒将FndCpf1和araB诱导元件的基因片段(araC-ParaB)与pACYDuet进行重组连接，构建利用阿拉伯糖诱导dCpf1表达的载体pACYDuet-ParaB-dCpf1；将GFP基因和线性化载体pRSFDuet进行重组连接，选用低拷贝复制子SC101ori替换初始载体pRSFDuet上的高拷贝复制子pRSF ori，构建pRSFDuet-GFP载体；将crRNA片段和线性化载体pETDuet进行重组连接，构建利用PJ23119表达crRNA的载体pETDuet-crRNA载体。随后以pACYDuet-ParaB-dCpf1为模板，将转录激活因子CRP和RpoZ分别融合在dCpf1蛋白的C端，构建了两种dCpf1激活质粒pACYDuet-ParaB-dCpf1-CRP和pACYDuet-ParaB-dCpf1-RpoZ。最后将dCpf1表达质粒pACYDuet-ParaB-dCpf1、pACYDuet-ParaB-dCpf1-CRP和pACYDuet-ParaB-dCpf1-RpoZ分别与pRSFDuet-GFP和pETDuet-crRNA载体共转化到E.coliMG1655感受态中，挑取单菌落到96孔板中培养，考察这两种转录因子的激活作用。
(2)CRISPR-dCpf1-CRP激活和抑制系统的构建
以上述步骤(1)中的pRSFDuet-GFP载体为模板，使用无缝克隆试剂盒将mCherry的基因片段与pRSFDuet-GFP进行重组连接，构建荧光激活和抑制的报告载体pRSFDuet-GFP-mCherry。将上述步骤(1)中荧光强度较高的转录因子CRP载体pACYDuet-ParaB-dCpf1-CRP与pRSFDuet-GFP-mCherry和pETDuet-crRNA载体共转化到E.coliMG1655感受态中，挑取单菌落到96孔板中培养，验证CRISPR-ParaB-dCpf1-CRP系统可以同时激活和抑制的效果。
(3)基于AI-2信号分子响应的细胞密度依赖型元件的构建
首先使用无缝克隆试剂盒将E.coliMG1655基因组上的ParaB启动子基因片段和PlsrA启动子基因片段(SEQ ID NO.2)分别与pACYDuet载体进行重组连接，构建对照组载体pACYDuet-ParaB-gfp和pACYDuet-PlsrR-LsrR-PlsrA-gfp。由于基于AI-2信号分子响应的天然细胞密度依赖型启动子PlsrA的荧光强度较低，进一步利用组成型启动子PJ23119表达转录因子LsrR，构建pACYDuet-PJ23119-LsrR-PlsrA-gfp载体。最后，将pACYDuet-ParaB-gfp、pACYDuet-PlsrR-LsrR-PlsrA-gfp和pACYDuet-PJ23119-LsrR-PlsrA-gfp质粒分别转化到E.coliMG1655感受态中，挑取单菌落到96孔板中培养，比较细胞密度依赖型启动子PlsrA相较于诱导型启动子ParaB的荧光强度。
(4)CRISPR-dCpf1-CRP动态调控系统的构建
将上述PlsrR-LsrR-PlsrA和PJ23119-LsrR-PlsrA的基因序列替换CRISPR-dCpf1系统中利用阿拉伯糖诱导的启动子ParaB，分别构建pACYDuet-PlsrR-LsrR-PlsrA-dCpf1-CRP和pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP质粒，并将这两个分别与pRSFDuet-GFP-mCherry和pETDuet-crRNA载体共转化到E.coliMG1655感受态中，挑取单菌落到96孔板中培养，验证CRISPR-PlsrA-dCpf1-CRP可以根据细胞密度的变化自发开启激活和抑制基因的效果。
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例对本发明进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
(1)CRISPR-dCpf1激活系统的构建
选用大肠杆菌常用的质粒pACYDuet、pRSFDuet和pETDuet作为出发载体，通过PCR分别将质粒pACYDuet、pRSFDuet和pETDuet线性化并使用DpnI酶消化模板DNA。随后以申请号为CN201911387447.8中pLCg6-dCpf1质粒为模板扩增FndCpf1基因，以E.coli基因组为模板扩增阿拉伯糖启动子ParaB，使用无缝克隆试剂盒将FndCpf1和ParaB的基因纯化回收片段连接到pACYDuet上，构建pACYDuet-ParaB-dCpf1载体；扩增申请号为CN201911387447.8中gfp基因，基因合成获得启动子PJ23117及其上游序列(来源于文献：Bikard,D.；Jiang,W.；Samai,P.；Hochschild,A.；Zhang,F.；Marraffini,L.A.,Programmable repression andactivation of bacterial gene expression using an engineered CRISPR-Cassystem.Nucleic Acids Res.2013,41(15),7429-7437.)，将这两个片段与线性化载体pRSFDuet进行重组连接；随后以质粒pSC101-Donor(Addgene#140630)为模板扩增复制子pSC101 ori，构建低拷贝报告蛋白质粒pRSFDuet-GFP；以申请号为CN201911387447.8中pcrF11载体为模板扩增crRNA片段，通过无缝克隆将crRNA片段与pETDuet进行重组连接，随后在CRISPR-DT网站(http://bioinfolab.miamioh.edu/CRISPR-DT/interface/Cpf1_design.php)上设计gfp基因启动子上游序列的crRNA用于激活报告蛋白GFP，构建激活gfp基因的pETDuet-crRNA载体。然后线性化pACYDuet-ParaB-dCpf1载体，以E.coli基因组为模板扩增转录激活因子CRP和RpoZ的基因片段，使用(GGGGS)2柔性Linker将转录因子分别融合在dCpf1蛋白的C端，构建dCpf1激活质粒pACYDuet-ParaB-dCpf1-CRP和pACYDuet-ParaB-dCpf1-RpoZ。最后将dCpf1表达质粒pACYDuet-ParaB-dCpf1、pACYDuet-ParaB-dCpf1-CRP和pACYDuet-ParaB-dCpf1-RpoZ分别与pRSFDuet-GFP和pETDuet-crRNA载体共同转化到E.coliMG1655感受态中，涂布带有氯霉素、卡那霉素和氨苄青霉素的抗性平板上，于30℃培养16-18h后，挑取阳性单菌落到96孔板中培养，使用Tecan酶标仪检测这两种转录因子(CRP和RpoZ)对绿色荧光蛋白的激活强度，设置绿色荧光蛋白的激发波长为490mm，发射波长为530mm。如图3所示，转录因子RpoZ对绿色荧光蛋白的激活效果不明显，而转录因子CRP对绿色荧光蛋白的激活效果明显，相比不融合转录因子时，绿色荧光强度提高了1.8倍。
(2)CRISPR-dCpf1-CRP激活和抑制系统的构建
通过PCR分别将质粒pRSFDuet-GFP线性化并使用DpnI酶消化模板DNA，扩增申请号为CN201911387447.8中的红色荧光蛋白基因mCherry，使用无缝克隆试剂盒将mCherry的基因片段与线性化载体pRSFDuet-GFP进行重组连接，构建荧光激活和抑制的报告载体pRSFDuet-GFP-mCherry。选用上述激活强度较高的转录因子CRP(图3)的载体pACYDuet-ParaB-dCpf1-CRP与报告蛋白质粒pRSFDuet-GFP-mCherry和pETDuet-crRNA载体共同转化到E.coli MG1655感受态中，涂布带有氯霉素、卡那霉素和氨苄青霉素的抗性平板上，于30℃培养16-18h后，挑取阳性单菌落到96孔板中培养12-15h后，添加终浓度为40-60mM的阿拉伯糖诱导GFP和mCherry的表达。使用Tecan酶标仪对绿色荧光蛋白和红色荧光蛋白的表达强度进行检测，设置绿色荧光蛋白的激发波长为490mm，发射波长为530mm；红色荧光蛋白激发波长为580mm，发射波长为615mm。如图4所示，当添加阿拉伯糖诱导dCpf1表达后，从15h开始，绿色荧光蛋白的表达强度有明显的提高，而红色荧光蛋白的表达强度也呈逐渐下降的趋势。说明构建的CRISPR-dCpf1调控系统在阿拉伯糖诱导的作用下可以同时激活和抑制不同基因的表达强度。
实施例2
pACYDuet-PlsrR-LsrR-PlsrA-gfp载体的构建
选择pACYDuet质粒为出发质粒，通过PCR将质粒pACYDuet线性化并使用DpnI酶消化模板DNA，随后以E.coli基因组为模板分别扩增野生型阿拉伯糖启动子ParaB和细胞密度依赖型启动子PlsrA，使用无缝克隆试剂盒将ParaB和PlsrA的基因纯化回收片段连接到线性化载体pACYDuet上，构建pACYDuet-ParaB-gfp和pACYDuet-PlsrR-LsrR-PlsrA-gfp绿色荧光蛋白报告载体。最后，将构建的pACYDuet-ParaB-gfp和pACYDuet-PlsrR-LsrR-PlsrA-gfp质粒分别转化到E.coli MG1655感受态中，挑取单菌落到96孔板中培养，比较细胞密度依赖型启动子PlsrA和诱导型启动子ParaB的荧光强度。使用Tecan酶标仪对绿色荧光蛋白的表达强度进行检测，设置绿色荧光蛋白的激发波长为490mm，发射波长为530mm。
实施例3
pACYDuet-PJ23119-LsrR-PlsrA-gfp载体的构建
由于利用野生型启动子PlsrR表达转录因子LsrR，pACYDuet-PlsrR-LsrR-PlsrA-gfp的荧光强度较弱，因此选用组成型启动子PJ23119来表达转录调控因子LsrR。以pACYDuet-PlsrR-LsrR-PlsrA-gfp质粒为模板，通过环化PCR将野生型PlsrR启动子替换为PJ23119启动子，并使用DpnI酶消化模板DNA，随后将纯化回收的片段转入转化到E.coli MG1655感受态中，涂布含有氯霉素的抗性平板，于37℃过夜培养，挑取阳性单菌落送测。将测序正确的pACYDuet-PJ23119-LsrR-PlsrA-gfp质粒与实施例2中构建正确的pACYDuet-ParaB-gfp和pACYDuet-PlsrR-LsrR-PlsrA-gfp质粒，再重新转化到E.coli MG1655感受态中，涂布含有氯霉素的抗性平板，挑取单菌落到96孔板中培养，设置绿色荧光蛋白的激发波长为490mm，发射波长为530mm，比较这三种质粒中绿色荧光蛋白的表达强度。如图5所示，野生型PlsrA启动子的强度最弱，几乎不表达，而当使用组成型启动子PJ23119表达转录因子LsrR后，GFP荧光强度相比诱导型启动子ParaB提高了2.2倍。
实施例4
CRISPR-dCpf1-CRP动态调控系统的构建
通过PCR线性化上述实施例1中构建的pACYDuet-ParaB-dCpf1-CRP质粒，并使用DpnI酶消化模板DNA，随后以上述实施例3中构建的pACYDuet-PJ23119-LsrR-PlsrA-gfp质粒为模板，扩增PJ23119-LsrR-PlsrA基因片段，使用无缝克隆试剂盒将PJ23119-LsrR-PlsrA的基因纯化回收片段连接到线性化载体pACYDuet-ParaB-dCpf1-CRP上，将上述实施例3中构建的细胞密度依赖型启动元件PJ23119-LsrR-PlsrA替换CRISPR-dCpf1系统中利用阿拉伯糖诱导的启动子ParaB，构建pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP质粒。最后将其与报告蛋白质粒pRSFDuet-GFP-mCherry和pETDuet-crRNA载体共同转化到E.coli MG1655感受态中，涂布含有氯霉素的抗性平板，挑取单菌落到96孔板中培养，设置绿色荧光蛋白的激发波长为490mm，发射波长为530mm，以及设置红色荧光蛋白激发波长为580mm，发射波长为615mm，验证CRISPR-PlsrA-dCpf1-CRP可以根据细胞密度的变化自发激活绿色荧光蛋白和抑制红色荧光蛋白的作用。如图6所示，当细胞培养到22h到26h时，CRISPR-dCpf1动态调控系统对绿色荧光蛋白和红色荧光蛋白有较明显的激活(1.3倍)和抑制(1.5倍)效果。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 光明乳业股份有限公司
<120> 基于AI-2分子响应的启动元件及其构建的大肠杆菌动态调控系统和方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1138
<212> DNA
<213> （人工序列）
<400> 1
ttaactacgt aaaatcgccg ctgctgtgtc ctgatcggta accagtgcgt tgatataacc 60
gcctttcatt gcagcggcaa ttgcttcggc tttattttct ccccctgcca cgccaacccg 120
gacgggtatg gtcttcagcg cgcttaaagg taagccaatc agttcgttat gtattttgat 180
attcgtgaca acgtcacctt ttgcatcaaa aaagtagcct aaaatgtcgc caaccgcccc 240
ttttcggcca atcattaact gttcgccctg gctgatataa ccggagcgaa tgattgtcgc 300
atcgtcctgt tgactcacag caccaatgcc gacaatcgcc acatccgctg cttgcgcggc 360
taacagaaca tctttgacgc aattttcatt ttttagcgta cgggcaatgt cagcggagga 420
tgcccgcaac ggagccggaa taatattcac actgcacgcc gcgttaagct gcccgattcc 480
cgtcatataa gaaccgacgc caccggagag cgtgaccagg cgaatttgct gtgacgaaat 540
aaaaccactt aagcgttgca gcgtattcat ggttgcctcg ccaaaaccaa tcgccagcat 600
ctgttgtggt tgaagtaaac tcatcaacat atgcgccgcg cctatcccca gtcgcccacc 660
gacatcagca tccgcaagcc cagggatcac ccggacatgt tgcagcgaaa actgacgacg 720
taattgagtt tcatattcca gacagccttc aaagcgagaa ttaatctgta cgcgaataat 780
gccggactga tgccctttct ccagcaatcg cgacactttc aaacgtgtca ggccgagacg 840
atcgctgatc tcgctctggg tcagcccgtc gtgatagtaa aaccacgcga tccgcgcgac 900
ctgttcttct tcacacattc cctgttctga aattgccgaa tcgttgattg tcatgaattc 960
attaaagagg agaaaggtac cgctagcatt atacctagga ctgagctagc tgtcaaaaat 1020
agcataaatt gtgatctatt cgtcggaaat atgtgcaatg tccacctaag gttatgaaca 1080
aattaaaagc agaaatacat ttgttcaaaa ctcacctgca aaactgaacg ggggaaat 1138
<210> 2
<211> 1202
<212> DNA
<213> （人工序列）
<400> 2
ttaactacgt aaaatcgccg ctgctgtgtc ctgatcggta accagtgcgt tgatataacc 60
gcctttcatt gcagcggcaa ttgcttcggc tttattttct ccccctgcca cgccaacccg 120
gacgggtatg gtcttcagcg cgcttaaagg taagccaatc agttcgttat gtattttgat 180
attcgtgaca acgtcacctt ttgcatcaaa aaagtagcct aaaatgtcgc caaccgcccc 240
ttttcggcca atcattaact gttcgccctg gctgatataa ccggagcgaa tgattgtcgc 300
atcgtcctgt tgactcacag caccaatgcc gacaatcgcc acatccgctg cttgcgcggc 360
taacagaaca tctttgacgc aattttcatt ttttagcgta cgggcaatgt cagcggagga 420
tgcccgcaac ggagccggaa taatattcac actgcacgcc gcgttaagct gcccgattcc 480
cgtcatataa gaaccgacgc caccggagag cgtgaccagg cgaatttgct gtgacgaaat 540
aaaaccactt aagcgttgca gcgtattcat ggttgcctcg ccaaaaccaa tcgccagcat 600
ctgttgtggt tgaagtaaac tcatcaacat atgcgccgcg cctatcccca gtcgcccacc 660
gacatcagca tccgcaagcc cagggatcac ccggacatgt tgcagcgaaa actgacgacg 720
taattgagtt tcatattcca gacagccttc aaagcgagaa ttaatctgta cgcgaataat 780
gccggactga tgccctttct ccagcaatcg cgacactttc aaacgtgtca ggccgagacg 840
atcgctgatc tcgctctggg tcagcccgtc gtgatagtaa aaccacgcga tccgcgcgac 900
ctgttcttct tcacacattc cctgttctga aattgccgaa tcgttgattg tcataattca 960
ttcttcactt tgaacatatt taaatcttta atgcaattgt tcagttcttg ctcatttata 1020
tctgtgatgg caaccacagt ttgactctac gagcatgaac aaacgcaacc gtgaaaatca 1080
aaatagcata aattgtgatc tattcgtcgg aaatatgtgc aatgtccacc taaggttatg 1140
aacaaattaa aagcagaaat acatttgttc aaaactcacc tgcaaaactg aacgggggaa 1200
at 1202

Claims (6)

1.一种基于AI-2分子响应的启动元件，其特征在于，该启动元件为细胞密度依赖型启动元件PJ23119-LsrR-PlsrA；所述细胞密度依赖型启动元件PJ23119-LsrR-PlsrA是将基于AI-2信号分子响应的野生型启动元件基因片段PlsrR-LsrR-PlsrA中的天然细胞密度依赖型启动子PlsrA进行优化，利用组成型启动子PJ23119表达转录调控因子LsrR，构建基于AI-2分子响应的细胞密度依赖型启动元件PJ23119-LsrR-PlsrA；所述启动元件PJ23119-LsrR-PlsrA的核苷酸序列如SEQ ID NO.1所示，所述野生型启动元件基因片段PlsrR-LsrR-PlsrA的核苷酸序列如SEQ IDNO.2所示。
2.一种基于AI-2分子响应的细胞密度依赖型启动元件PJ23119-LsrR-PlsrA的应用，其特征在于，用于自动触发CRISPR-dCpf1调控系统。
3.一种基于AI-2分子响应的大肠杆菌CRISPR-dCpf1动态调控系统，其特征在于，包括dCpf1蛋白表达载体pACYDuet-dCpf1、报告荧光蛋白GFP和mCherry表达载体pRSFDuet-GFP-mCherry和报告蛋白的crRNA表达载体pETDuet-crRNA；所述pACYDuet-dCpf1表达载体包括细胞密度依赖型启动元件PJ23119-LsrR-PlsrA。
4.根据权利要求3所述的基于AI-2分子响应的大肠杆菌CRISPR-dCpf1动态调控系统，其特征在于，选用大肠杆菌中转录激活因子CRP融合在dCpf1蛋白的C端。
5.一种基于AI-2分子响应的大肠杆菌CRISPR-dCpf1动态调控系统的构建方法，其特征在于，包括以下步骤：
(1)选择质粒pACYDuet、pRSFDuet和pETDuet作为出发载体，将FndCpf1和PJ23119-LsrR-PlsrA基因片段与pACYDuet进行重组连接，构建pACYDuet-PJ23119-LsrR-PlsrA-dCpf1载体；将GFP基因和线性化载体pRSFDuet进行重组连接，构建pRSFDuet-GFP载体；将crRNA片段和线性化载体pETDuet进行重组连接，构建crRNA基因的pETDuet-crRNA载体；
(2)以pACYDuet-PJ23119-LsrR-PlsrA-dCpf1为模板，将转录激活因子CRP融合在dCpf1蛋白的C端，构建dCpf1激活质粒pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP；
(3)以步骤(1)中的pRSFDuet-GFP载体为模板，将mCherry的基因片段与pRSFDuet-GFP进行重组连接，构建荧光激活和抑制的报告载体pRSFDuet-GFP-mCherry；
(4)将pACYDuet-PJ23119-LsrR-PlsrA-dCpf1-CRP质粒与pRSFDuet-GFP-mCherry和pETDuet-crRNA载体共转化到E.coliMG1655感受态中。
6.一种基于AI-2分子响应的大肠杆菌CRISPR-dCpf1动态调控系统在大肠杆菌合成途径基因的表达调控中的应用。
CN202110968581.8A 2021-08-23 2021-08-23 基于ai-2分子响应的启动元件及其构建的大肠杆菌动态调控系统和方法 Active CN113667687B (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202110968581.8A CN113667687B (zh) 2021-08-23 2021-08-23 基于ai-2分子响应的启动元件及其构建的大肠杆菌动态调控系统和方法
US17/972,463 US20230167435A1 (en) 2021-08-23 2022-10-24 Autoinducer-2 (ai-2) molecular response-based starting element and escherichia coli (e. coli) dynamic regulation system and method constructed thereby

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110968581.8A CN113667687B (zh) 2021-08-23 2021-08-23 基于ai-2分子响应的启动元件及其构建的大肠杆菌动态调控系统和方法

Publications (2)

Publication Number Publication Date
CN113667687A true CN113667687A (zh) 2021-11-19
CN113667687B CN113667687B (zh) 2023-08-01

Family

ID=78545124

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110968581.8A Active CN113667687B (zh) 2021-08-23 2021-08-23 基于ai-2分子响应的启动元件及其构建的大肠杆菌动态调控系统和方法

Country Status (2)

Country Link
US (1) US20230167435A1 (zh)
CN (1) CN113667687B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116732075B (zh) * 2023-06-09 2024-03-08 江南大学 一种生产2′-岩藻糖基乳糖的多层动态调控系统及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100021942A1 (en) * 2008-07-25 2010-01-28 Paul Freemont Cell-free expression system for the detection of bacterial biofilms
US20190144852A1 (en) * 2017-11-13 2019-05-16 The Board Of Trustees Of The University Of Illinois Combinatorial Metabolic Engineering Using a CRISPR System

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100021942A1 (en) * 2008-07-25 2010-01-28 Paul Freemont Cell-free expression system for the detection of bacterial biofilms
US20190144852A1 (en) * 2017-11-13 2019-05-16 The Board Of Trustees Of The University Of Illinois Combinatorial Metabolic Engineering Using a CRISPR System

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JINZHONG TIAN, ET AL.: "Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in Streptomyces." *
PRICILA HAUK, ET AL.: "Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression." *
XIANGYU JI, ET AL.: "CRISPRi/dCpf1-mediated dynamic metabolic switch to enhance butenoic acid production in Escherichia coli." *

Also Published As

Publication number Publication date
CN113667687B (zh) 2023-08-01
US20230167435A1 (en) 2023-06-01

Similar Documents

Publication Publication Date Title
Valdez-Cruz et al. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters
US20200010867A1 (en) Method of homologous recombination of dna
US7179644B2 (en) Recombinase-based methods for producing expression vectors and compositions for use in practicing the same
WO2018045630A1 (zh) 一种为克鲁维酵母优化的CRISPR/Cas9高效基因编辑系统
WO2017043656A1 (ja) 標的化したｄｎａ配列の核酸塩基を特異的に変換する、グラム陽性菌のゲノム配列の変換方法、及びそれに用いる分子複合体
Olmedo-Verd et al. Localized induction of the ntcA regulatory gene in developing heterocysts of Anabaena sp. strain PCC 7120
Sun et al. A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35
Roberts et al. Applications of CRISPR-Cas systems in lactic acid bacteria
Deaner et al. Modular ligation extension of guide RNA operons (LEGO) for multiplexed dCas9 regulation of metabolic pathways in Saccharomyces cerevisiae
KR20220054434A (ko) 신규한 crispr dna 표적화 효소 및 시스템
WO2019120193A1 (zh) 拆分型单碱基基因编辑系统及其应用
CN111718885B (zh) 一种枯草芽孢杆菌高效稳定的双质粒系统
US10435716B2 (en) Hybrid proteins and uses thereof
WO2020124830A1 (zh) 基于人工串联启动子的枯草芽孢杆菌高效诱导表达系统
CN101978057B (zh) 修饰宿主dna中的对象区域的方法和选择性标记盒
US20230167435A1 (en) Autoinducer-2 (ai-2) molecular response-based starting element and escherichia coli (e. coli) dynamic regulation system and method constructed thereby
US20110281362A1 (en) Electrotransformation of Gram-Positive, Anaerobic, Thermophilic Bacteria
Li et al. Targeted transcriptional activation in plants using a potent dead Cas9–derived synthetic gene activator
CN109929788B (zh) 一种具有ccdB负筛作用的菌株及其构建方法
CN106086025B (zh) 一种具有启动子功能的dna片段及其应用
WO2021088944A1 (zh) 大肠杆菌分子伴侣GroEL/ES在协助合成植物Rubisco中的应用
Ehsaan et al. Clostridium difficile genome editing using pyrE alleles
WO2022075419A1 (ja) Ｃｒｉｓｐｒタイプｉ－ｄシステムを利用した標的ヌクレオチド配列改変技術
Wen et al. A CRISPR/dCas9-assisted system to clone toxic genes in Escherichia coli
WO2020036181A1 (ja) 細胞を単離又は同定する方法及び細胞集団

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