CN114107155B - 一种中长链烷烃诱导型生物传感器及其应用 - Google Patents
一种中长链烷烃诱导型生物传感器及其应用 Download PDFInfo
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Abstract
一种中长链烷烃诱导型生物传感器及其应用,涉及基因工程生物传感器技术领域,生物传感器具体为含有pUC19‑ep3alks‑EGFP载体的Top10工程菌,其中ep3alks具有如SEQ ID NO:9所示的核苷酸序列。本发明所采用的烷烃诱导型操纵子是野生型烷烃诱导型启动子的突变子,基于该操纵子优化的烷烃诱导型细菌生物传感器,其检测碳谱提高,可以同时实现对辛烷至十七烷等烷烃的检测。该生物传感器对烷烃的检测易于操作,制样过程简单快捷。其检测限提高,具有良好的重复性。大大提高了检测效率,而且降低试剂成本。
Description
技术领域
本发明涉及基因工程生物传感器技术领域,具体是涉及一种中长链烷烃诱导型生物传感器及其应用。
背景技术
随着人类对石油及其产品需求量的日益增加,油田附近的湿地面临着石油污染加剧的严重威胁,其污染物主要是在原油开采、运输、石化燃料的生产使用及事故性泄露中所产生的石油污染物(Chih-Wen Liua,Mao-Shih Lianga,Yun-Chi Chena,etal.Biodegradation of n-alkanes at high concentration and correlation totheaccumulation of H+ions in Rhodococcuserythropolis NTU-1.BiochemicalEngineering Journal,2012,63:124-128)。被石油污染的湿地的水体质量下降、水生生物多样性降低、土壤总氮和总磷含量减少、湿地生态功能退化(Leichang Huang,Shuhong Ye,Yu Zhang,et al.Microbial Remediation and Optimization of Oil PollutedWetlands at Dalian Bay in China.Biomedical and environmental sciences,2007,20:414-419.)。石油污染物主要来自于油井生产过程和钻井过程中产生的含油废水、钻井岩屑、钻井泥浆和钻井机械运转过程中使用的各种润滑剂。大量研究表明,石油污染物的溢入对世界著名石油产区墨西哥湾地区和苏丹地区的湿地生态环境都产生了严重影响;中国的长江口、黄河口和大连湾滨海湿地、吉林省西部的莫莫格湿地、大庆地区的内陆湿地等也已经受到不同程度的石油污染(Xiangliang Pan,Daoyong Zhang,Liu Quan.Interactivefactorsleading to dying-off Carextato in Momoge wetland polluted bycrude oil,Western Jilin,China.Chemosphere,2006,65(10):1772-1777)。
烃类化合物是石油产品的主要成分,按照其骨架结构的不同分为饱和链烃(烷烃)、不饱和链烃(烯烃和炔烃)、脂环烃和芳香烃,其中烷烃占石油产品主要成分的50%以上。因此,在石油污染物中,烷烃是最常见的污染物。在自然环境中,短链烷烃易挥发,中链烷烃不易挥发、低溶解性的长链烷烃则属于很难被生物降解的污染物,对环境的危害较持久(Rosa Margesin,Christoph Moertelmaier,Johannes Mair.Low-temperaturebiodegradation of petroleum hydrocarbons(n-alkanes,phenol,anthracene,pyrene)by four actinobacterial strains.International Biodeterioration&Biodegradation,2012,6:1-7.)。因此,亟需寻找可靠的检测手段,来检测和控制中长链烷烃类的污染。
鉴于中长链烷烃污染的广泛性和对人类健康的严重危害,实现对烷烃的快速检测成为防止烷烃污染的关键问题。传统的烷烃的检测主要依赖物理化学手段,包括气相色谱-质谱联用仪(GC-MS)、高效液相色谱(HPLC-MS)和电感耦合等离子体质谱(ICP-MS)等。这些方法虽然精确灵敏,却需要复杂的预处理且操作复杂,更需要专业人员投入及高昂的设备资金。因此,发展出更加便捷、廉价且易操作的新型烷烃检测手段变得尤为迫切。
近年来,随着生物技术的快速发展及基因工程手段的日趋成熟,生物传感器技术的出现为烷烃的检测提供了一种新的手段。生物传感器可通过生物感应元件将待测物浓度与可测信号建立浓度梯度关系,在污染物的分析中具有极大的发展潜力和前景。烷烃的生物传感器利用细菌内烷烃的天然降解系统,即细菌内烷烃诱导型启动子及防御调控基因作为生物感应元件。在烷烃诱导型启动子的下游连接报告基因,通过建立报告基因信号与烷烃浓度梯度对应关系,实现对烷烃的检测。相较于传统的物理化学检测方法,细菌生物传感器不仅反应时间快,可在一小时甚至数分钟内实现对目标物质浓度的分析,而且容易操作。由于细菌生物传感器的核心即是细菌本身,其成本极其的廉价利于推广和应用。最重要的是,细菌生物传感器不仅检测环境中烷烃的含量,更可以准确反映环境中烷烃进入生物的量及其生物毒性,对烷烃的毒理作出指导性评估。
烷烃的细菌生物传感器的工作主要依赖于其烷烃诱导型启动子对烷烃的响应。在没有烷烃存在的条件下,烷烃诱导型启动子需要微量启动,启动合成下游alkS蛋白,该蛋白结合在下游烷烃结合位点(abs),阻止自身及下游基因的进一步表达;在烷烃存在的条件下,诱导高效启动烷烃诱导型启动子,烷烃与alkS蛋白结合并造成其构象变化,alkS蛋白从烷烃结合位点上解离,从而使下游基因得以表达。
研究发现利用细菌的天然烷烃降解系统构建的烷烃的生物传感器仅能对链长为十碳(正癸烷)及以下链长的烷烃产生响应,即野生型烷烃响应生物传感器检测限仅能检测癸烷及以下链长的烷烃,对中长链的烷烃不响应,因此研究和实现对中长链烷烃的检测尤为重要。定向进化技术,即在实验室中模拟自然进化的人工进化过程。定向进化技术无需了解DNA或蛋白的结构和作用机制,而是通过随机突变和定向选择获得理想的突变体,从而实现对目标物的优化(Stephen Lutz,Beyond directed evolution-semi-rationalproteinengineering and design,CurrOpinBiotechnol.2010December;21(6):734–743.)。将定向进化技术应用于烷烃诱导型启动子的进化,可直接有效的实现对烷烃细菌生物传感器的优化,筛选出更加灵敏、响应更长碳链烷烃的突变子。
鉴于中长链烷烃污染的严重性,以及烷烃细菌生物传感器对于烷烃污染检测的巨大前景和优越性。通过定向进化手段获得检测限更加广泛的烷烃诱导型启动子从而建立能够检测中长链烷烃的细菌生物传感器具有非常重要的意义。
发明内容
本发明的目的在于克服现有技术的不足,提供一种中长链烷烃诱导型生物传感器及其应用,以解决现有技术的基于烷烃诱导型启动子构建的细菌生物传感器检测限低的问题。
为了实现上述目的,本发明所采用的技术方案为:一种中长链烷烃诱导型生物传感器,具体为含有pUC19-ep3alks-EGFP载体的Top10工程菌,其中ep3alks具有如SEQ IDNO:9所示的核苷酸序列。
本发明还提出了该中长链烷烃诱导型生物传感器的制备方法,步骤如下:
1)、野生型烷烃诱导型操纵子基因的获得:
以质粒pCOM8-Alks为模板,以SEQ ID NO:1和SEQ ID NO:2所述的引物做PCR扩增,获得含有烷烃结合位点ABS,启动子Palks和调节蛋白alkS基因的野生型烷烃诱导型操纵子alks;
2)、野生型烷烃诱导型重组载体的构建
利用EcoRI和XhoI双酶切pUC19载体和步骤1)扩增获得的野生型烷烃诱导型操纵子,T4连接酶连接,使野生型烷烃诱导型操纵子替代pUC19载体中原本的lac启动子,获得pUC19-Alks载体;再在pUC19-Alks载体的调节蛋白alkS基因的下游引入绿色荧光蛋白EGFP基因,获得野生型烷烃诱导型重组载体pUC19-Alks-EGFP,所述EGFP基因是以SEQ ID NO:3和SEQ ID NO:4为引物,以质粒pPRars-GFP为模板克隆获得的;
利用EcoRI和XhoI双酶切pUC19载体同步构建pUC19-AID-EGFP空白对照质粒,用无功能序列AID(activation-induced cytidine deaminase)替换pUC19-Alks-EGFP质粒中的烷烃诱导型操纵子,所述AID序列是以SEQ ID NO:5和SEQ ID NO:6为引物,以pCI-mAID为模板克隆获得的;
3)、定向进化获得烷烃诱导型生物传感器
以pUC19-Alks-EGFPP质粒为模板,对烷烃诱导型操纵子的烷烃结合位点ABS,启动子Palks和调节蛋白alkS基因进行易错PCR,获得随机突变体文库,对随机突变体文库进行流式高通量筛选;
利用EcoRI和XhoI进行双酶切,用获得的随机突变体替换pUC19-AID-EGFP中的AID基因,构建重组突变体文库,其中连接体系中,插入片段和载体的摩尔比为4:1,或在每100ul连接体系中加入50ng载体以及200ng片段,连接反应条件为22℃连接30min;连接产物经电转化,导入Top10感受态细胞,获得流式筛选文库,进行流式高通量筛选;所述易错PCR的引物如SEQ ID NO:7和SEQ ID NO:8所示;
经过三轮流式高通量筛选,最终获得进化后的含有pUC19-ep3alks-EGFP载体的Top10工程菌,即为进化后的细菌生物传感器,其中,ep3alks具有如SEQ ID NO:9所示的核苷酸序列。
优选地,制备方法中所述易错PCR的反应体系为:
所述易错PCR的反应程序为:94℃预变性5min,94℃变性30s,62℃退火45s,72℃延伸2.5min,25个循环后,再在72℃下继续延伸10min后,置于4℃下保存备用。
本发明还提出了该诱导型生物传感器在检测中长链烷烃中的应用,步骤如下:
1)将细菌生物传感器pUC19-ep3alks-EGFP接种于含氨苄青霉素抗性的LB固体培养基平板上,37℃培养过夜;
同时,接种一份野生型烷烃诱导型传感器pUC19-AID-EGFP,作对照;
2)分别挑取野生型和进化后的传感器单菌落,接种于1mL含有氨苄青霉素抗性的LB液体培养基中,在37℃,200rpm下过夜培养,获得检测菌液;
3)将检测菌液用上述含有氨苄青霉素抗性的LB液体培养基稀释50倍,获得稀释菌液,继续培养至对数期;
4)准备链长为C8-C17一系列烷烃标准品;
5)对数期菌液加入终浓度为100μM的烷烃标准品,作为诱导组;同步取对数期菌液加入等量去离子水,作为空白对照;在37℃,200rpm下培养1h,获得诱导菌液;
6)将诱导后的菌液置于离心机中5000rpm离心3min,弃上清;
7)1×M9缓冲液重悬后再次离心,反复漂洗3次最后使用1×PBS重悬,流式细胞仪检测荧光表达。
与现有技术相比,本发明的有益效果表现在:
1、本发明所采用的烷烃诱导型操纵子是野生型烷烃诱导型启动子的突变子,基于该操纵子优化的烷烃诱导型细菌生物传感器,其检测碳谱提高,可以同时实现对辛烷至十七烷等烷烃的检测。
2、该生物传感器对烷烃的检测易于操作,制样过程简单快捷。其检测限提高,具有良好的重复性。大大提高了检测效率,而且降低试剂成本。
附图说明
图1分别为pUC19-Alks-EGFP(A)、pUC19-AID-EGFP(B)的质粒图谱。
图2分别为野生型细菌生物传感器(A)和进化后的细菌生物传感器(B)对中长链烷烃的响应情况。
图3为进化后的细菌生物传感器对中长链烷烃(以十五烷为例)的诱导浓度梯度响应情况。
图4为进化后的细菌生物传感器对中长链烷烃(以十五烷为例)的诱导时间梯度响应情况。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,以便本领域的技术人员更了解本发明,但本发明的保护范围不限于下述的实施例。
其中,LB培养基的制备方法为:
LB液体培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠;
LB固体培养基:每升LB液体培养基中加入琼脂15g;
氨苄青霉素抗性的LB固体培养基:将配置好的LB固体培养基加热完全溶解,待温度降至55℃左右加入总重量1‰的氨苄青霉素。
实施例1
中长链烷烃诱导型生物传感器的制备,步骤如下:
1)、野生型烷烃诱导型操纵子基因的获得:
以质粒pCOM8-Alks为模板,以SEQ ID NO:1和SEQ ID NO:2所述的引物做PCR扩增,获得含有烷烃结合位点ABS,启动子Palks和调节蛋白alkS基因的野生型烷烃诱导型操纵子alks。
2)、野生型烷烃诱导型重组载体的构建
利用EcoRI和XhoI双酶切pUC19载体和步骤1)扩增获得的野生型烷烃诱导型操纵子,T4连接酶连接,使野生型烷烃诱导型操纵子替代pUC19载体中原本的lac启动子,获得pUC19-Alks载体;再在pUC19-Alks载体的调节蛋白alkS基因的下游引入绿色荧光蛋白EGFP基因,获得野生型烷烃诱导型重组载体pUC19-Alks-EGFP,所述pUC19-Alks-EGFP的质粒图谱如图1A所示,所述EGFP基因是以SEQ ID NO:3和SEQ ID NO:4为引物,以质粒pPRars-GFP为模板克隆获得的。
利用EcoRI和XhoI双酶切pUC19载体同步构建pUC19-AID-EGFP空白对照质粒,用无功能序列AID(activation-induced cytidine deaminase)替换pUC19-Alks-EGFP质粒中的烷烃诱导型操纵子,所述pUC19-AID-EGFP的质粒图谱如图1B所示,所述AID序列是以SEQ IDNO:5和SEQ ID NO:6为引物,以pCI-mAID为模板克隆获得的,所述pCI-mAID的构建获得方法参见Wu等人于2005年发表的论文(Wu,X.,Geraldes,P.,Platt,J.L.,andCascalho,M.(2005).The double-edged sword of activation-induced cytidinedeaminase.J Im-munol 174,934–941.)。
3)、定向进化获得烷烃诱导型生物传感器
以pUC19-Alks-EGFPP质粒为模板,对烷烃诱导型操纵子的烷烃结合位点ABS,启动子Palks和调节蛋白alkS基因进行易错PCR,获得随机突变体文库,对随机突变体文库进行流式高通量筛选。
利用EcoRI和XhoI进行双酶切,用获得的随机突变体替换pUC19-AID-EGFP中的AID基因,构建重组突变体文库,其中连接体系中,插入片段和载体的摩尔比为4:1,或在每100ul连接体系中加入50ng载体以及200ng片段,连接反应条件为22℃连接30min;连接产物经电转化,导入Top10感受态细胞,获得流式筛选文库,进行流式高通量筛选;流式筛选文库构建时,库容量达到2x107个克隆,以保证能有足够突变基因型以供筛选;所述易错PCR的引物如SEQ ID NO:7和SEQ ID NO:8所示;所述易错PCR的反应体系如下表所示:
所述易错PCR的反应程序为:94℃预变性5min,94℃变性30s,62℃退火45s,72℃延伸2.5min,25个循环后,再在72℃下继续延伸10min后,置于4℃下保存备用。
经过三轮流式高通量筛选,最终获得进化后的含有pUC19-ep3alks-EGFP载体的Top10工程菌,即为进化后的细菌生物传感器,其中,ep3alks具有如SEQ ID NO:9所示的核苷酸序列,该ep3alks序列也可以通过人工合成的方式获得。
实施例2
进化后的细菌生物传感器对不同链长的烷烃诱导试验:
1)将进化后的细菌生物传感器pUC19-ep3alks-EGFP接种于含氨苄青霉素抗性的LB固体培养基平板上,37℃培养过夜;同时,接种一份野生型烷烃诱导型传感器pUC19-AID-EGFP,作对照。
2)分别挑取野生型和进化后的传感器单菌落,接种于1mL含有氨苄青霉素抗性的LB液体培养基中,在37℃,200rpm下过夜培养,获得检测菌液。
3)将检测菌液用上述含有氨苄青霉素抗性的LB液体培养基稀释50倍,获得稀释菌液,继续培养至对数期。
4)准备链长为C8-C17等一系列烷烃标准品,标准品购自西格玛奥德里奇(上海)贸易有限公司。
5)对数期菌液加入终浓度为100μM的烷烃标准品,作为诱导组;同步取对数期菌液加入等量去离子水,作为空白对照;在37℃,200rpm下培养1h,获得诱导菌液;
6)将诱导后的菌液置于离心机中5000rpm离心3min,弃上清。
7)1×M9缓冲液重悬后再次离心,反复漂洗3次最后使用1×PBS重悬,流式细胞仪检测荧光表达。
获得的响应情况如图2所示,由图2A可以看出,野生型烷烃响应生物传感器仅对癸烷及以下链长的烷烃有所响应,且对癸烷的响应较低。而进化后的烷烃响应生物传感器(图2B)不仅有更广泛的检测碳谱,能够对癸烷以上链长的烷烃产生响应,而且荧光响应有了更大的提高。
实施例3
进化后的细菌生物传感器对中长链烷烃(以十五烷为例)的梯度浓度诱导试验:
1)挑取pUC19-alks-EGFP单菌落,接种于1mL含有氨苄青霉素抗性的LB液体培养基中,在37℃,200rpm下过夜培养,获得检测菌液。
2)将检测菌液用上述含有氨苄青霉素抗性的LB液体培养基稀释50倍,获得稀释菌液,继续培养至对数期。
3)对数期菌液加入终浓度为0μM-50μM的十五烷标准品,作为诱导组;同步取对数期菌液加入等量去离子水,作为空白对照;在37℃,200rpm下培养1h,获得诱导菌液。
4)将诱导后的菌液置于离心机中5000rpm离心3min,弃上清。
5)1×M9缓冲液重悬后再次离心,反复漂洗3次最后使用1×PBS重悬,流式细胞仪检测荧光表达。
检测结果如图3所示,结果表明,进化后的细菌生物传感器的荧光响应随着烷烃浓度的升高而增强。
实施例4
进化后的细菌生物传感器对中长链烷烃(以十五烷为例)的梯度时间诱导试验:
1)挑取单菌落,接种于1mL含有氨苄青霉素抗性的LB液体培养基中,在37℃,200rpm下过夜培养,获得检测菌液。
2)将检测菌液用上述含有氨苄青霉素抗性的LB液体培养基稀释50倍,获得稀释菌液,继续培养至对数期;
3)对数期菌液加入终浓度为10μM的十五烷标准品,作为诱导组;同步取对数期菌液加入等量去离子水,作为空白对照;在37℃,200rpm分别下培养30、60、90、120,150,180分钟获得诱导菌液。
4)将诱导后的菌液置于离心机中5000rpm离心3min,弃上清。
5)1×M9缓冲液重悬后再次离心,反复漂洗3次最后使用1×PBS重悬,流式细胞仪检测荧光表达。
检测结果如图4所述,结果表明,进化后的细菌生物传感器的荧光响应随着烷烃诱导时间的增加而增强。
以上内容仅仅是对本发明的构思所作的举例和说明,所属本技术领域的技术人员对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代,只要不偏离发明的构思或者超越本权利要求书所定义的范围,均应属于本发明的保护范围。
序列表
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<400> 4
tcagctaatt gaatccttat ttgtatagtt catccat 37
<210> 5
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atcgccgaat tcatgtatcc atatgatg 28
<210> 6
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aagccgctcg agtcaaaatc ccaacatac 29
<210> 7
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gctagccatg cactcgagtt agataattcc ttgacgc 37
<210> 8
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
agtggacctt tctcctcttt aaatggaatt ctccaa 36
<210> 9
<211> 3089
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tccaattttt attaaattag tcgctacgag atttaagacg taattttatg cctaactgag 60
aaagttaagc cgcccactct cactctcgac atcttaaacc tgagctaatc ggacgcttgc 120
gccaactaca cctacgggta gtttttgctc cgtcgtctgc tggaaaaaca cgagctggcc 180
gcaagcatgc caggtaccgc gagctactcg cgacggctga aagcaccgaa atgagcgagc 240
tatctggtcg attttgaccc ggtgcccgtc ttcaaaatcg gcgaaggccg aagtcggcca 300
gaaatagcgg cctacttcag accttcccta gtaaatattt tgcaccaccg atcatgccga 360
ctacacttaa gtgtagtttt aatatttaac accgtaacct atggtgagaa tttccagtca 420
gctggcgcta gaattgcata atgaaaataa taataaataa tgatttcccg gtcgctaagg 480
tcggagcgga tcaaattacg actctagtaa gtgccaaagt tcatagttgc atatatcggc 540
caagattgag tatcgcggat ggagccgctc ccagagtatg cctttacaga gccccacctg 600
gatatgggaa aaccgttgct cttgcgttcg agtggctacg ccacagaaca gccggacgtc 660
ctgcagtgtg gctttcttta agagccagtt cttacagtga atttgatatc tgcgcagaga 720
ttattgagca gcttgaaact ttcgaaatgg taaaattcag ccgtgtgaga gagggtgtga 780
gcaagcctgc gctcttgcga gaccttgcat ctagtctttg gcagagcacc tcgaataacg 840
agatagaaac gctagtttgt ttggataata ttaatcatga cttagacttg ccgttgttgc 900
acgcacttat ggagtttatg ttaaatacac caaaaaatat caggtttgca gttgcaggca 960
atacaataaa agggttctcg cagcttaaac ttgcaggcgc tatgcgggag tacaccgaga 1020
aagacttggc ctttagcgca gaagaggcgg tggcgttagc ggaggcagag tctgttcttg 1080
gagttcctga agaacagata gagaccttgg tgcaagaagt tgaggggtgg cctgctcttg 1140
tagttttttt gttaaagcgt gagttgccgg ccaagcatat ttcagcagta gttgaagtag 1200
acaattactt tagggatgaa atatttgagg cgattcccga gcgctatcgt gtttttcttg 1260
caaattcttc attgctcgat ttcgtgacgc ctgatcaata caattatgta ttcaaatgcg 1320
tcaatggggt cacatgtatt aagtatttaa gcactaatta catgttgctt cgccatgtga 1380
gcggtgagcc agcgcagttt acactgcatc cagtactgcg taattttcta cgagaaatta 1440
cttggactga aaatcctgct aaaagatcct acctgcttaa gcgtgcagct ttctggcatt 1500
ggcgtagagg tgaataccag tatgcaatac gaatatccct acgggcgaat gactgtcgct 1560
gggcagtcag catgtctgag agaataattt tagatttgtc atttcgtcag ggcgaaatag 1620
atgcgctgag acagtggctg ttagagctgc cgaagcaggc ctggcacaaa aaacccatag 1680
tgcttattag ttacgcgtgg gtattgtatt tcagtcagca aggcgcgcga gcagagaagt 1740
taattaaaga cttatcttca caatccgata aaaaaaataa atggcaagaa aaggaatggc 1800
tgcagcttgt gcttgcaata ggtaaagcaa cgaaagatga aatgcttacg agtgaggagc 1860
tctgtaataa gtggattagt ttatttgggg attcaaacgc agttggaaaa ggggccgcgc 1920
taacctgttt ggctttaatt tttgccagtg agtatagatt tgcagagttg gagagggtgc 1980
tggctcaggc ccaagccgtg aataaatttg caaaacaaaa ttttgctttt ggttggctgt 2040
atgtcgcgag gtttcaacaa gccctagcaa gcggagaaat gggctgggcg aggcagatta 2100
taactcaagc gcgcacagac tgtcgcgcgc agatgatgga atccgagttt acttcgaaaa 2160
tgtttgacgc tctagagctt gagttacatt atgaattgcg ctgcttggac acctcagaag 2220
aaaagctctc caaaatttta gagttcattt ccaatcacgg ggtgacagac gtgttttttt 2280
ccgtatgccg tgctgtgtca gcttggcggc ttggaaggag tgacctaaat ggctccattg 2340
agatattgga gtgggcgaag gcgcatgcgg ttgaaaaaaa tctaccaaga ttggaagtta 2400
tgagccaaat tgagatctat cagcgcttag tctgtcaagg cataacgggc ataaataatt 2460
taaaaactct tgaagatcat aagattttct ccggacagca ctcagccccc ctaaaagcac 2520
gcctgctgct tgttcaatca ctagtgcttt cccgagatcg gaactttcat agtgccgcgc 2580
acagtgcgtt attggctatt cagcaagccc gtaaaattaa cgcgggccag ctggaagtcc 2640
gtggattatt gtgtttggcc ggagcgcagg caggtgccgg tgatttaaaa aaggctcagc 2700
ttaacattgt ttatgcagtg gagatagcaa aacagcttca atgctttcaa acagttcttg 2760
atgaagtatg tttaattgag cgaataatac cggcttcatg tgaagccttc acagcagtta 2820
atttagatca agcgattggg gcttttagtc ttccgcgaat agttgagatt ggaaagtccg 2880
cagagaataa agctgacgct ttattgacac ggaagcagat tgctgtcttg aggcatgtaa 2940
aagaggggtg ctcaaacaaa caaatagcaa gaaatatgta tgtcaccgaa gatgctataa 3000
agtggcacat gaggaaaata tttgccacct tgaatgtagt gaatcgcacg caagcaacaa 3060
ttgaagctga gcgtcaagga attatctaa 3089
Claims (2)
1.一种中长链烷烃诱导型生物传感器,具体为含有pUC19-ep3alks-EGFP载体的Top10工程菌,其中ep3alks具有如SEQ ID NO:9所示的核苷酸序列。
2.如权利要求1所述诱导型生物传感器在检测中长链烷烃中的应用,其特征在于,步骤如下:
1)将细菌生物传感器pUC19-ep3alks-EGFP接种于含氨苄青霉素抗性的LB固体培养基平板上,37℃培养过夜;同时,接种一份野生型烷烃诱导型传感器pUC19-AID-EGFP,作对照;
所述野生型烷烃诱导型传感器pUC19-AID-EGFP的制备步骤为:
利用EcoRI和XhoI双酶切pUC19载体和扩增获得的野生型烷烃诱导型操纵子,T4连接酶连接,使野生型烷烃诱导型操纵子替代pUC19载体中原本的lac启动子,获得pUC19-Alks载体;再在pUC19-Alks载体的调节蛋白alkS基因的下游引入绿色荧光蛋白EGFP基因,获得野生型烷烃诱导型重组载体pUC19-Alks-EGFP,所述EGFP基因是以SEQ ID NO:3和SEQ ID NO:4为引物,以质粒pPRars-GFP为模板克隆获得的;利用EcoRI和XhoI双酶切pUC19载体同步构建pUC19-AID-EGFP空白对照质粒,用无功能序列AID替换pUC19-Alks-EGFP质粒中的烷烃诱导型操纵子,所述AID序列是以SEQ ID NO:5和SEQ ID NO:6为引物,以pCI-mAID为模板克隆获得的;
2)分别挑取野生型和进化后的传感器单菌落,接种于1mL含有氨苄青霉素抗性的LB液体培养基中,在37℃,200rpm下过夜培养,获得检测菌液;
3)将检测菌液用上述含有氨苄青霉素抗性的LB液体培养基稀释50倍,获得稀释菌液,继续培养至对数期;
4)准备链长为C8-C17一系列烷烃标准品;
5)对数期菌液加入终浓度为100μM的烷烃标准品,作为诱导组;同步取对数期菌液加入等量去离子水,作为空白对照;在37℃,200rpm下培养1h,获得诱导菌液;
6)将诱导后的菌液置于离心机中5000rpm离心3min,弃上清;
7)1×M9缓冲液重悬后再次离心,反复漂洗3次最后使用1×PBS重悬,流式细胞仪检测荧光表达。
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