CN116121287A - 一种检测水体铜离子的荧光报告质粒载体及其应用 - Google Patents
一种检测水体铜离子的荧光报告质粒载体及其应用 Download PDFInfo
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Abstract
本发明公开了一种检测水体铜离子浓度水平的荧光报告质粒载体及其应用。本发明所提供的质粒pBCOPgfp,携带了恶臭假单胞菌的copAB1启动子和绿色荧光蛋白基因(gfp),copAB1启动子控制gfp的转录,质粒上还携带了恶臭假单胞菌的双组分系统copRS1。当无Cu2+时,copAB1启动子处于沉默状态,当Cu2+存在时,CopRS1可激活copAB1启动子,启动gfp转录,gfp的表达效率在一定范围内随着Cu2+浓度的提高而提高。以恶臭假单胞菌为宿主菌时,菌株培养物与水体样品混合孵育后,可通过检测GFP的荧光强度鉴定水体样品中的Cu2+浓度水平。
Description
技术领域
本发明属于生物技术领域,具体涉及一种检测水体铜离子(Cu2+)的荧光报告质粒载体及其应用。
背景技术
铜是自然资源丰富而且使用最为广泛的有色金属之一,具有优良的导电性、导热性、延展性和耐腐蚀性等特点,被应用在电力、电子、机械制造、交通运输、建筑和电镀等行业。铜的活动性较弱,但在潮湿或水体环境中容易被腐蚀,转化为铜离子溶于水中,进一步释放到环境中。铜离子是生物体的必需微量元素,对于维持正常的造血功能、维护中枢神经系统、促进骨骼发育以及抗氧化过程等有重要作用,但是过量摄入铜可引起肝脾肿胀等疾病。在畜牧养殖业上,铜元素常被作为添加剂添加到饲料中,可提高反刍动物对草料的利用效率以及促进动物的健康发育。由于动物对铜元素的吸收效率低,饲料中添加的铜元素大部分随着粪便排到环境中,在水体和土壤中积累。环境中过量的铜元素除了会干扰环境微生物群落结构的稳定性外,还会通过影响叶绿素的合成来危害植物的生长。目前检测铜离子的方法主要有原子吸收光谱法和电感耦合等离子体质谱法,这些方法能准确定量样品中的铜离子含量,但是对仪器设备要求较高,且操作繁琐,不利于对环境水体中铜离子浓度水平的日常监测。
铜离子对微生物具有一定的毒性,细菌也因此进化出了响应铜离子的抗性系统,例如Cop系统和Cus系统等。在典型环境微生物恶臭假单胞菌中,Cop系统是赋予菌株铜抗性的主要系统,其包含响应铜离子的双组分调控系统CopRS和外排泵CopAB。组氨酸激酶CopS在感应铜离子后会将ATP的磷酸基团转移到反应调节子CopR上使其磷酸化,磷酸化的CopR结合copAB的启动子并激活基因的表达,合成的CopA和CopB组成外排泵,将胞内过量的铜离子排出。恶臭假单胞菌中有两个Cop系统,均能响应铜离子,但是我们发现只有copAB1的表达受到严格调控,在无添加铜离子的情况下处于完全沉默状态,而当加入铜离子时表达水平显著提高,并且过表达CopRS1可显著提高copAB1的表达水平。因此,将copAB1基因簇的启动子与报告基因相连,同时将copRS1也一同连接到质粒载体中,可得到高灵敏度响应铜离子的报告载体,应用于检测水体中的铜离子浓度水平。
发明内容
本发明提出了一种用于指示水体中重金属Cu2+的荧光报告质粒载体及其应用,该报告载体含有绿色荧光蛋白报告基因(gfp),该报告基因的转录受copAB1启动子控制,载体上还含有双组分系统调控基因copRS1。当无Cu2+时,copAB1启动子处于沉默状态;当Cu2+存在时,CopRS1能够响应Cu2+激活copAB1启动子,启动gfp的转录,Cu2+浓度较高时,转录效率也随之提高。
第一方面,本发明提供一种检测水体中Cu2+的荧光报告质粒载体pBCOPgfp,其序列如SEQ ID NO.1所示。
所述荧光报告质粒载体的骨架为pBBR1MCS-5。
所述荧光报告质粒载体上携带了来源于质粒pCdrA-gfpC的gfp基因及SD序列和终止子片段。
优选地,从pCdrA-gfpC上PCR扩增gfp基因的引物如下:
gfpSc:AAAGAGGAGAAATTAACTAT;
gfpA-B5B:AATTCCTGCAGCCCGGGGGATCCCAACGGTGGTATATCCAGTG。
所述荧光报告质粒载体上的gfp基因的转录由来源于恶臭假单胞菌KT2440的copAB1基因启动子控制。
优选地,从恶臭假单胞菌KT2440(ATCC 47054)基因组中PCR扩增copAB1基因启动子的引物如下:
copA1S-B5X:CACCGCGGTGGCGGCCGCTCTAGAGGGCGACCATTTGGAACTG;
gfp-copA1A:ATAGTTAATTTCTCCTCTTTGGCGAGGCCTCATGCGGAAT。
优选地,所述的copAB1启动子片段与gfp片段通过无缝克隆连接到pBBR1MCS-5骨架的XbaI和BamHI酶切位点处。
所述荧光报告质粒载体上还携带了来源于恶臭假单胞菌KT2440的调控基因copRS1及其启动子。
优选地,从恶臭假单胞菌KT2440基因组中PCR扩增copRS1基因的引物如下:
copRS1s-B5X:TGGGTACCGGGCCCCCCCTCGAGCCAGGGCAAGGGTGGCGGT;
copRS1a-B5E:ATCCCCCGGGCTGCAGGAATTC CCTACCGGGCGTCGCAGGT。
优选地,所述荧光报告质粒载体上的copRS1基因通过无缝克隆连接到pBBR1MCS-5骨架的XhoI和EcoRI酶切位点处。
第二方面,本发明还提供了荧光报告质粒载体pBCOPgfp在制备检测Cu2+的全细胞生物传感器中的应用。
第三方面,本发明还提供了一种检测Cu2+的全细胞生物传感器,包括宿主细胞和位于宿主细胞内的荧光报告质粒载体pBCOPgfp。
优选地,所述的宿主细胞为恶臭假单胞菌。
第四方面,本发明还提供了所述的荧光报告质粒载体pBCOPgfp或全细胞生物传感器在检测水体中Cu2+浓度水平中的应用。
第五方面,本发明还提供了一种荧光报告质粒载体pBCOPgfp的构建方法,包括以下步骤:
a、以恶臭假单胞菌KT2440基因组为模板,使用引物copA1S-B5X和gfp-copA1A扩增得到copAB1启动子片段;以质粒pCdrA-gfpC为模板,使用引物gfpSc和gfpA-B5B扩增得到gfp片段;
b、使用限制性核酸内切酶XbaI和BamHI对质粒pBBR1MCS-5进行酶切,得到线性化pBBR1MCS-5;将copAB1启动子片段、gfp片段和线性化pBBR1MCS-5进行连接,得到质粒pBBR1-copA1gfp;
c、对质粒pBBR1-copA1gfp进行XhoI和EcoRI酶切,得到线性化pBBR1-copA1gfp;
d、以恶臭假单胞菌KT2440基因组为模板,使用引物copRS1s-B5X和copRS1a-B5E扩增得到copRS1基因片段;
e、将copRS1基因片段和线性化pBBR1-copA1gfp进行连接,得到质粒pBCOPgfp。
第六方面,本发明还提供了一种水体中Cu2+浓度水平的检测方法,包括以下步骤:将携带pBCOPgfp的恶臭假单胞菌培养至OD600≈1;然后将菌液与待测水体样品混合培养4h,使用酶标仪检测绿色荧光蛋白的荧光信号;以无菌水为阴性对照,以含不同浓度Cu2+的水溶液为阳性对照,通过比较荧光信号辨别水体样品中的Cu2+浓度水平。
具体步骤为:
(1)用含40μg/mL庆大霉素的LB培养基将携带pBCOPgfp的恶臭假单胞菌KT2440在28℃摇床以180rpm震荡培养至OD600≈1;
(2)在96孔黑色酶标板中加入50μL菌液和50μL待测水体样品,混合后置于28℃培养4h,在多功能酶标仪中检测绿色荧光蛋白的荧光信号;以纯水为阴性对照,以不同浓度的Cu2+溶液为阳性对照,通过比较荧光信号辨别水体样品中的Cu2+浓度水平。
本发明具有以下有益效果:
本发明提供的一种荧光报告质粒载体pBCOPgfp,可作为生物传感器用于检测水体样品中Cu2+浓度水平。采用本发明方法对水体中Cu2+浓度水平进行检测,Cu2+终浓度在1/128mM(7.8μM)至1mM范围内,荧光信号随着Cu2+浓度的提高而逐渐提高,且与空白对照相比均具有统计学显著性(p<0.05),可区分检测样品中的Cu2+浓度水平,因此本发明为初步检测水体的Cu2+浓度水平提供一种简便、灵敏且易操作的方法。
附图说明
图1是荧光报告质粒载体pBCOPgfp的图谱。
图2是携带pBCOPgfp的恶臭假单胞菌KT2440在不同浓度Cu2+条件下的GFP荧光信号,*p<0.5,**p<0.01,n=3。
具体实施方式
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
以下实施例中所用实验方法均为常规方法。所用大肠杆菌感受态细胞购自唯地生物有限公司,所用PCR试剂、限制性核酸内切酶和无缝克隆试剂盒均购自Takara公司,质粒提取和纯化DNA的胶回收试剂盒购自生工生物有限公司,试剂详细使用方法参照使用说明书。如无特殊说明,所用培养基均为LB培养基(10g/L胰蛋白胨、5g/L酵母浸粉和10g/LNaCl,溶剂为水),大肠杆菌的培养温度均为37℃,恶臭假单胞菌的培养温度均为28℃。检测荧光所用多功能酶标仪型号为BioTek Synergy H1。实施例中所使用的恶臭假单胞菌KT2440保藏编号是ATCC 47054,基因组序列信息是NCBI数据库NC_002947.4。
实施例1:荧光报告质粒载体pBCOPgfp的构建
本实施例中构建一种含有copAB1启动子、gfp报告基因和copRS1调控基因的报告载体,具体步骤如下:
(1)使用引物copA1S-B5X和gfp-copA1A,以恶臭假单胞菌KT2440基因组DNA为模板用高保真PCR酶扩增copAB1启动子区域,扩增产物序列如SEQ ID NO.2所示。使用引物gfpSc和gfpA-B5B,以质粒pCdrA-gfpC(核苷酸序列如SEQ ID NO.5所示)为模板扩增gfp片段,扩增产物序列如SEQ ID NO.3所示。PCR扩增反应条件为:95℃1min;95℃20s,55℃20s,72℃1min,32个循环;72℃5min。获得PCR产物后,进行琼脂糖凝胶电泳并使用胶回收试剂盒回收DNA片段。
引物序列如下:
copA1S-B5X:CACCGCGGTGGCGGCCGCTCTAGAGGGCGACCATTTGGAACTG;
gfp-copA1A:ATAGTTAATTTCTCCTCTTTGGCGAGGCCTCATGCGGAAT;
gfpSc:AAAGAGGAGAAATTAACTAT;
gfpA-B5B:AATTCCTGCAGCCCGGGGGATCCCAACGGTGGTATATCCAGTG。
质粒pCdrA-gfpC来源参考文献:Rybtke MT,Borlee BR,Murakami K,Irie Y,Hentzer M,Nielsen TE,Givskov M,Parsek MR,Tolker-Nielsen T.Fluorescence-basedreporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa.ApplEnviron Microbiol.2012Aug;78(15):5060-9。
质粒pCdrA-gfpC的序列如SEQ ID NO.5所示,具体为:
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATACGAATTGGCCGCGGCGTTGTGACAATTTACCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTAGGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGCCCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGCACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATTGATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGAGATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACGTAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCGCGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGCTGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATCAAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGCGAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCGCATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAATTCGCCTCTCAGGCGCCGCTGGTGCCGCTGGTTGGACGCCAAGGGTGAATCCGCCTCGATACCCTGATTACTCGCTTCCTGCGCCCTCTCAGGCGGCGATAGGGGACTGGTAAAACGGGGATTGCCCAGACGCCTCCCCCGCCCCTTCAGGGGCACAAATGCGGCCCCAACGGGGCCACGTAGTGGTGCGTTTTTTGCGTTTCCACCCTTTTCTTCCTTTTCCCTTTTAAACCTTTTAGGACGTCTACAGGCCACGTAATCCGTGGCCTGTAGAGTTTAAAAAGGGACGGATTTGTTGCCATTAAGGGACGGATTTGTTGTTAAGAAGGGACGGATTTGTTGTTGTAAAGGGACGGATTTGTTGTATTGTGGGACGCAGATACAGTGTCCCCTTATACACAAGGAATGTCGAACGTGGCCTCACCCCCAATGGTTTACAAAAGCAATGCCCTGGTCGAGGCCGCGTATCGCCTCAGTGTTCAGGAACAGCGGATCGTTCTGGCCTGTATTAGCCAGGTGAAGAGGAGCGAGCCTGTCACCGATGAAGTGATGTATTCAGTGACGGCGGAGGACATAGCGA
CGATGGCGGGTGTCCCTATCGAATCTTCCTACAACCAGCTCAAAGAAGCGGCCCTGCGC
CTGAAACGGCGGGAAGTCCGGTTAACCCAAGAGCCCAATGGCAAGGGGAAAAGACCG
AGTGTGATGATTACCGGCTGGGTGCAAACAATCATCTACCGGGAGGGTGAGGGCCGTGT
AGAACTCAGGTTCACCAAAGACATGCTGCCGTACCTGACGGAACTCACCAAACAGTTC
ACCAAATACGCCTTGGCTGACGTGGCCAAGATGGACAGCACCCACGCGATCAGGCTTTA
CGAGCTGCTCATGCAATGGGACAGCATCGGCCAGCGCGAAATAGAAATTGACCAGCTGC
GAAAGTGGTTTCAACTGGAAGGCCGGTATCCCTCGATCAAGGACTTCAAGTTGCGAGTG
CTTGATCCAGCCGTGACGCAGATCAACGAGCACAGCCCGCTACAGGTGGAGTGGGCGC
AGCGAAAGACCGGGCGCAAGGTCACACATCTGTTGTTCAGTTTTGGACCGAAGAAGCC
CGCCAAGGCGGTGGGTAAGGCCCCAGCGAAGCGCAAGGCCGGGAAGATTTCAGATGCT
GAGATCGCGAAACAGGCTCGCCCTGGTGAGACATGGGAAGCGGCCCGCGCTCGACTAA
CCCAGATGCCGCTGGATCTGGCCTAGAGGCCGTGGCCACCACGGCCCGGCCTGCCTTTC
AGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCT
GGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAG
TCACGACGTTGTAAAACGACGGCCAGTGAATTCGAGCTCGGTACCCGGGGATCCTCTAG
ACATTGTCGGTTTTTTGACGGTATTACATATTTAACCTCCGTAGTGTCATTTTTGAGTCAC
TCCGACGAACGGTCGCCCTTTCAAGTGGACAATTTTTGATCTGAAAGGCATTTTTTTGGC
ACACAAGGCGCAATGTGAATTGCTTCTCAGATTTTCGTCATTTAACTGACGAAATGCAGT
GCTGGAAAACTGACGTGCGAAAGAGGAGAAATTAACTATGAGCATGCGTAAAGGAGAA
GAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCAC
AAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAA
ATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGG
TTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAA
GAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGA
ACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAG
TTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAAC
TATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAA
CTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAAC
AAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACAC
AATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTT
GTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAGCTTAATTAGCT
GAGCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTC
AGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAAGCTAGCTTGGCGA
GATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTT
GATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTA
CCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATA
AGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGA
ATTTCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTA
CACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACG
ATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGG
CCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAG
TTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCAC
CATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCA
TCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTG
CGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGC
CTGGGGTAATGACTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGA
CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCC
GCCGCTAGGAGCTTGCGGCCGCAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAAT
TGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTG
GGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCA
GTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGC
GGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT
CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATC
AGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCG
TAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACA
AAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC
GTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATA
CCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTA
TCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC
AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC
GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG
CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTAT
TTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGAT
CCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG
CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA
GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCA
CCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAAC
TTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT
CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTA
CCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA
TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTAT
CCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTA
ATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTG
GTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGT
TGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC
GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCG
TAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGC
GGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGA
ACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTT
ACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC
TTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAA
AAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATT
GAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAA
TAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC。
copAB1启动子序列如SEQ ID NO.2所示,具体为:
CACCGCGGTGGCGGCCGCTCTAGAGGGCGACCATTTGGAACTGATGACCCCGCGCGGCAACTACCACTTCGACCTGCACCGCCTGTGCAACCGCCAGCAGCACGCCATCGAGGTCGCACCGGGCGATGGCCATGTGGTGTATCTGCCGATCCCGGAACAGGTTGCCCTCGACTACGGCCTGCTGATGCGCGACCTGCGCATCGACGAAGCAGCAAGCTGACAACACTGTAATGTGCACCTCAGCTACCTGACAGCAGGCGGCAGCGAACATCGTGGCTATTCCGCATGAGGCCTCGCCAAAGAGGAGAAATTAACTAT。
gfp片段序列如SEQ ID NO.3所示,具体为:
AAAGAGGAGAAATTAACTATGAGCATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAGCTTAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATTGGTGAGAATCCAAGCTAGCTTGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTG。
(2)使用限制性核酸内切酶XbaI和BamHI对商业化质粒pBBR1MCS-5(GenBank:U25061.1)进行酶切,并进行琼脂糖凝胶电泳和切胶回收,得到线性化质粒。
(3)使用无缝克隆试剂盒将上述经过纯化的copAB1启动子片段、gfp片段和线性化pBBR1MCS-5进行连接。通过热激转化的方法将连接产物导入大肠杆菌S17-1λpir,将10μL连接产物与大肠杆菌S17-1λpir感受态细胞混合,冰上孵育30min,放入42℃水浴锅中热激90s,加入LB培养基复苏培养1h后,收集菌体涂布在含20μg/mL庆大霉素的LB平板上,过夜培养后挑取单菌落,使用引物copA1S-B5X和gfpA-B5B进行PCR验证,得到质粒pBBR1-copA1gfp。
(4)扩大培养携带pBBR1-copA1gfp的大肠杆菌S17-1λpir,使用质粒提取试剂盒提取质粒,质粒经XhoI和EcoRI酶切后,进行切胶回收,得到线性化的pBBR1-copA1gfp。
(5)使用引物copRS1s-B5X和copRS1a-B5E以恶臭假单胞菌KT2440基因组DNA为模板,使用高保真酶进行PCR扩增,扩增产物序列如SEQ ID NO.4所示,得到copRS1基因片段后,进行切胶回收纯化DNA片段。
引物序列如下:
copRS1s-B5X:TGGGTACCGGGCCCCCCCTCGAGCCAGGGCAAGGGTGGCGGT;
copRS1a-B5E:ATCCCCCGGGCTGCAGGAATTC CCTACCGGGCGTCGCAGGT。
copRS1基因的序列如SEQ ID NO.4所示,具体为:
TGGGTACCGGGCCCCCCCTCGAGCCAGGGCAAGGGTGGCGGTAATGAACAGGTGTTTCATGATGACTCCGGGAATACACAGCAGGATGAGGCCACATTAGCCGGGGGCGACTGGCAGCTACCTGACAGGCACATTACAACTTTGTCAGCTTGGGCGAGCGGGCACATCGTCACGTATCATTTCAGCCATCCCATTGCCCCTGTGCTAGCGGTAAGGCGGCGCCATAGGCTACGCACAATCTTTCAGAATATGGACAGCGCATGAAACTGCTGATCGTCGAAGACCAGGCCCGCACCGGCCAGTACCTGAGCCAAGGCCTGAGCGAGGCCGGGTTTGCCACCGAATTGGCCACCGATGGCGAGACCGGCCAGTTCCTCGCCCTGACCGGCGACCACGACCTGCTGATCCTCGACGTGATGCTGCCCGGCCGCGACGGCTGGCAGATCCTTCAGGCCGTGCGCCAGGCTGGCCTGGATACGCCAGTGCTGTTCCTGACCGCCCGCGACGCCGTCGAGGATCGCGTGCATGGCCTGGAACTGGGCGCCGACGACTACCTGGTCAAGCCGTTCGCCTTTTCCGAGCTGCTGGCCCGGGTACGCAGCCTGCTGCGCCGTGGCACCAGCCCGAGCCAGGACACGATCCTGAGCCTGGCTGACCTGCGCCTGGACCTGATCCGCCGCCGCGCCGAGCGTGCCGGGCTGCGCATCGACCTGACCGCCAAGGAGTTCTCACTCCTTGAGCTGCTGCTGCGCCGCCAGGGTGAGGTGCTGCCAAAATCGCTGATCGCCTCGCAGGTGTGGGACATGAACTTCGACAGCGACACCAATGTGATCGAAGTGGCCATCCGCCGCCTGCGCCTGAAAATCGACGACCCGCACCCCAACAAGCTGATCCACACCGTCCGCGGCATGGGTTACGTACTCGAAGAGCGCACCGAGTGATGCGCCGGGTTTCCCTCGGCAGCCGCCTGGCCCTGCTGTTCGCCGCCTGCACCGCCACCGTCTCGCTGGGCGCCGGTCTGCTGTTCAGCCGGGCCAGCGAGCAGCACTTCGTCGAACTCGACCAACAACTGCTGGACTCGCGCCTGTCATTGTTTCGGACGCAACTGGCTGGCGTCAGCACGGCTGATGAGCTCCAGGCCCGCCTGCCGGCCCTGCGCGATGAACTGAGTCACCAGGCCGACCTGGCCCTGCGCATCAGCGCCAGCAATGGCGCCACCTGGTTCGAAAGCCGCAGCGGGCTGCCTCATGCAGCGCAGGCCACCGGGCTGGCCACCCTGCATGCGCCAGGCATCGACTACCGCAGCCTGTCCGTGCCCCTGACACAGGGCGCCATGCAGTCGCCGCGACTGACCCTGTACCTCGACATCACCCACCACCAGCACTTCCTGCAGGGCATGCAACGACTGATCTGGCTGACGGTCGGCCTGTCGGCGCTAATCACCGCACTATTGGGTGCCTGGGCCGCCCGCAGCGGCCTGCGCCCATTGCGGCAGATGGGCCAGGTGGCCGCCAGCGTGTCAGCGCGCTCCCTCACCACCCGTCTGCCGGTGGCGCAGATGCCCGAAGAGTTGGCCGAGCTGGCCAGCAGCATGAACGCGATGCTGCAGCGCCTGGACGATGCCTTCCAGCGCCTGTCAGCGTTTTCGGCCGATATCGCCCACGAGTTACGTACGCCGCTGTCCAACCTGCTGACGCACACCCAGGTCACCCTCACCCGCCCACGCAGCCTCGAAGAATACCGAGAGGCACTGCATGGCAACCTGGAAGAACTGCAATGGATGGCGCAGATGATCAACGACATGCTGTTTCTGGCCAAGGCCGACCACGGCCTGCTGGTACCGGGGGACGCGCCTTTGGCGCTGCATGACGAGGTGGATGCGCTGCTGGAGTACTACGCGCCTCTGGCAGAAGACAGCGACGTGCAGATGCTGCGCGAGGGTGAAGCGGTGCTGCACGGCGACCAGCATATGCTGCGCCGGGCGCTGTCCAACCTGCTGGACAACGCCATGCGTTTTACCCCGGCAGGAGGACAGATCAAGGTAACCCTGGGGCCGGGGCCGACGATCAACGTGGCCAATACCGGGCTGGCTATCGACCCGGCCGCACTACCGCGGCTTTTCGACCGCTTCTACCGGGTGGACCCGGCTCGACGGGAAGGCAGCAGCGAGCATGCGGGGCTGGGCCTGGCGATTACCCGGTCGATCGTGCAGGCCCATGGCGGATGCATACGGGCGGAGTGCGAGGGTGGGTGGACCCGATTCGTGATCGAGTTCACTCAAGACCGGTAATCCTGGGGCGCTTTACGCCCCCAGGATCTCAGGCAGACATCAAACCGCCAGGCCAATCGCCGGCTGCACCTGCGACGCCCGGTAGGGAATTCCTGCAGCCCGGGGGAT。
(6)使用无缝克隆试剂盒将上述经过纯化的copRS1片段和线性化pBBR1-copA1gfp进行连接。将连接产物通过热激转化导入大肠杆菌S17-1λpir感受态细胞中,涂布在含20μg/mL庆大霉素的LB平板上,过夜培养后挑取单菌落,使用引物copRS1s-B5X和copRS1a-B5E进行PCR验证,得到质粒pBCOPgfp(图1),序列如SEQ ID NO.1所示。
质粒pBCOPgfp的序列如SEQ ID NO.1所示,具体为:
CTCGGGCCGTCTCTTGGGCTTGATCGGCCTTCTTGCGCATCTCACGCGCTCCTGCGGCGGCCTGTAGGGCAGGCTCATACCCCTGCCGAACCGCTTTTGTCAGCCGGTCGGCCACGGCTTCCGGCGTCTCAACGCGCTTTGAGATTCCCAGCTTTTCGGCCAATCCCTGCGGTGCATAGGCGCGTGGCTCGACCGCTTGCGGGCTGATGGTGACGTGGCCCACTGGTGGCCGCTCCAGGGCCTCGTAGAACGCCTGAATGCGCGTGTGACGTGCCTTGCTGCCCTCGATGCCCCGTTGCAGCCCTAGATCGGCCACAGCGGCCGCAAACGTGGTCTGGTCGCGGGTCATCTGCGCTTTGTTGCCGATGAACTCCTTGGCCGACAGCCTGCCGTCCTGCGTCAGCGGCACCACGAACGCGGTCATGTGCGGGCTGGTTTCGTCACGGTGGATGCTGGCCGTCACGATGCGATCCGCCCCGTACTTGTCCGCCAGCCACTTGTGCGCCTTCTCGAAGAACGCCGCCTGCTGTTCTTGGCTGGCCGACTTCCACCATTCCGGGCTGGCCGTCATGACGTACTCGACCGCCAACACAGCGTCCTTGCGCCGCTTCTCTGGCAGCAACTCGCGCAGTCGGCCCATCGCTTCATCGGTGCTGCTGGCCGCCCAGTGCTCGTTCTCTGGCGTCCTGCTGGCGTCAGCGTTGGGCGTCTCGCGCTCGCGGTAGGCGTGCTTGAGACTGGCCGCCACGTTGCCCATTTTCGCCAGCTTCTTGCATCGCATGATCGCGTATGCCGCCATGCCTGCCCCTCCCTTTTGGTGTCCAACCGGCTCGACGGGGGCAGCGCAAGGCGGTGCCTCCGGCGGGCCACTCAATGCTTGAGTATACTCACTAGACTTTGCTTCGCAAAGTCGTGACCGCCTACGGCGGCTGCGGCGCCCTACGGGCTTGCTCTCCGGGCTTCGCCCTGCGCGGTCGCTGCGCTCCCTTGCCAGCCCGTGGATATGTGGACGATGGCCGCGAGCGGCCACCGGCTGGCTCGCTTCGCTCGGCCCGTGGACAACCCTGCTGGACAAGCTGATGGACAGGCTGCGCCTGCCCACGAGCTTGACCACAGGGATTGCCCACCGGCTACCCAGCCTTCGACCACATACCCACCGGCTCCAACTGCGCGGCCTGCGGCCTTGCCCCATCAATTTTTTTAATTTTCTCTGGGGAAAAGCCTCCGGCCTGCGGCCTGCGCGCTTCGCTTGCCGGTTGGACACCAAGTGGAAGGCGGGTCAAGGCTCGCGCAGCGACCGCGCAGCGGCTTGGCCTTGACGCGCCTGGAACGACCCAAGCCTATGCGAGTGGGGGCAGTCGAAGGCGAAGCCCGCCCGCCTGCCCCCCGAGCCTCACGGCGGCGAGTGCGGGG
GTTCCAAGGGGGCAGCGCCACCTTGGGCAAGGCCGAAGGCCGCGCAGTCGATCAACAA
GCCCCGGAGGGGCCACTTTTTGCCGGAGGGGGAGCCGCGCCGAAGGCGTGGGGGAAC
CCCGCAGGGGTGCCCTTCTTTGGGCACCAAAGAACTAGATATAGGGCGAAATGCGAAAG
ACTTAAAAATCAACAACTTAAAAAAGGGGGGTACGCAACAGCTCATTGCGGCACCCCC
CGCAATAGCTCATTGCGTAGGTTAAAGAAAATCTGTAATTGACTGCCACTTTTACGCAAC
GCATAATTGTTGTCGCGCTGCCGAAAAGTTGCAGCTGATTGCGCATGGTGCCGCAACCG
TGCGGCACCCTACCGCATGGAGATAAGCATGGCCACGCAGTCCAGAGAAATCGGCATTC
AAGCCAAGAACAAGCCCGGTCACTGGGTGCAAACGGAACGCAAAGCGCATGAGGCGT
GGGCCGGGCTTATTGCGAGGAAACCCACGGCGGCAATGCTGCTGCATCACCTCGTGGCG
CAGATGGGCCACCAGAACGCCGTGGTGGTCAGCCAGAAGACACTTTCCAAGCTCATCG
GACGTTCTTTGCGGACGGTCCAATACGCAGTCAAGGACTTGGTGGCCGAGCGCTGGATC
TCCGTCGTGAAGCTCAACGGCCCCGGCACCGTGTCGGCCTACGTGGTCAATGACCGCGT
GGCGTGGGGCCAGCCCCGCGACCAGTTGCGCCTGTCGGTGTTCAGTGCCGCCGTGGTG
GTTGATCACGACGACCAGGACGAATCGCTGTTGGGGCATGGCGACCTGCGCCGCATCCC
GACCCTGTATCCGGGCGAGCAGCAACTACCGACCGGCCCCGGCGAGGAGCCGCCCAGC
CAGCCCGGCATTCCGGGCATGGAACCAGACCTGCCAGCCTTGACCGAAACGGAGGAAT
GGGAACGGCGCGGGCAGCAGCGCCTGCCGATGCCCGATGAGCCGTGTTTTCTGGACGA
TGGCGAGCCGTTGGAGCCGCCGACACGGGTCACGCTGCCGCGCCGGTAGCACTTGGGT
TGCGCAGCAACCCGTAAGTGCGCTGTTCCAGACTATCGGCTGTAGCCGCCTCGCCGCCC
TATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCT
GAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCGTTTTTATCAGGCTCTGGGAGG
CAGAATAAATGATCATATCGTCAATTATTACCTCCACGGGGAGAGCCTGAGCAAACTGGC
CTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAAC
CAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAA
TTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCACCAGG
CGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGC
AGTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACA
AAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCG
ATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC
GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT
GAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGGTGGCGGCCG
CTCTAGAGGGCGACCATTTGGAACTGATGACCCCGCGCGGCAACTACCACTTCGACCTG
CACCGCCTGTGCAACCGCCAGCAGCACGCCATCGAGGTCGCACCGGGCGATGGCCATG
TGGTGTATCTGCCGATCCCGGAACAGGTTGCCCTCGACTACGGCCTGCTGATGCGCGAC
CTGCGCATCGACGAAGCAGCAAGCTGACAACACTGTAATGTGCACCTCAGCTACCTGAC
AGCAGGCGGCAGCGAACATCGTGGCTATTCCGCATGAGGCCTCGCCAAAGAGGAGAAA
TTAACTATGAGCATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTT
GAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGA
TGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCC
ATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGA
TCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAA
GAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAA
GGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAAC
ATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGAC
AAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAA
GCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTT
TACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAG
AGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGAT
GAACTATACAAATAAGCTTAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATG
ACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATT
GGTGAGAATCCAAGCTAGCTTGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAG
AAAAAAATCACTGGATATACCACCGTTGGGATCCCCCGGGCTGCAGGAATTCCCTACCG
GGCGTCGCAGGTGCAGCCGGCGATTGGCCTGGCGGTTTGATGTCTGCCTGAGATCCTGG
GGGCGTAAAGCGCCCCAGGATTACCGGTCTTGAGTGAACTCGATCACGAATCGGGTCCA
CCCACCCTCGCACTCCGCCCGTATGCATCCGCCATGGGCCTGCACGATCGACCGGGTAAT
CGCCAGGCCCAGCCCCGCATGCTCGCTGCTGCCTTCCCGTCGAGCCGGGTCCACCCGGT
AGAAGCGGTCGAAAAGCCGCGGTAGTGCGGCCGGGTCGATAGCCAGCCCGGTATTGGC
CACGTTGATCGTCGGCCCCGGCCCCAGGGTTACCTTGATCTGTCCTCCTGCCGGGGTAA
AACGCATGGCGTTGTCCAGCAGGTTGGACAGCGCCCGGCGCAGCATATGCTGGTCGCCG
TGCAGCACCGCTTCACCCTCGCGCAGCATCTGCACGTCGCTGTCTTCTGCCAGAGGCGC
GTAGTACTCCAGCAGCGCATCCACCTCGTCATGCAGCGCCAAAGGCGCGTCCCCCGGTA
CCAGCAGGCCGTGGTCGGCCTTGGCCAGAAACAGCATGTCGTTGATCATCTGCGCCATC
CATTGCAGTTCTTCCAGGTTGCCATGCAGTGCCTCTCGGTATTCTTCGAGGCTGCGTGGG
CGGGTGAGGGTGACCTGGGTGTGCGTCAGCAGGTTGGACAGCGGCGTACGTAACTCGT
GGGCGATATCGGCCGAAAACGCTGACAGGCGCTGGAAGGCATCGTCCAGGCGCTGCAG
CATCGCGTTCATGCTGCTGGCCAGCTCGGCCAACTCTTCGGGCATCTGCGCCACCGGCA
GACGGGTGGTGAGGGAGCGCGCTGACACGCTGGCGGCCACCTGGCCCATCTGCCGCAA
TGGGCGCAGGCCGCTGCGGGCGGCCCAGGCACCCAATAGTGCGGTGATTAGCGCCGAC
AGGCCGACCGTCAGCCAGATCAGTCGTTGCATGCCCTGCAGGAAGTGCTGGTGGTGGG
TGATGTCGAGGTACAGGGTCAGTCGCGGCGACTGCATGGCGCCCTGTGTCAGGGGCAC
GGACAGGCTGCGGTAGTCGATGCCTGGCGCATGCAGGGTGGCCAGCCCGGTGGCCTGC
GCTGCATGAGGCAGCCCGCTGCGGCTTTCGAACCAGGTGGCGCCATTGCTGGCGCTGAT
GCGCAGGGCCAGGTCGGCCTGGTGACTCAGTTCATCGCGCAGGGCCGGCAGGCGGGCC
TGGAGCTCATCAGCCGTGCTGACGCCAGCCAGTTGCGTCCGAAACAATGACAGGCGCG
AGTCCAGCAGTTGTTGGTCGAGTTCGACGAAGTGCTGCTCGCTGGCCCGGCTGAACAG
CAGACCGGCGCCCAGCGAGACGGTGGCGGTGCAGGCGGCGAACAGCAGGGCCAGGCG
GCTGCCGAGGGAAACCCGGCGCATCACTCGGTGCGCTCTTCGAGTACGTAACCCATGCC
GCGGACGGTGTGGATCAGCTTGTTGGGGTGCGGGTCGTCGATTTTCAGGCGCAGGCGG
CGGATGGCCACTTCGATCACATTGGTGTCGCTGTCGAAGTTCATGTCCCACACCTGCGA
GGCGATCAGCGATTTTGGCAGCACCTCACCCTGGCGGCGCAGCAGCAGCTCAAGGAGT
GAGAACTCCTTGGCGGTCAGGTCGATGCGCAGCCCGGCACGCTCGGCGCGGCGGCGGA
TCAGGTCCAGGCGCAGGTCAGCCAGGCTCAGGATCGTGTCCTGGCTCGGGCTGGTGCC
ACGGCGCAGCAGGCTGCGTACCCGGGCCAGCAGCTCGGAAAAGGCGAACGGCTTGAC
CAGGTAGTCGTCGGCGCCCAGTTCCAGGCCATGCACGCGATCCTCGACGGCGTCGCGGG
CGGTCAGGAACAGCACTGGCGTATCCAGGCCAGCCTGGCGCACGGCCTGAAGGATCTG
CCAGCCGTCGCGGCCGGGCAGCATCACGTCGAGGATCAGCAGGTCGTGGTCGCCGGTC
AGGGCGAGGAACTGGCCGGTCTCGCCATCGGTGGCCAATTCGGTGGCAAACCCGGCCT
CGCTCAGGCCTTGGCTCAGGTACTGGCCGGTGCGGGCCTGGTCTTCGACGATCAGCAGT
TTCATGCGCTGTCCATATTCTGAAAGATTGTGCGTAGCCTATGGCGCCGCCTTACCGCTA
GCACAGGGGCAATGGGATGGCTGAAATGATACGTGACGATGTGCCCGCTCGCCCAAGCT
GACAAAGTTGTAATGTGCCTGTCAGGTAGCTGCCAGTCGCCCCCGGCTAATGTGGCCTC
ATCCTGCTGTGTATTCCCGGAGTCATCATGAAACACCTGTTCATTACCGCCACCCTTGCC
CTGGCTCGAGGGGGGGCCCGGTACCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCG
CGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTC
CACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAG
CTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTG
CCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCA
TGCATAAAAACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGG
CATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGC
CCATGGACGCACACCGTGGAAACGGATGAAGGCACGAACCCAGTTGACATAAGCCTGT
TCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTT
GACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTT
TTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGAT
GTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGG
CAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGC
TCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCG
GAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTC
CGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCT
CGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCT
CGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCA
AGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGAC
GATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGAT
ATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGC
GGAGTTGTTCGGTAAATTGTCACAACGCCGCCAGGTGGCACTTTTCGGGGAAATGTGCG
CGCCCGCGTTCCTGCTGGCGCTGGGCCTGTTTCTGGCGCTGGACTTCCCGCTGTTCCGT
CAGCAGCTTTTCGCCCACGGCCTTGATGATCGCGGCGGCCTTGGCCTGCATATCCCGATTCAACGGCCCCAGGGCGTCCAGAACGGGCTTCAGGCGCTCCCGAAGGT。
(7)通过接合转移将pBCOPgfp从大肠杆菌S17-1λpir导入恶臭假单胞菌KT2440。离心收集含pBCOPgfp的大肠杆菌S17-1λpir(供体菌)和恶臭假单胞菌KT2440(受体菌)菌体,用无抗LB培养基洗去抗生素并悬浮,将供体菌和受体菌以体积比3:1混合,置于28℃孵育5h,涂布含25μg/mL氯霉素和40μg/mL庆大霉素的LB平板上,过夜培养,挑取单菌落,使用引物copA1S-B5X和copRS1s-B5X进行PCR验证,得到含pBCOPgfp的恶臭假单胞菌KT2440。
实施例2:使用含pBCOPgfp的恶臭假单胞菌KT2440检测Cu2+浓度水平
本实施例中在LB培养基中检测Cu2+浓度水平,具体步骤如下:
(1)在含20μg/mL庆大霉素的LB中活化培养携带pBCOPgfp的恶臭假单胞菌KT2440,以180rpm震荡培养,培养至OD600≈1,或以新鲜LB培养基调整到OD600≈1。
(2)准备梯度浓度的Cu2+样品。使用无菌水对1mM的CuCl2溶液进行2倍的梯度稀释,浓度范围为从8mM至1/128mM(约7.8μM),取50μL各浓度的CuCl2溶液到96孔黑色酶标板中,以50μL无菌水作为对照。
(3)取50μL步骤(1)中的菌液加到每个检测孔中,Cu2+的终浓度为4mM至1/256mM,放置于28℃静置培养4h,取酶标板在多功能酶标仪中测量荧光值(激发光波长为485nm,发射波长为528nm),以荧光值指示不同溶液中Cu2+浓度的相对高低。
实验结果:如图2所示,本实施例中检测的Cu2+终浓度在1/128mM(7.8μM)至1mM范围内,荧光信号随着Cu2+浓度的提高而逐渐提高,且与空白对照相比均具有统计学显著性(p<0.05),可区分检测样品中的Cu2+浓度水平。在此浓度范围之上,Cu2+对细菌有毒性,影响蛋白的正常表达,因此GFP荧光信号未能进一步增强。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种检测水体中Cu2+的荧光报告质粒载体pBCOPgfp,其特征在于,其序列如SEQ IDNO.1所示。
2.根据权利要求1所述的荧光报告质粒载体,其特征在于,所述荧光报告质粒载体携带gfp报告基因,gfp基因的前端与恶臭假单胞菌KT2440的copAB1基因启动子相连;还携带来源于恶臭假单胞菌KT2440的调控基因copRS1。
3.根据权利要求2所述的荧光报告质粒载体,其特征在于,所述荧光报告质粒载体的骨架为pBBR1MCS-5。
4.权利要求1-3任一项所述的荧光报告质粒载体在制备检测Cu2+的全细胞生物传感器中的应用。
5.一种检测Cu2+的全细胞生物传感器,其特征在于,包括宿主细胞和位于宿主细胞内的如权利要求1-3任一项所述的荧光报告质粒载体。
6.根据权利要求5所述的全细胞生物传感器,其特征在于,所述的宿主细胞为恶臭假单胞菌。
7.权利要求1所述的荧光报告质粒载体或权利要求5所述的全细胞生物传感器在检测水体中Cu2+浓度水平中的应用。
8.一种权利要求1所述的荧光报告质粒载体的构建方法,其特征在于,包括以下步骤:
a、以恶臭假单胞菌KT2440基因组为模板,使用引物copA1S-B5X和gfp-copA1A扩增得到copAB1启动子片段;以质粒pCdrA-gfpC为模板,使用引物gfpSc和gfpA-B5B扩增得到gfp片段;
b、使用限制性核酸内切酶XbaI和BamHI对质粒pBBR1MCS-5进行酶切,得到线性化pBBR1MCS-5;将copAB1启动子片段、gfp片段和线性化pBBR1MCS-5进行连接,得到质粒pBBR1-copA1gfp;
c、对质粒pBBR1-copA1gfp进行XhoI和EcoRI酶切,得到线性化pBBR1-copA1gfp;
d、以恶臭假单胞菌KT2440基因组为模板,使用引物copRS1s-B5X和copRS1a-B5E扩增得到copRS1基因片段;
e、将copRS1基因片段和线性化pBBR1-copA1gfp进行连接,得到质粒pBCOPgfp。
9.根据权利要求8所述的构建方法,其特征在于,
所述引物copA1S-B5X和gfp-copA1A的序列为:
copA1S-B5X:CACCGCGGTGGCGGCCGCTCTAGAGGGCGACCATTTGGAACTG;
gfp-copA1A:ATAGTTAATTTCTCCTCTTTGGCGAGGCCTCATGCGGAAT;
所述引物gfpSc和gfpA-B5B的序列为:
gfpSc:AAAGAGGAGAAATTAACTAT;
gfpA-B5B:AATTCCTGCAGCCCGGGGGATCCCAACGGTGGTATATCCAGTG;
所述引物copRS1s-B5X和copRS1a-B5E的序列为:
copRS1s-B5X:TGGGTACCGGGCCCCCCCTCGAGCCAGGGCAAGGGTGGCGGT;
copRS1a-B5E:ATCCCCCGGGCTGCAGGAATTC CCTACCGGGCGTCGCAGGT。
10.一种水体中Cu2+浓度水平的检测方法,其特征在于,包括以下步骤:将携带权利要求1所述质粒pBCOPgfp的恶臭假单胞菌培养至OD600≈1;然后将菌液与待测水体样品混合培养4h,使用酶标仪检测绿色荧光蛋白的荧光信号;以无菌水为阴性对照,以含不同浓度Cu2+的水溶液为阳性对照,通过比较荧光信号辨别水体样品中的Cu2+浓度水平。
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