CN112375771B - 一种高丝氨酸生物传感器及其构建方法和应用 - Google Patents
一种高丝氨酸生物传感器及其构建方法和应用 Download PDFInfo
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Abstract
本发明提供了一种高丝氨酸生物传感器，其是基于谷氨酸棒状杆菌转录调节因子NCgl0581构建的，含有NCgl0581开放阅读框和NCgl0580启动子的基因组区域被克隆在GFP的前面，使得GFP的表达受NCgl0580启动子的控制。本发明传感器将L‑高丝氨酸浓度与荧光信号强度联系起来，使L‑高丝氨酸浓度可视化，实现对目标产物L‑高丝氨酸的灵敏检测。该生物传感器可在0~5g/L的L‑高丝氨酸浓度下表现良好的线性关系。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种生物传感器及其构建方法，和在筛选氨基酸高产菌株中的应用。
背景技术
L-高丝氨酸是一种天然存在的重要功能性氨基酸，是甲硫氨酸、赖氨酸和苏氨酸的前体，参与体内多种生理生化反应和多个生物代谢过程，具有重要的生理功能和应用价值。L-高丝氨酸作为中间体或原料合成多种化工产品、农药及饲料，广泛应用于食品、化妆品、医药和饲料等领域，具有广阔的应用前景。例如，L-高丝氨酸是用来合成新型农药L-草胺磷的主要前体原料，已在国外发达国家得到广泛应用，且需求量逐年递增。
目前，高丝氨酸的合成方法主要是化学法和生物酶法。化学法以L-甲硫氨酸或天冬氨酸为原料，需要使用碘化物和大量有机溶剂，并且生成硫化物，对环境造成污染。生物酶法是以丙酮酸和醛类化合物为原料，在醛缩酶、甲酸脱氢酶和L-氨基酸脱氢酶复合酶的共同催化下反应得到高丝氨酸，成本高，需要使用有毒原料甲醛和甲酸，并需要使用价格昂贵的辅酶等。微生物发酵法具有成本低、条件温和、环境污染少等许多优点，近年来已成为生产各类氨基酸的首选工艺。
生物传感器已广泛应用于多种氨基酸高产菌株的途径优化和高效筛选。代谢物生物传感器是基于转录调控因子、RNA 开关等生物识别元件和荧光信号、酶活力等信号输出元件构建的一类生物传感器装置，能够感应细胞内特定代谢物浓度的变化，转化为可识别信号输出，在构建高效微生物细胞工厂中具有巨大应用潜力。通过后续偶联高通量筛选装置建立相应的小分子代谢物筛选技术，可以为快速筛选获得性能优异的菌种资源提供重要保障。然而目前为止，尚没有关于L-高丝氨酸生物传感器的报道。由于缺乏简便快捷的检测方法，极大地限制了L-高丝氨酸高产菌株的高通量筛选和选育。本发明通过设计和构建基于转录调控因子的L-高丝氨酸生物传感器，并将其偶联流式细胞分选等高通量筛选体系，可以高效快速的筛选出L-高丝氨酸高产候选菌。
发明内容
本发明目的在于提供一种L-高丝氨酸生物传感器及其构建方法，并使用该生物传感器建立高效的L-高丝氨酸高产菌株筛选方法。
本发明依据L-高丝氨酸结构特性分析，选择能够响应结构相似的丝氨酸的转录调控因子为候选元件，首选提供了一种基于转录调控因子NCgl0581的L-高丝氨酸生物传感器的构建方法，该生物传感器可以将L-高丝氨酸浓度转化为荧光强度信号。所述生物传感器是由谷氨酸棒杆菌来源的转录调控因子NCgl0581编码基因、NCgl0581启动子区、NCgl0580启动子区、标记基因和质粒骨架组成。本发明提供的生物传感器能特异性响应L-高丝氨酸，在0~5 g/L L-高丝氨酸浓度下表现出优良的线性关系。
因此，本发明首先提供一种L-高丝氨酸生物传感器，其是含有NCgl0581启动子及与其可操作连接的NCgl0581开放阅读框，和NCgl0580启动子及与其可操作连接荧光蛋白GFP基因的表达载体。
优选地，所述转录调控因子NCgl0581编码基因来源于谷氨酸棒杆菌，和/或基因NCgl0580的启动子区域来源于谷氨酸棒杆菌。
在一个具体实施方案中，所述标记基因优选为编码绿色荧光蛋白的GFP基因。
在具体实施方式中，所述表达载体适合于在大肠杆菌、或谷氨酸棒杆菌中表达。更优选地，所述表达载体为大肠杆菌-谷氨酸棒杆菌穿梭载体pTRCmob表达质粒。
在优选实施方式中，所述传感器是将转录调控因子NCgl0581编码基因、NCgl0581启动子、NCgl0580启动子、以及标志基因组成的片段连接到表达载体得到，其中NCgl0581启动子和NCgl0580启动子在表达载体中方向相反。
在一个更具体的实施方式中，所述转录调控因子NCgl0581编码基因的核苷酸序列如SEQ ID No：1；NCgl0581启动子的核苷酸序列如SEQ ID No：2所示；NCgl0580启动子的核苷酸序列如SEQ ID No：3所示；GFP的核苷酸序列如SEQ ID No：4所示。
本发明还提供一种利用上述的生物传感器在检测L-高丝氨酸中的应用。
本发明进一步提供一种筛选或鉴定L-高丝氨酸高产菌株的方法，具体步骤是对候选菌株进行培养，得到培养悬浮液后，采用所述的生物传感器进行筛选方法，荧光强度达到设定水平的候选菌株即为L-高丝氨酸高产菌株。在具体实施方式中，可以是筛选L-高丝氨酸高产菌株的方法，其包括下述步骤：对菌株进行随机诱变，获得随机突变体菌株库，培养得到培养菌悬液后，采用本发明所述的生物传感器，利用高通量筛选方法获得目标突变体，荧光强度达到设定水平的诱变菌株即为L-高丝氨酸生产能力获得提升的候选突变菌株。
优选地，还包括对所述候选突变菌株做进一步验证，获得L-高丝氨酸生产能力获得提升的突变菌株。
在具体的实施方式中，通过常压室温等离子体(ARTP)诱变、化学诱变、实验室适应性进化等方法对上述重组菌株进行随机诱变，获得随机突变体菌株库。将诱变菌株接种于CGXII基本培养基（20 g/L硫酸铵, 1 g/L K2HPO4, 1 g/L KH2PO4, 42 g/L MOPS, 13 mg/LCaCl2·2H2O, 10 mg FeSO4·7H2O, 14 mg/L MnSO4·5H2O, 1 mg/L ZnSO4·7H2O, 0.3 mg/L CuSO4·5H2O, 0.02 mg/L NiCl2·6H2O, 0.5 mg/L biotin, 30 mg/L PCA and 0.5 mg/L thiamine），在32 oC，200 r/min条件下培养24 h，得到培养菌悬液。将上述菌悬液使用Beckman MoFlo XDP流式细胞仪分选，荧光信号获得明显提升的细胞即为L-高丝氨酸生产能力提高的候选诱变菌株。收集分选获得的菌体细胞，悬浮于新鲜的发酵培养基中（50~60g/L葡萄糖，0.1~0.2 g/L尿素，5~10 g/L玉米浆，15~20 g/L硫酸铵，5~6 g/L KH2PO4，2 ~5g/L MgSO4·7H2O，0.2~0.5 g/L FeSO4·7H2O，0.2~0.4 g/L MnCl2·4H2O，0.2~0.4 mg/L生物素，1~2 mg/L维生素B1，1~2 mg/L维生素B6），在32 oC，200 r/min条件下进行L-高丝氨酸生产性能测试和验证。
本发明具有以下优点：本发明利用转录调控因子翻译的蛋白质可以与L-高丝氨酸形成蛋白复合物，结合到启动子区域，激活下游基因的表达，可以将L-高丝氨酸浓度与荧光信号强度联系起来，使L-高丝氨酸浓度可视化，实现对目标产物L-高丝氨酸的灵敏检测。该传感器可以与高通量筛选体系相结合，实现可以区分在低浓度范围内氨基酸内部积累略有差异的细胞亚群，并以此为基础可以构建一种快速检测胞内高丝氨酸浓度的高通量筛选方法，以便于通过荧光强度可以筛选高丝氨酸高产菌株。
附图说明
图1为L-高丝氨酸生物传感器的质粒图谱；
图2为L-高丝氨酸生物传感器的荧光信号响应曲线；
图3为L-高丝氨酸生物传感器对谷氨酸等氨基酸的荧光信号响应曲线；
图4为利用L-高丝氨酸生物传感器筛选菌株的流程示意图。
具体实施方式
实施例1：L-高丝氨酸生物传感器质粒的构建
本实施例对L-高丝氨酸生物传感器各组成模块进行PCR扩增，得到核苷酸片段，并通过分子克隆技术将各片段按一定顺序连接，完成L-高丝氨酸生物传感器质粒的构建。其中，所述生物传感器是由谷氨酸棒杆菌来源的转录调控因子NCgl0581编码基因（Genebank：NP_599842）、NCgl0581启动子区、NCgl0580（Genebank：NP_599841）启动子区、标记基因和质粒骨架组成（如图1所示，转录调控因子Ncgl0581由自身启动子Ncgl0581启动子控制表达，而NCgl0580启动子控制标记基因GFP的表达。转录调控因子NCgl0581能够响应胞内L-高丝氨酸浓度，从而激活启动NCgl0580启动子的表达，利用GFP标记基因，可实现L-高丝氨酸浓度和荧光信号的偶联。
其中，NCgl0581编码基因核苷酸序列如下（SEQ ID NO：1）：
atgctcaatctcaaccgcttacacatcctgcaggaattccaccgcctgggaacgattacagcagtggcggaatccatgaactacagccgctctgccatctcccaacaaatggcgctgctggaaaaagaaattggtgtgaaactctttgaaaaaagcggccgaaacctctacttcacagaacaaggcgaagtgttggcctcagaaacacatgcgatcatggcagcagtcgaccatgcccgcgcagccgttctagattcgctgtctgaagtgtccggaacgctgaaagtcacctccttccaatccctgctgttcacccttgccccgaaagccatcgcgcgcctgaccgagaaatacccacacctgcaagtagaaatctcccaactagaagtcaccgcagcgctcgaagaactccgcgcccgccgcgtcgacgtcgcactcggcgaggaataccccgtggaagtcccccttgttgaggccagcattcaccgcgaagtcctcttcgaagaccccatgctgctcgtcaccccagcaagcggcccatactctggcctcaccctgccagaactccgcgacatccccatcgccatcgatccacccgaccttcccgcgggcgaatgggtccataggctctgccggcgcgccgggtttgagccccgcgtgacctttgaaaccagcgatcccatgctccaagcacacctcgtgcgtagcggcttggccgtgacattttcccccacactgctcaccccgatgctggaaagcgtgcacatccagccgctgcccggcaaccccacgcgcacgctctacaccgcggtcagggaagggcgccaggggcatccagccattaaagcttttcgacgagccctcgcccatgtggccaaagaatcttatttggaggctcgtctagtagagtga。
NCgl0581启动子的核苷酸序列如下（SEQ ID NO：2）
attgccaaagaaacctttaaggactagatcgaaaaacagccaactatagttaagtaatactgaacaattttggaggtgtc
NCgl0580启动子的核苷酸序列如下（SEQ ID NO：3）
Aatcaagggcatgaataaacagtccgctgcagtgttgatggtgatgggttccgccctatccctgcaatttggtgctgccattggaacgtga
荧光蛋白GFP基因的核苷酸序列如下：
atgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgacgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactctgacttatggtgttcaatgcttttcaagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtcctttcaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagatcacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaatag
具体步骤如下：
以谷氨酸棒杆菌（Corynebacterium glutamicum ATCC 13032）基因组为模板，使用的引物为F1和R1，扩增获得含有NCgl0581编码基因（SEQ ID NO：1）、NCgl0581启动子（SEQID NO：2）以及NCgl0580启动子（SEQ ID NO：3）的整个序列片段；以pXMJ19-GFP质粒(见Weiliang et. al.Promoter library-based module combination (PLMC) technology foroptimization of threonine biosynthesis in Corynebacterium glutamicum.ApplMicrobiol Biotechnol. 2018,102:4117-4130)模板使用引物F2和R2扩增获得荧光报告GFP基因片段(SEQ ID NO:4)，使用引物F3和R3，以质粒pTRCmob为模板，扩增获得pTRCmob质粒骨架；通过Goldengate组装方法将上述获得的核苷酸片段和质粒骨架进行连接，将GFP置于Ncgl0580启动子控制之下，并转化至大肠杆菌DH5α感受态中，最终获得质粒PhomBio，并进行公司测序确认。质粒构建过程所使用的引物序列如下：
F1：5’-caccaggtctcagaactcactctactagacgagcctccaaataag-3’
R1：5’-caccaggtctcattcacgttccaatggcagcaccaaattg-3’
F2：5’-caccaggtctcatgaaactttaagaaggagatatacatatgagtaaaggagaagaacttttcactgg-3’
R2：5’-caccaggtctcactatttgtatagttcatccatgccatgtgtaatc-3’
F3：5’-caccaggtctcaataggaattcgagctcggtacccggggatc-3’
R3：5’-caccaggtctcagttccagctcatttcagaatatttgccagaac-3’
质粒构建过程的PCR扩增体系：10uL 5×HF Phusion buffer，4uL 2.5mM dNTPMix，2.5 uL 10 uM上游引物，2.5 uL 10 uM下游引物，1.5 uL DMSO，1 uL模板，0.5 uLPhusion聚合酶，28 uL ddH2O。PCR扩增条件：98 oC预变性30 s；98 oC变性10 s，60 oC退火10s，72 oC延伸2 k/min，35个循环；72 oC延伸5min。Goldengate组装体系：1.5 uL T4 DNALigase buffer，1.5 uL 10×BSA，1 uL T4 DNA Ligase，1 uL BsaI，100 ng片段或骨架，补足15 uL体系。PCR连接条件：37 oC 3 min，22 oC 4 min，40个循环；22 oC 30 min；80 oC 5min；4 oC 10 min。
实施例2 L-高丝氨酸生物传感器对L-高丝氨酸的响应测试
将实施例1所述的PhomBio质粒电转入谷氨酸棒杆菌ATCC 13032中，获得含有L-高丝氨酸生物传感器的重组菌。挑取单菌落接种到含有终浓度25 ug/L卡那霉素的LBHIS液体培养基中（2.5 g/L 酵母提取物；5 g/L 胰蛋白胨；5 g/L 氯化钠；18 g/L脑心浸液，91 g/L山梨醇），32℃，200 rpm过夜培养16 h。将过夜培养菌液按1%接种量接种到含终浓度0~5 g/L L-高丝氨酸的新鲜CGXII基本液体培养基中，32℃，200 rpm，孵育培养6 h。离心收集菌体，使用等体积0.9%NaCl重悬后，用多功能酶标仪测定其荧光强度，设定激发波长为488nm，发射波长为520 nm，同时测量菌体OD600值，将荧光强度和OD600的比值作定义为相对荧光强度的数值。以外源L-高丝氨酸浓度为横坐标，以相对荧光强度为纵坐标，绘制L-高丝氨酸浓度-荧光强度的关系曲线。
结果显示，该生物传感器可在0~5 g/L L-高丝氨酸浓度下表现良好的线性关系，随着L-高丝氨酸浓度的增加，含有该生物传感器的重组菌株具有相应提高的荧光强度。同时，评估测定了该生物传感器或含有所述生物传感器的重组菌株对其他氨基酸的响应情况，5 g/L谷氨酸、苏氨酸、脯氨酸、缬氨酸、组氨酸、苯丙氨酸、精氨酸、异亮氨酸和蛋氨酸等均不会引起宿主菌荧光强度的明显变化，说明该生物传感器具有较好的特异性（图3）。
实施例3 L-高丝氨酸高产菌株的筛选
将实施例1所述的PhomBio质粒电转入具有一定L-高丝氨酸合成能力的谷氨酸棒杆菌中，获得含有L-高丝氨酸生物传感器的重组菌。挑取单菌落接种到含有终浓度25 ug/L卡那霉素的LBHIS液体培养基中，32℃，200 rpm过夜培养16 h。采用物理、化学或生物的方法，对上述重组菌株进行随机诱变，获得随机突变体菌株库。在本实施例中，使用常压室温等离子体诱变仪ARTP进行菌体诱变，具体步骤为：使用生理盐水洗涤菌体并重悬至OD600＝1～2，取10 μL菌液涂布在贴片上，调整照射距离2mm，功率100W，进行诱变处理。将诱变后的菌悬于LBHIS液体培养基中，32℃、200 rpm过夜培养16 h。将过夜培养的菌液离心收集菌体并用PBS溶液洗涤重悬至OD为0.1，用流式细胞仪进行初筛，筛选出荧光信号增强且位于前0.1%的菌体细胞。收集分选获得的菌体细胞，接种于LBHIS液体培养基，过夜培养后按5％接种量接种至发酵培养基中，在32 oC，200 r/min条件下振荡培养96 h，利用高效液相色谱法分析发酵液中L-高丝氨酸含量。
经过流式细胞初筛分选和发酵检测复筛（如图4），最终筛选获得两株突变菌，命名为HSE-16和HSE-81，其L-高丝氨酸的积累能力（8.4±0.6 g/L和7.9±0.4 g/L）比原始出发菌株OAH3-2均提高50%以上。通过上述实施例表明，本发明设计构建的L-高丝氨酸生物传感器在高产菌株筛选中具有较好应用潜力，通过结合随机诱变和高通量筛选策略，可以快速高效的筛选获得L-高丝氨酸高产菌株。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、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> 中国科学院天津工业生物技术研究所
<120> 一种高丝氨酸生物传感器及其构建方法和应用
<130>
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 912
<212> DNA
<213> 谷氨酸棒杆菌（Corynebacterium glutamicum ATCC 13032）
<400> 1
atgctcaatc tcaaccgctt acacatcctg caggaattcc accgcctggg aacgattaca 60
gcagtggcgg aatccatgaa ctacagccgc tctgccatct cccaacaaat ggcgctgctg 120
gaaaaagaaa ttggtgtgaa actctttgaa aaaagcggcc gaaacctcta cttcacagaa 180
caaggcgaag tgttggcctc agaaacacat gcgatcatgg cagcagtcga ccatgcccgc 240
gcagccgttc tagattcgct gtctgaagtg tccggaacgc tgaaagtcac ctccttccaa 300
tccctgctgt tcacccttgc cccgaaagcc atcgcgcgcc tgaccgagaa atacccacac 360
ctgcaagtag aaatctccca actagaagtc accgcagcgc tcgaagaact ccgcgcccgc 420
cgcgtcgacg tcgcactcgg cgaggaatac cccgtggaag tcccccttgt tgaggccagc 480
attcaccgcg aagtcctctt cgaagacccc atgctgctcg tcaccccagc aagcggccca 540
tactctggcc tcaccctgcc agaactccgc gacatcccca tcgccatcga tccacccgac 600
cttcccgcgg gcgaatgggt ccataggctc tgccggcgcg ccgggtttga gccccgcgtg 660
acctttgaaa ccagcgatcc catgctccaa gcacacctcg tgcgtagcgg cttggccgtg 720
acattttccc ccacactgct caccccgatg ctggaaagcg tgcacatcca gccgctgccc 780
ggcaacccca cgcgcacgct ctacaccgcg gtcagggaag ggcgccaggg gcatccagcc 840
attaaagctt ttcgacgagc cctcgcccat gtggccaaag aatcttattt ggaggctcgt 900
ctagtagagt ga 912
<210> 2
<211> 80
<212> DNA
<213> 谷氨酸棒杆菌（Corynebacterium glutamicum ATCC 13032）
<400> 2
attgccaaag aaacctttaa ggactagatc gaaaaacagc caactatagt taagtaatac 60
tgaacaattt tggaggtgtc 80
<210> 3
<211> 91
<212> DNA
<213> 谷氨酸棒杆菌（Corynebacterium glutamicum ATCC 13032）
<400> 3
aatcaagggc atgaataaac agtccgctgc agtgttgatg gtgatgggtt ccgccctatc 60
cctgcaattt ggtgctgcca ttggaacgtg a 91
<210> 4
<211> 717
<212> DNA
<213>
<400> 4
atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60
gacgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120
aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180
gtcactactc tgacttatgg tgttcaatgc ttttcaagat acccagatca tatgaaacag 240
catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc 300
aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360
aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa 420
ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga 480
atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540
cattatcaac aaaatactcc aattggcgat ggccctgtcc tttcaccaga caaccattac 600
ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagatca catggtcctt 660
cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaatag 717
<210> 5
<211> 45
<212> DNA
<213>
<400> 5
caccaggtct cagaactcac tctactagac gagcctccaa ataag 45
<210> 6
<211> 40
<212> DNA
<213>
<400> 6
caccaggtct cattcacgtt ccaatggcag caccaaattg 40
<210> 7
<211> 67
<212> DNA
<213>
<400> 7
caccaggtct catgaaactt taagaaggag atatacatat gagtaaagga gaagaacttt 60
tcactgg 67
<210> 8
<211> 46
<212> DNA
<213> 人工序列
<400> 8
caccaggtct cactatttgt atagttcatc catgccatgt gtaatc 46
<210> 9
<211> 42
<212> DNA
<213> 人工序列
<400> 9
caccaggtct caataggaat tcgagctcgg tacccgggga tc 42
<210> 10
<211> 44
<212> DNA
<213> 人工序列
<400> 10
caccaggtct cagttccagc tcatttcaga atatttgcca gaac 44
Claims (8)
1.一种生物传感器在检测L-高丝氨酸中的应用，其特征在于，所述生物传感器是含有NCgl0581启动子及与其可操作连接的NCgl0581编码基因，和NCgl0580启动子及与其可操作连接荧光蛋白GFP基因的表达载体。
2.根据权利要求1所述的应用，其特征在于，所述NCgl0581编码基因来源于谷氨酸棒杆菌，和/或基因NCgl0580的启动子区域来源于谷氨酸棒杆菌。
3.根据权利要求1所述的应用，其特征在于：表达载体适合于在大肠杆菌、或谷氨酸棒杆菌中表达。
4.根据权利要求3所述的应用，其特征在于：所述表达载体适合在细菌中扩增和表达。
5.根据权利要求4所述的应用，其特征在于：所述细菌为大肠杆菌或谷氨酸棒杆菌。
6.根据权利要求4所述的应用，其特征在于：所述表达载体为pTRCmob表达质粒。
7.根据权利要求1所述的应用，其特征在于，其中NCgl0581启动子和NCgl0580启动子在表达载体中为相反的方向，分别可操作连接NCgl0581编码基因和荧光蛋白GFP基因。
8.根据权利要求1至7任一项所述的应用，其特征在于， NCgl0581编码基因的核苷酸序列如SEQ ID No：1；NCgl0581启动子的核苷酸序列如SEQ ID No：2所示；NCgl0580启动子的核苷酸序列如SEQ ID No：3所示；GFP的核苷酸序列如SEQ ID No：4所示。
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (5)
| Title |
|---|
| A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level;Binder, S等;《Genome Biology》;20121231;第13卷(第5期);摘要，正文第4页左栏第3段-右栏第1段，第10页左栏第6-7段，图2，补充文件1 * |
| Binder, S等.A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.《Genome Biology》.2012,第13卷(第5期),1-12. * |
| Biosensor-Mediated Adaptive Laboratory Evolution of Escherichia coli for L-Serine Overproduction from Glycerol;Xiaomei Zhang等;《Research & Reviews: Journal of Microbiology and Biotechnology》;20200127;第9卷(第1期);摘要 * |
| Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum;Xin Zhang等;《Applied Microbiology and Biotechnology》;20180503;第102卷(第14期);摘要，正文第5941页左栏第3段-右栏第1段，第5943页左栏第3-6段，图1-2 * |
| Xin Zhang等.Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum.《Applied Microbiology and Biotechnology》.2018,第102卷(第14期),5939-5951. * |
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