CN116121285B - 一种2-吡咯烷酮生物传感器的构建及应用 - Google Patents
一种2-吡咯烷酮生物传感器的构建及应用 Download PDFInfo
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Abstract
本发明公开了一种2‑吡咯烷酮生物传感器的构建及应用，属于基因工程技术领域。本发明提供一种2‑吡咯烷酮的生物传感器，在原有基础上对其启动子区域中调控蛋白的结合位点突变，增强了该传感器对高浓度的2‑吡咯烷酮的信号响应，可更好用于对2‑吡咯烷酮合成相关酶和菌的定向进化。本发明通过对原有启动子进行改造，并非单纯的提高基因的转录水平，而是通过改造使生物传感器在不同浓度的2‑吡咯烷酮条件下荧光强度的差异性更大，以才能更好的区分开提高2‑吡咯烷酮产量的菌株或酶，减少假阳性的筛选。
Description
技术领域
本发明涉及提供一种2-吡咯烷酮生物传感器的构建及应用，属于基因工程技术领域。
背景技术
生物传感器(biosensor)是以生物学组件作为主要功能元件，能够感应特定的待测物质，并按照一定规律将其转换成可识别信号的器件或装置。生物传感功能元件中的启动子是决定转录起始、影响转录速率的一段关键DNA序列，启动子强度直接影响报告基因的转录水平。
荧光基因(绿色荧光蛋白gfp、红色荧光蛋白mCherry等)是目前应用最为广泛的一类报告基因，在特定的激发光下可以快速准确测量荧光强度，结合荧光激活细胞分选技术更是能够实现目的菌株的高通量筛选。构建基于荧光检测的生物传感器，将2-吡咯烷酮浓度与荧光强度偶联，有望用于2-吡咯烷酮合成酶的高通量筛选。
发明内容
本发明提供一种响应2-吡咯烷酮的生物传感器，含有启动子Pcon、ChnR编码基因，启动子Pb-E1和荧光蛋白编码基因；所述启动子Pb-E1上具有转录因子结合位点，所述启动子Pb-E1调控荧光蛋白编码基因的表达；所述启动子Pcon调控ChnR基因的表达；所述启动子Pcon和启动子Pb-E1的转录方向相反。
在一种实施方式中，所述启动子Pcon、ChnR编码基因、启动子Pb-E1和荧光蛋白编码基因位于同一质粒或同一基因组DNA上。
在一种实施方式中，所述转录因子结合位点具有SEQ ID NO.6所示的核苷酸序列。
在一种实施方式中，所述荧光蛋白编码基因包括但不限于mCherry。
在一种实施方式中，所述启动子Pcon的核苷酸序列如SEQ ID NO.1所示，所述ChnR编码基因的核苷酸序列如SEQ ID NO.2所示；所述启动子Pb-E1的核苷酸序列如SEQ IDNO.3所示；所述荧光蛋白编码基因mCherry的核苷酸序列如SEQ ID NO.4所示。
在一种实施方式中，所述生物传感器以pBbS5C-RFP质粒为质粒骨架。
本发明还提供了含有所述生物传感器的重组微生物细胞。
在一种实施方式中，所述微生物包括但不限于大肠杆菌或谷氨酸棒杆菌。
在一种实施方式中，所述大肠杆菌为大肠杆菌BW25113。
本发明还提供了所述生物传感器在筛选2-吡咯烷酮高产菌株、高活力的2-吡咯烷酮合成关键性酶等方面的应用。
在一种实施方式中，所述筛选2-吡咯烷酮高产菌株是将所述生物传感器转入目的菌株细胞内，将待筛选的菌株在一定条件下培养一段时间，根据菌株发酵液的荧光强度筛选2-吡咯烷酮高产菌株。
本发明还提供了一种新的启动子，含有SEQ ID NO.3所示的核苷酸序列。
有益效果：
本发明提供一种2-吡咯烷酮的生物传感器，在原有基础上对其启动子区域中调控蛋白的结合位点突变，增强了该传感器对高浓度的2-吡咯烷酮的信号响应，可更好用于对2-吡咯烷酮合成相关酶和菌的定向进化。本发明对启动子的改造不是单纯的提高基因的转录水平，是通过改造后的生物传感器在不同浓度的2-吡咯烷酮条件下荧光强度的差异性更大，以更好的区分开提高2-吡咯烷酮产量的菌株或酶，减少假阳性的筛选。
附图说明
图1为响应2-吡咯烷酮的生物传感器示意图。
图2为对启动子进行优化后的生物传感器在添加不同浓度2-吡咯烷酮后的荧光强度变化。
图3为生物传感器在流式细胞仪(FACS)分选测试。
具体实施方式
培养基：
LB培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，用NaOH调节该培养基的pH达到7.4，121℃高压蒸汽灭菌15min。
M9培养基：M9 salts(5X)200mL，20％葡萄糖20mL，1M MgSO4 2mL，1M CaCl20.1mL，H2O 780mL。其中M9 Salts(5X)：Na2HPO4 33.9g/L，KH2PO4 15g/L，NaCl 2.5g/L，NH4Cl 5g/L，用1000mL去离子水重悬上述粉末，加热搅动使其完全溶解，121℃高压蒸汽灭菌15min；20％葡萄糖溶液过滤除菌，4℃保存；1M MgSO4溶液和1M CaCl2分别121℃灭菌。
实施例1生物传感器的构建及启动子优化
图1为2-吡咯烷酮诱导的生物传感器系统，以pBbS5C-RFP质粒为骨架，含有启动子Pcon、ChnR编码基因，启动子Pb-E1和荧光蛋白编码基因mCherry；所述启动子Pcon的核苷酸序列如SEQ ID NO.1所示，所述ChnR编码基因的核苷酸序列如SEQ ID NO.2所示；所述启动子Pb-E1的核苷酸序列如SEQ ID NO.3所示；所述荧光蛋白编码基因mCherry的核苷酸序列如SEQ IDNO.4所示；所述启动子Pb-E1上具有转录因子结合位点，所述启动子Pb-E1调控荧光蛋白编码基因mCherry的表达；所述启动子Pcon调控ChnR基因的表达；所述启动子Pcon和启动子Pb-E1的转录方向相反。
本发明构建的生物传感器的工作原理为：在环境中含有2-吡咯烷酮的情况下，受Pcon启动子调控表达的转录因子ChnR结合环境中的2-吡咯烷酮诱发DNA结合域的构象变化，并结合在位于启动子Pb-E1上的转录因子结合位点TGTAGCCCACC，激活转录，从而调控启动子Pb-E1表达荧光蛋白。2-吡咯烷酮浓度越高，Pb-E1调控转录的强度越强，从而荧光信号越强。
在质粒pBbSLactamC-mCherry(公开于论文《Development of a TranscriptionFactor-Based Lactam Biosensor》)的基础上，对启动子中的动子区域中调控蛋白的结合位点ttgtttggatc(SEQ ID NO.5所示)进行了优化，突变为TGTAGCCCACC(SEQ ID NO.6所示)，通过设计定点突变引物对：
Pb-E1F:tgggtaactGGTGGGCTACAtctcttttagttgcaagcttc；
Pb-E1R:ctaaaagagaTGTAGCCCACCagttacccaaaatcgttg；
以pBbSLactamC-mCherry质粒(公开于《Development of a TranscriptionFactor-Based Lactam Biosensor》)的基因作为模板，通过PCR进行扩增得到带有序列优化的启动子片段的质粒片段，直接转化至E.coli DH5α中，测序验证其构建成功，命名为pBbS-E1。
实施例2添加不同浓度的2P作为底物，检测荧光强度的变化
将实施例1中构建的pBbS-E1质粒转入大肠杆菌BW25113(WT)中，以转入原始质粒pBbSLactamC-mCherry(pBbS-mCherry)的大肠杆菌BW25113为对照。用于检测启动子优化后添加不同底物浓度对荧光强度的响应。将菌株WT pBbS-E1和WT pBbS-mCherry分别在LB培养基中，用试管于37℃培养12小时，获得菌浓为OD600＝5.0的种子液；将种子液离心后用M9培养基重悬洗涤2次，最后加0.5mL的M9培养基重悬，随后将50mL M9培养基加到500mL摇瓶中，按10％的接种量接种，使接种后的初始OD600＝0.5，同时分别加入0mM、0.2mM、0.4mM、0.6mM和1.2mM的2-吡咯烷酮，培养温度为37℃，摇床转速200rpm，培养24小时。24小时后取0.2mL液体转入96孔板，进行荧光强度的检测。
利用酶标仪检测荧光强度：酶标仪型号为Infifinite F200 multimode reader(TECAN,San Jose,CA)，96孔板在酶标仪的线性模式摇动2分钟，检测600nm吸光度波长下的OD和mCherry荧光强度(激发光波长＝575nm，发射光波长＝620nm)；如图2所示，对照没有进行启动子优化的质粒pBbS-mCherry，荧光强度低，显示构建的pBbS-E1质粒能提高荧光强度，且不同浓度的2-吡咯烷酮，荧光强度差异性更大，底物浓度的差异变化直观体现在荧光强度的变化上，荧光强度差异更显著意味着在将来的筛选工作中，不仅能够提高检测效率，还可以有效去除假阳性的存在。
实施例3生物传感器在流式细胞仪(FACS)分选测试
辅酶A转移酶ACT可以将γ-氨基丁酸转化生成2-吡咯烷酮。应用过表达该酶来测试实施例1构建的2-吡咯烷酮生物传感器pBbS-E1，在流式细胞仪中能否区分可以生产和不可以生产的2-吡咯烷酮菌株。选择来自Butyricicoccus faecihominis菌株的act基因(Genbank登录号为MCQ5130945.1)设计扩增引物：
ACTF:CATGTGTCAATTGAAAGGACATCAACGATGCGTTCTCTGGAGGGAGTCCG；
ACTR:CTACTGCCGCCAGGCAGCGGCCGCTTTAAATCGCACCGCAGGCTGCCAG。
合成核苷酸序列如Gene ID：MCQ5130945.1所示的基因，以该基因为模板，通过PCR进行扩增得到目的片段，经DNA纯化试剂盒纯化后，通过Gibson将PCR扩增产物和质粒pCES(质粒公开于论文《Development of a high-copy-number plasmid via adaptivelaboratory evolution of Corynebacterium glutamicum》)的主干片段相连，转化至E.coli DH5α中，测序验证其构建成功。将携带ACT编码序列的表达载体pCES-ACT共转化到实施例2构建的菌株WT pBbS-E1和WT pBbS-mCherry中。
应用LB培养基，在试管中于37℃培养12小时，获得种子液；将50mL M9培养基加到500mL摇瓶中，添加5g/Lγ-氨基丁酸，按10％的接种率接种，培养温度为37℃，摇床转速200rpm，培养24小时。根据mCherry红色荧光蛋白信号通过流式细胞仪FACS确认荧光强度。
图3分别显示了流式细胞仪对菌株WT pBbS-E1和WT pBbS-mCherry加入和不加入pCES-ACT情况下红色荧光蛋白mCherry的荧光强度对比。结果显示：未优化的生物传感器不能很好地区分可以生产和不可以生产的2-吡咯烷酮菌株，两者信号强度差异性不大，优化后的生物传感器的荧光强度差异更显著，能很好地区分可以生产和不可以生产的2-吡咯烷酮菌株，也更有利于筛选出关键酶定向进化后的酶活提高的阳性突变。
对比例1：
具体实施方式同实施例1，区别在于，对启动子中调控蛋白的结合位点ttgtttggatc序列进行了另一种优化，将SEQ ID NO.5所示的序列突变为ATACAATCGGAG(SEQID NO.7)，具体步骤为：
设计定点突变引物对：
Pb-E2F:tgggtaactATACAATCGGAGtctcttttagttgcaagcttc；
Pb-E2R:ctaaaagagaCTCCGATTGTATagttacccaaaatcgttg；
以pBbSLactamC-mCherry质粒的基因作为模板，通过PCR进行扩增得到带有序列优化的启动子片段的质粒片段，直接转化至E.coli DH5α中，测序验证其构建成功，命名为pBbS-E2。
按照实施例2的方法，检测启动子的不同优化序列对传感器所能提供的荧光强度进行验证，如图2所示，对照没有进行启动子优化的质粒pBbS-mCherry，荧光强度低，而pBbS-E2在不添加2-吡咯烷酮时，就能够检测到较高的荧光强度，本底的荧光表达就很强，会极大可能导致筛选过程中假阳性。虽然在含有2-吡咯烷酮的发酵液中检测到的荧光强度很大，但是不同浓度2-吡咯烷酮的发酵液之间荧光信号差异性不大，说明提高检测灵敏度的方法并非单纯提高启动子强度就能实现；同样是加入0.6mM 2-吡咯烷酮，pBbS-E1与不加底物相比，有2.4倍的差异，pBbS-E2仅为1.1倍，pBbS-E1更有利于筛选2-吡咯烷酮高产菌株或高活力的2-吡咯烷酮合成关键性酶。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (8)
1.一种响应2-吡咯烷酮的生物传感器，其特征在于，以pBbS5C-RFP质粒为质粒骨架，含有启动子Pcon、ChnR编码基因、启动子Pb-E1和荧光蛋白编码基因；所述启动子Pb-E1上具有转录因子结合位点，所述启动子Pb-E1调控荧光蛋白编码基因的表达；所述启动子Pcon调控ChnR基因的表达；所述启动子Pcon和启动子Pb-E1的转录的方向相反；所述转录因子结合位点具有SEQ ID NO.6所示的核苷酸序列；所述启动子Pcon的核苷酸序列如SEQ ID NO.1所示，所述ChnR编码基因的核苷酸序列如SEQ ID NO.2所示；所述启动子Pb-E1的核苷酸序列如SEQ ID NO.3所示。
2.根据权利要求1所述的生物传感器，其特征在于，所述启动子Pcon、ChnR编码基因、启动子Pb-E1和荧光蛋白编码基因位于同一质粒或同一基因组DNA上。
3.根据权利要求1或2所述的生物传感器，其特征在于，所述荧光蛋白编码基因包括mCherry。
4.根据权利要求3所述的生物传感器，其特征在于，所述荧光蛋白编码基因mCherry的核苷酸序列如SEQ ID NO.4所示。
5.含有权利要求1~4任一所述生物传感器的重组微生物，其特征在于，所述微生物为大肠杆菌或谷氨酸棒杆菌。
6.根据权利要求5所述的重组微生物，其特征在于，所述大肠杆菌为大肠杆菌BW25113。
7.权利要求1~4任一所述生物传感器在筛选2-吡咯烷酮高产菌株中的应用，其特征在于，所述菌株为大肠杆菌或谷氨酸棒杆菌。
8.启动子，其特征在于，核苷酸序列如SEQ ID NO.3所示。
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