具体实施方式
1.细菌菌株和生长条件:
大肠杆菌DH5α和枯草芽抱杆菌1A751(BGSC,USA)用作主要宿主,用于克隆和表达。大肠杆菌于37℃生长于LB液体培养基(Luria S.E.等人,Virology12,1960,348-390)和LB琼脂平板,其中补充了100μg/ml氨苄青霉素。枯草芽孢杆菌于37℃生长于LB液体培养基和SR发酵培养基。液体培养基和琼脂平板分别补充了20μg/ml卡那霉素或5μg/ml氯霉素。为了诱导麦芽糖启动子,加入无菌过滤的或高压灭菌的D-麦芽糖至1%(w/v)的最终浓度。
2.实验材料:
所有的化学品都获自Sigma-Aldrich或Merck。合成的DNA寡核昔酸购自金唯智Genewiz。限制性酶和DNA修饰酶购自New England Biolabs.。以来自Fermentas的高保真DNA聚合酶在来自Eppendorf的热循环仪上进行PCR。
3.DNA的制备和转化:
使用Tiangen或Omega的DNA制备试剂盒,根据生产商的说明书,从大肠杆菌和枯草芽抱杆菌或从琼脂糖凝胶分离DNA。在所有实施例中均使用标准的分子技术。使用如ChungC.T.等人,Proc.Natl.Acad.Sci.USA 86,1989,21722175描述的质粒DNA转化大肠杆菌。根据改进的“Paris方法”(HarwoodC.R.Molecular Biological Methods for Bacillus,1990,John Wiley&Sons Ltd.,England),使用质粒DNA或DNA片段转化枯草芽抱杆菌。
4.绿色荧光蛋白荧光强度RFU的测定:
将培养至OD600约为0.6-0.8的待测菌株于4℃,4000rpm离心10分钟收集菌体,用预冷的PBS溶液洗涤菌体2次,转移150μL至96孔黑色底透酶标板(Corning,USA)中,放置于微孔板SpectraMax M2酶标仪(Molecular Devices,USA)于室温条件下483nm/525nm激发并检测荧光发射光强度RFU,于600nm测定菌体浓度,荧光强度计算按照RFU/OD600计算。
5.枯草芽孢杆菌基因无痕敲除:
根据改进的“基于AraR的枯草芽孢杆菌基因敲除方法”(Liu S,Endo K,etal.Microbiology.2008;154(Pt 9):2562–2570.)进行基因无痕敲除。首先利用同源重组原理将携带同源臂的受阿拉伯糖启动子调控的壮观霉素抗性基因片段“Para-spc”通过转化方法转入枯草芽孢杆菌1A751菌株中,替换基因组上的AraR基因,构建原始的基因敲除原始菌,其带有壮观霉素抗性。需要敲除基因X时,分别克隆X基因的上游1Kb片段(命名为UP)、下游1Kb片段(命名为DN)、待敲除基因X片段(命名为G)以及带有AraR基因和氯霉素抗性基因cat的筛选片段(命名为CR),利用overlap-PCR方法将上述4个片段以“UP-DN-CR-G”的顺序融合为一个片段,并通过转化方法转入枯草芽孢杆菌1A751中进行同源重组,以氯霉素抗性筛选转化子。将得到的转化子在壮观霉素抗性LB平板上验证,选取在氯霉素上生长、而在壮观霉素上不生长的转化子作为阳性转化子。将阳性转化子在无抗性的LB培养基中以37℃,200rpm培养16小时,取10uL菌液涂布于壮观霉素抗性平板上进行筛选,将得到的转化子通过菌落PCR进行验证,确定基因是否敲除。将菌落PCR验证正确的菌株作为敲除X基因的菌株进行保存。
表1
表2
实施例1枯草芽孢杆菌malR基因缺失菌株的构建
为了筛选具有低渗漏表达、高诱导表达强度的MalR转录激活因子突变体,需要构建敲除枯草芽孢杆菌基因组上野生型malR基因的工程菌株,用于排除野生型MalR转录激活因子对筛选体系的干扰。因此,本发明在前文所述的基因组无痕敲除法的基础上,利用malR-KO-UP.FOR/malR-KO-UP.REV引物扩增待敲除基因malR的上游重组片段UP,利用malR-KO-DN.FOR/malR-KO-DN.REV引物扩增待敲除基因malR的下游重组片段DN,利用malR-KO-neo.FOR/malR-KO-neo.REV引物扩增抗性筛选基因neo,将上述UP、neo、DN三个DNA片段利用PCR进行重叠延伸后,扩增得到UP-neo-DN的DNA拼接片段。将上述拼接片段转化枯草芽孢杆菌后,利用含有卡那霉素的抗性LB平板进行筛选,以malR-KO-vali-s/malR-KO-vali-a引物进行PCR验证,最终得到敲除malR基因的枯草芽孢杆菌菌株△R00。
实施例2枯草芽孢杆菌pDRG载体的构建
为了在敲除malR基因的枯草芽孢杆菌菌株△R00中进行malR基因突变体的筛选,构建了含有来源于枯草芽孢杆菌的麦芽糖操纵子PmalA启动子及其调控的gfp基因以及反向插入受组成型启动子PhpaII启动子调控的malR基因,能够整合在枯草芽孢杆菌基因组amyE基因位点的整合型表达载体,命名为pDRG。上述整合型表达载体的构建方法为:以pDL质粒(BGSC,USA)为基础,以PmalA-insert.for/rev引物扩增来源于枯草芽孢杆菌的麦芽糖操纵子MalA启动子或其突变体DNA片段并通过KpnI/BamHI酶切位点连入pDL质粒。使用CPECPCR cloning方法(Nature Protocols,6,242–251,2011),将pDL质粒上的bgaB基因替换为枯草芽孢杆菌PmalA启动子调控的gfp基因以及反向插入受组成型启动子HpaII启动子调控的malR基因,构建出带有PmalA-gfp表达盒以及PhpaII-malR表达盒的pDRG整合型质粒,质粒图谱见图1,质粒构建过程见图2,所使用的寡核苷酸如表1所示。将pDRG载体通过转化导入枯草芽孢杆菌△R00中,利用带有氯霉素抗性以及卡那霉素抗性的LB固体培养基平板进行筛选,所获得的转化子经过PCR验证正确后保存,获得遗传改造菌株△R01,其携带有来源于枯草芽孢杆菌的野生型MalR蛋白,其蛋白序列如SEQ ID NO:1所示。
实施例3枯草芽孢杆菌麦芽糖转录激活因子MalR的随机突变文库构建
以实施例2中的pDRG载体为基础,通过易错PCR产生的随机突变构建来源于枯草芽孢杆菌的麦芽糖转录激活因子MalR突变体文库,转化枯草芽孢杆菌△R00后通过检测启动子诱导表达的绿色荧光蛋白基因gfp来评价MalR突变体对麦芽糖malA启动子的激活作用。具体方法为:采用北京天恩泽基因科技有限公司的即用型易错PCR试剂盒(Beijing,China)对实施例2构建的pDRG质粒中的malR全长基因通过引物pDRG_malR.FOR/pDRG_malR.REV进行PCR扩增,利用添加Mn2+来引入随机突变,退火温度设为40℃-57℃;Mn2+终浓度设为0.25mM-1.25mM。整个实验过程采用多次易错PCR的方法来提高突变率,即将第一轮的PCR产物PCR片段进行胶回收用于第二轮易错PCR的模板。将扩增得到的malR插入片段和pEASY-T1Cloning Vector按照摩尔比7:1的比例添加到反应体系中,轻轻混合,室温反应5分钟,反应结束后,将离心管置于冰上,直接转化大肠杆菌,利用氨苄青霉素抗性筛选阳性转化子,将甘油菌送金唯智公司进行测序鉴定,确定引入的突变比率。测序正确后经计算平均突变比率为每700个核苷酸有3.4个突变。按照退火温度为51℃、Mn2+终浓度为0.75mM的条件进行两轮易错PCR,将扩增得到的MalR随机突变库片段和pDRG载体片段切胶回收后,按照摩尔比1:1的比例添加到PCR体系中进行CPEC-PCR反应,所得的PCR产物直接转化已构建的枯草芽孢杆菌基因敲除突变体△R00,利用带有氯霉素抗性以及卡那霉素抗性的LB固体培养基平板进行筛选,得到单拷贝整合于枯草芽孢杆菌基因组上的,受不同MalR突变体调控的GFP表达菌株文库。
实施例4枯草芽孢杆菌麦芽糖转录激活因子MalR的突变体筛选
将实施例3中构建的菌株突变体文库经含有1%(w/v)麦芽糖诱导物的LB培养活化后,取1mL新鲜菌液于4℃,12000rpm离心2分钟收集菌体,以磷酸盐缓冲溶液PBS重悬洗涤菌体3次,用等体积PBS缓冲液重悬后稀释50倍,利用流式细胞仪(Beckman,USA)进行FACS单细胞绿色荧光分选,选取RFU荧光值最高的1%细胞进行收集。将收集到的细胞重复上述FACS分选过程2次以获得稳定的受不同malR调控蛋白突变体调控的GFP表达菌株。将此单细胞菌株接种于含有150μL新鲜LB培养基的96孔板(Corning,USA)中过夜培养,次日,吸取5μL过夜培养物分别接种于不含/含有1%(w/v)麦芽糖诱导物的新鲜LB培养基中进行发酵,而后以携带野生型malR基因的pDRG表达菌株△R01作为对照,进行荧光强度的测定。根据测定结果,选取诱导后荧光强度明显增高、未诱导下本底表达明显降低的突变菌株,最终筛选获得4株符合条件的突变菌株,分别命名为△R02、△R03、△R04、△R05。将上述4株突变菌株提取基因组,PCR扩增malR基因并进行测序,分析malR基因序列的突变位点信息。将此4个malR突变基因命名为mut01malR、mut02malR、mut03malR、mut04malR。上述突变体菌株的荧光强度测定结果见图3,MalR突变体的序列如SEQ ID NO:2-SEQ ID NO:5所示。所使用的寡核苷酸如表1所示。从图3结果可知,上述突变体在未添加麦芽糖诱导物的条件下具有更低的渗漏表达水平,在添加麦芽糖诱导物的条件下诱导表达强度明显提升,其中△R04、△R05具有更低的渗漏表达,△R03具有最高的诱导后表达强度。
序列表
<110> 中国科学院天津工业生物技术研究所
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