L.paracasei HD1.7 prcR定位突变及pMG36e对该菌的转化
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摘要
Lactobacillus paracasei HD1.7是本实验室在2003年从乳酸酸菜发酵液中分离出的一株乳酸杆菌。在对其的研究中发现,该菌的发酵液中含有一种能够抑制多种革兰氏阳性菌,革兰氏阴性菌以及酿酒酵母的肽类物质,且该类物质的生成具有群体效应的特征。Jiro Nakayama et al.(2003)在L.paracasei E93490基因中发现了群体效应组件的身影,并阐明其信号分子可能具有抗菌活性。由此推测,在L.paracasei HD1.7中也有可能存在与L.paracasei E93490相同或相似的情况。至今为止,人们对L.paracasei该方面知之甚少,所以本研究具有非常重要的意义。
参考现有的文献数据,本实验首先要通过PCR方法对实验菌株中的拟群体效应反应调节因子编码基因prcR的存在情况进行摸索,证实了该基因的存在。然后利用现代的生物信息学分析手段,对该基因片段的核苷酸组成,蛋白质构象等方面进行预测和对比分析,找寻prcR与其它已报道的反应调节子之间的联系。
其次,以窄宿主范围的pUC18质粒为主体,构建可用于插入失活实验的自杀质粒pUC18-prcR-tet。在该质粒上,引入了四环素抗性基因做筛选标记,其上下游均有大约400bp的prcR片段作为同源重组的途径。
电转化条件的摸索是将自杀质粒导入实验菌株的重要一环。本实验从感受态细胞的处理与制备,电击电压的大小以及复苏培养基的选择等方面对L.paracasei HD1.7电转化情况进行了探索,获得了最佳的电转条件:用含1%甘氨酸的MRS培养基培养的菌体,在A600为0.78时,经AEB1电转缓冲液处理后,在9kV/cm电压下完成电击,并迅速加入含10%蔗糖的高渗培养基中,复苏2~3h,即可达到最佳的电转化效果。
最后,根据以上几方面的数据,通过电转化的途径,将自杀质粒pUC18-prcR-tet导入L.paracasei HD1.7,以期获得prcR基因敲除菌株。对能在Tet平板上生长的菌株进行PCR验证。
Lactobacillus paracasei HD1.7 was isolated from Chinese sauerkraut juice in 2003. It had been proved that the strain can produce non-acid antimicrobial substance. It was a kind of peptide which had broad spectrum of inhibition. At the same time, it was found that the process of this peptide production showed the feature of quorum-sensing ,just like the others in Gram-positive bacteria. Jiro Nakayama et al.
(2003)got a series of genes (prcA、prcK、prcR and prcB) which was defined the putitive quorum-sensing components. They predicted that quorum-sensing system was related with the production of antibacterial peptide, and regulated by PrcR. Just like that, the similar condition was supposed in L.paracasei HD1.7 here. A lot of data in this study would supported the idea and this work will be very significant because the data in this field was few.
First of all, according to the data of NCBI, the presence of prcR in L.paracasei HD1.7 was examined by PCR, using the primers designed by ourselves. Then, the information of prcR was revealed and forecasted by bioinformatics method.
Secondly, the suicide plasmid, pUC18-prcR-tet, was constructioned by inserting a tetracycline-resistant gene into prcR, whose single site was breakaged by enzymatic digestion of MunI. It was the preparation to getting the mutant strain of prcR deletion by gene knock-out technique.
Electrotransformation technique was introducted because it had high effect of transformation and suited all kinds of bacteria. Meanwhile, this technology was influenced by many factors, such as the method of competent cell preparation. So the optimal conditions were researched here. When the recipient cell was treated by 1% of glycine and washed by electrotransformation buffer of AEB1, as well as the 9 kV/cm of field strength and one time of pulse with the A600 value of 0.78, the best result could been got.
Eventually, the pUC18-prcR-tet plasmid was imported to the L.paracasei HD1.7. And the mutant strain wad screened by cultureing with tetracycline. PCR would be done to exmain the result.
参考现有的文献数据,本实验首先要通过PCR方法对实验菌株中的拟群体效应反应调节因子编码基因prcR的存在情况进行摸索,证实了该基因的存在。然后利用现代的生物信息学分析手段,对该基因片段的核苷酸组成,蛋白质构象等方面进行预测和对比分析,找寻prcR与其它已报道的反应调节子之间的联系。
其次,以窄宿主范围的pUC18质粒为主体,构建可用于插入失活实验的自杀质粒pUC18-prcR-tet。在该质粒上,引入了四环素抗性基因做筛选标记,其上下游均有大约400bp的prcR片段作为同源重组的途径。
电转化条件的摸索是将自杀质粒导入实验菌株的重要一环。本实验从感受态细胞的处理与制备,电击电压的大小以及复苏培养基的选择等方面对L.paracasei HD1.7电转化情况进行了探索,获得了最佳的电转条件:用含1%甘氨酸的MRS培养基培养的菌体,在A600为0.78时,经AEB1电转缓冲液处理后,在9kV/cm电压下完成电击,并迅速加入含10%蔗糖的高渗培养基中,复苏2~3h,即可达到最佳的电转化效果。
最后,根据以上几方面的数据,通过电转化的途径,将自杀质粒pUC18-prcR-tet导入L.paracasei HD1.7,以期获得prcR基因敲除菌株。对能在Tet平板上生长的菌株进行PCR验证。
Lactobacillus paracasei HD1.7 was isolated from Chinese sauerkraut juice in 2003. It had been proved that the strain can produce non-acid antimicrobial substance. It was a kind of peptide which had broad spectrum of inhibition. At the same time, it was found that the process of this peptide production showed the feature of quorum-sensing ,just like the others in Gram-positive bacteria. Jiro Nakayama et al.
(2003)got a series of genes (prcA、prcK、prcR and prcB) which was defined the putitive quorum-sensing components. They predicted that quorum-sensing system was related with the production of antibacterial peptide, and regulated by PrcR. Just like that, the similar condition was supposed in L.paracasei HD1.7 here. A lot of data in this study would supported the idea and this work will be very significant because the data in this field was few.
First of all, according to the data of NCBI, the presence of prcR in L.paracasei HD1.7 was examined by PCR, using the primers designed by ourselves. Then, the information of prcR was revealed and forecasted by bioinformatics method.
Secondly, the suicide plasmid, pUC18-prcR-tet, was constructioned by inserting a tetracycline-resistant gene into prcR, whose single site was breakaged by enzymatic digestion of MunI. It was the preparation to getting the mutant strain of prcR deletion by gene knock-out technique.
Electrotransformation technique was introducted because it had high effect of transformation and suited all kinds of bacteria. Meanwhile, this technology was influenced by many factors, such as the method of competent cell preparation. So the optimal conditions were researched here. When the recipient cell was treated by 1% of glycine and washed by electrotransformation buffer of AEB1, as well as the 9 kV/cm of field strength and one time of pulse with the A600 value of 0.78, the best result could been got.
Eventually, the pUC18-prcR-tet plasmid was imported to the L.paracasei HD1.7. And the mutant strain wad screened by cultureing with tetracycline. PCR would be done to exmain the result.
引文
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