维生素C混合发酵中产酸菌基因文库的构建  

CONSTRUCTION OF GENOMIC LIBRARY OF GLUCONOBACTER OXYDANS SCB329 IN THE L-SORBOSE UTILIZING MIXED FERMENTATION

作　　者：刘娟[1] 尹光琳[1] 

机构地区：[1]中国科学院上海生物工程研究中心,上海200233

出　　处：《微生物学通报》1999年第5期314-319,共6页Microbiology China

摘　　要：在对氧化葡萄糖酸杆菌ＳＣＢ３２９的纯培养方法进行了探索并获得了一定量的纯培养的ＳＣＢ３２９菌体的前提下，用常规方法抽提得到Ｇｌｕｃｏｎｏｂａｃｔｅｒ　ｏｘｙｄａｎｓ　ＳＣＢ３２９染色体ＤＮＡ。选用质粒ｐＫＳ作为载体，该载体具有氨苄抗性以及ｌａｃＺ基因．用限制酶对染色体进行部分消化，将一定范围内的消化片段回收后与载体连接，连接产物转化Ｅ.ｃｏｌｉＤＨ５ａ感受态细胞，利用蓝白斑特性选出重组子构建ＳＣＢ３２９基因组文库。用低熔点琼脂糖将ＳＣＢ３２９菌体包埋起来，对包埋在凝胶块中的细菌菌体进行细胞裂解、去蛋白等操作，防止染色体因机械剪切作用而断裂，以测出ＳＣＢ３２９染色体的完整长度。本工作为进行菌株改造以及构建以Ｌ－山梨糖为底物发酵产生维生素Ｃ前体２－酮基－Ｌ－古龙酸的基因工程菌株打下基础。Improvements were made on pure culture of Gluconobacter oxydans SCB329 and certainquantities of bacteria were obtained. Chromosome DNA of SCB329 was extracted by common method.PlasmidpKS was used as vector, on which there are one Amp marker gene and one lacZ gene. The total chromosomeDNA was partially digested by restricted endonuclease. Fragments of certain size were recovered from gel toligate with the vector. Ligation mixture was used to transform DH5a competant cell, with the result of largequantities of positive transformants. A genomic library can be constructed. Bacterial cells of SCB 329 wereburied in low melting-point agarose gel. Lysis and elimination of proteins were performed to the cells buried inthe gel. Prevented from breaking under mechanic forces, complete chromosome size can be determined.

关 键 词：维生素C 氧化 葡萄糖酸杆菌 基因文库 

分 类 号：Q939.11[生物学—微生物学] TQ924[轻工技术与工程—发酵工程]