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【摘要】：基因工程菌是通过转基因技术，定向构建重组质粒，从而达到稳定遗传，优势表达的菌株.这类菌株相对于野生菌而言，具有针对性，易培养，易繁殖，能表达特定产物等优点.如何大规模培养基因工程菌，积累目的产物是实验的最终目的，也是整个实验控制过程的难点和重点.本实验通过对基因工程菌的验证，以及在单一条件（如温度，补料速度等）改变的情况下，大肠杆菌生长情况的比对，初步得出大肠杆菌最适发酵条件.流程如下：（1）重组菌通过质粒提取，酶切，电泳图谱的分析，挑选出构建成功的重组大肠杆菌.（2）通过培养条件的改变，优化其最适生长条件.（3）合适的诱导，促使目的基因最大化存在与表达.（3）离心收集菌种，为进一步的纯化做准备.本实验通过对生长条件的优化，可以最大程度的发挥基因工程菌的优势，也能够使发酵生产效益最大化，就其中几个单一因素进行了优化，初步得出：温度37℃情况下，接种2 mL作为二级种子活化量，在补料pH为6.9，诱导pH为7.0时，高溶氧诱导6 h，低溶氧诱导2 h，工程菌不仅可以实现菌体的相对最大产量，在外源基因表达上也较为有利.26549
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毕业论文关键词：发酵，基因工程菌，优化
Fermentation Optimization for the Recombinant Escherichia coli with Enterokinase gene
【Abstract】：Genetically engineered bacteria are genetically engineered to construct the recombinant plasmid, and thus to achieve the stable heredity and the dominant strains.This strain has the advantages of targeting, easy to culture, and easy to breed, and it can express certain products, etc... How to cultivate genetically engineering bacteria, accumulate the target product is the ultimate goal of the experiment, but also the difficulty and the key of the experiment control process.. Through the experimental verification of gene engineering bacteria, as well as in the single condition (such as temperature, feeding rate), comparison of the growth of Escherichia coli. The results showed that Escherichia coli were the most optimum fermentation conditions. The process is as follows: (1) recombinant bacteria were extracted by plasmid, and analyzed by enzyme digestion and electropHoresis, and the recombinant E. coli was selected to construct the recombinant E. coli. (2) By changing the conditions of the cultivation, optimizing the optimum conditions for its optimum growth. (3) The right induction, the maximum of the purpose of the gene to promote the existence and expression of gene. (3) The centrifugal collection is prepared for further purification. This experiment through optimization of the growth conditions can be the greatest degree of play of gene engineering bacteria advantage, but also to maximize the efficiency of fermentation production, among them a few single factors were optimized, the preliminary conclusions can be drawn: under temperature of 37℃, inoculated with 2 ml as secondary seed volume of activation, filling material in pH 6.9 and induced pH 7.0, high dissolved oxygen induced 6h, 2h induced by low dissolved oxygen, engineering bacteria can not only achieve maximum relative yield of cell and on the expression of exogenous gene is also more favorable.
Keywords: Fermentation, Gene engineering bacteria, Optimization
目录
引言 1
1. REK大肠杆菌的鉴定 2
1.1 仪器和试剂 2
1.2 REK重组质粒提取流程 3
1.3 REK重组质粒酶切流程（70ul体系）： 4
1.4 REK 重组大肠杆菌质粒电泳: 4
2.REK大肠杆菌的发酵培养 4
2.1仪器和试剂 4
2.2发酵前准备： 7
2.3发酵操作流程 7
2.4 收集菌种 8