碩士 / 國立臺灣海洋大學 / 食品科學系 / 96 / Site-directed mutagenesis is a very important method in the study of the structure-function relationships of proteins. The QuikChange site-directed mutagenesis (QCM), which was developed by Stratagene, is one of the most widely used in site-directed mutagenesis. However, most of the amplified products are linear and blunt-end products that are incapable of yielding any transformants, for this reason, we attempt to improve this method. We used pMAL-p2X plasmid as a working plasmid, and introduce a stop codon in the internal of the malE gene and also could disrupt lacZα expression. In light of the megaprimer method, one mutagenic primer and one universal flanking primer were used to produce the megaprimers, and then whole mutated plasmid were extended as original QCM method. At last, we isolated the mutant by the blue-white screen following the transformation. The megaprimed QCM method yields a mutagenesis efficiency about 90% similar to that of QCM but uses only one mutagenic oligonucleotide instead of two of them. Moreover, we applied this method to multiple site-directed mutagenesis further, and the result reveals that the mutagenic efficiencies of quadruple and sextuple mutation is 80 and 40%, respectively.
Maltooligosyltrehalose trehalohydrolase (MTHase) will hydrolyze maltooligosyltrehalose to produce trehalose. The thermophile archaebacteria Sulfolobus acidocaldarius and Sulfolobus solfataricus both can produce the thermostable MTHase that is encoded by the treZ gene. Their nucleic acid sequences were 68% identical, we attempt to recombine these two gene. The pET-21b-SStreZ and pET-21b-SAtreZ recombinant plasmid were used as template DNA during recombination. The directed evolution was carried out by using Staggered extension process (StEP) to obtain the recombinant MTHase gene, and then the recombinant genes were transformated into E. coli Rosetta (DE3) after inserting into an expression vector pET21b, and the transformants were screened with activity test finally. However, we did not obtain any recombinant that recombined successfully and retained the MTHase activity.
| Identifer | oai:union.ndltd.org:TW/096NTOU5253070 |
| Date | January 2008 |
| Creators | Jing-Wei Lin, 林敬瑋 |
| Contributors | Tsuei-Yun Fang, 方翠筠 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 104 |
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