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DNA transformation and fermentation study of Acremonium chrysogenum.

Lau, Shong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Abstract of thesis --- p.i / 碩士論文摘要 --- p.iii / Acknowledgement --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Genetic symbols --- p.viii / Table of content --- p.ix / List of figures --- p.xiii / List of tables --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Thesis outline --- p.1 / Chapter 1.2 --- A. chrysogenum --- p.3 / Chapter 1.3 --- Antibiotic industry --- p.4 / Chapter 1.4 --- Cephalosporins --- p.4 / Chapter 1.5 --- CPC biosynthetic pathway --- p.5 / Chapter 1.6 --- DAC --- p.8 / Chapter 1.7 --- Aims of study --- p.10 / Chapter Chapter 2 --- Construction of transformation cassettes --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.2 --- Materials and Methods --- p.13 / Chapter 2.2.1 --- Construction of cassette RTCAH --- p.13 / Chapter 2.2.1.1 --- Cultivation of R. toruloides --- p.13 / Chapter 2.2.1.2 --- Preparation of R. toruloides genomic DNA --- p.13 / Chapter 2.2.1.3 --- PCR of R. toruloides CAH gene --- p.14 / Chapter 2.2.1.4 --- Construction of pRTCAHhyr --- p.16 / Chapter 2.2.2 --- Construction of cassette GHG --- p.17 / Chapter 2.3 --- Results and Discussion --- p.18 / Chapter 2.3.1 --- pGHG construction --- p.18 / Chapter 2.3.2 --- pRTCAHhyr construction --- p.18 / Chapter Chapter 3 --- Optimization of integrative transformation of A. chrysogenum --- p.22 / Chapter 3.1 --- Introduction --- p.22 / Chapter 3.2 --- Materials and Methods --- p.24 / Chapter 3.2.1 --- Strain and culture medium --- p.24 / Chapter 3.2.2 --- Reagents --- p.24 / Chapter 3.2.3 --- Standard transformation procedures --- p.25 / Chapter 3.2.3.1 --- Cell cultivation --- p.25 / Chapter 3.2.3.2 --- Protoplast preparation --- p.25 / Chapter 3.2.3.3 --- PEG mediated protoplast fusion --- p.27 / Chapter 3.2.4 --- Examination of transformation parameters --- p.28 / Chapter 3.3 --- Results and Discussion --- p.29 / Chapter 3.3.1 --- Cell growth period --- p.30 / Chapter 3.3.2 --- DNA concentration --- p.32 / Chapter 3.3.3 --- PEG molecular weight --- p.35 / Chapter 3.3.4 --- Transformation additives --- p.37 / Chapter 3.3.5 --- Modified transformation protocol --- p.39 / Chapter Chapter 4 --- Metabolic engineering of A. chrysogenum --- p.42 / Chapter 4.1 --- Introduction --- p.42 / Chapter 4.2 --- Materials and Methods --- p.43 / Chapter 4.2.1 --- Transformation of A. chrysogenum --- p.43 / Chapter 4.2.2 --- Screening of transformants --- p.43 / Chapter 4.2.3 --- HPLC analysis --- p.43 / Chapter 4.2.4 --- A. chrysogenum genomic DNA preparation --- p.44 / Chapter 4.2.5 --- Genotyping by PCR --- p.45 / Chapter 4.2.5.1 --- GHG transformants --- p.45 / Chapter 4.2.5.2 --- RTCAH transformants --- p.47 / Chapter 4.2.6 --- Genotyping of GHG transformants by Southern hybridization --- p.47 / Chapter 4.2.6.1 --- Preparation of DIG-labeled GHG probe by PCR --- p.48 / Chapter 4.2.6.2 --- PCR preparation of DIG-labeled Cef-EF probe --- p.48 / Chapter 4.2.6.3 --- Restriction digestion of genomic DNA of GHG transformants --- p.49 / Chapter 4.2.6.4 --- Agarose gel electrophoresis and DNA transfer to positively charged nylon membrane --- p.50 / Chapter 4.2.6.5 --- Pre-hybridization and hybridization --- p.52 / Chapter 4.2.6.6 --- Membrane washing and blocking --- p.52 / Chapter 4.2.6.7 --- Membrane detection --- p.53 / Chapter 4.2.7 --- Genotyping of RTCAH transformants by Southern hybridization --- p.53 / Chapter 4.2.7.1 --- PCR preparation of DIG-labeled RTCAH probe --- p.54 / Chapter 4.2.7.2 --- Restriction digestion of genomic DNA of RTCAH transformants --- p.54 / Chapter 4.2.7.3 --- Agarose gel electrophoresis and Southern hybridization of RTCAH transformants --- p.55 / Chapter 4.3 --- Results and Discussion --- p.56 / Chapter 4.3.1 --- Hygromycin B screening and HPLC analysis of GHG transformants --- p.56 / Chapter 4.3.2 --- Genotyping of GHG232 by PCR --- p.56 / Chapter 4.3.3 --- Genotyping of GHG232 by Southern hybridization --- p.58 / Chapter 4.3.4 --- Hygromycin B screening and HPLC analysis of RTCAH transformant --- p.61 / Chapter 4.3.5 --- Genotyping of RTCAH transformants by PCR --- p.61 / Chapter 4.3.6 --- Genotyping of RTCAH transformants by Southern hybridization --- p.63 / Chapter Chapter 5 --- Characterization of modified strains and fermentation study --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- Materials and Methods --- p.67 / Chapter 5.2.1 --- Comparison of DAC production in GHG232 and RTCAH transformants by small-scale fermentation --- p.67 / Chapter 5.2.2 --- CPC conversion assay --- p.67 / Chapter 5.2.3 --- Evaluation of fermentation media --- p.68 / Chapter 5.2.4 --- Factorial design for medium formulation --- p.68 / Chapter 5.3 --- Results and Discussion --- p.71 / Chapter 5.3.1 --- Evaluation of DAC production profiles of GHG232 and RTCAH transformants --- p.71 / Chapter 5.3.2 --- CPC conversion assay --- p.79 / Chapter 5.3.3 --- Medium formulation --- p.81 / Chapter 5.3.4 --- Factorial design for medium formulation --- p.85 / Chapter 5.3.4.1 --- CSL and NH4OAc --- p.85 / Chapter 5.3.4.2 --- CaC03 --- p.91 / Chapter 5.3.4.3 --- Sucrose --- p.94 / Chapter 5.3.4.4 --- Starch and soy oil --- p.97 / Chapter 5.3.4.5 --- Methionine --- p.99 / Chapter Chapter 6 --- Conclusive remarks --- p.101 / Appendix: Study of reusability of commercial plasmid extraction kit --- p.103 / Chapter A1 --- Introduction --- p.103 / Chapter A2 --- Materials and Methods --- p.105 / Chapter A2.1 --- Preparation of competent E. coli DH5a cells --- p.105 / Chapter A2.2 --- Transformation and cultivation ofE. coli DH5a cells --- p.106 / Chapter A2.3 --- Column and solutions for plasmid DNA preparation --- p.107 / Chapter A2.4 --- Plasmid preparation --- p.108 / Chapter A2.5 --- Regeneration and storage of DNA extraction column --- p.109 / Chapter A2.6 --- Yield and quality assessment of the prepared plasmid DNA --- p.109 / Chapter A3 --- Results and Discussion --- p.111 / Chapter A3.1 --- Plasmid DNA yield and purity comparison --- p.111 / Chapter A3.2 --- Column regeneration and EtOH storage --- p.111 / Chapter A3.2.1 --- DNA yield after column regeneration --- p.111 / Chapter A3.2.2 --- Purity of plasmid DNA --- p.112 / Chapter A3.2.3 --- Restriction digestion --- p.117 / Chapter A3.2.4 --- E. coli DH5α cells transformation --- p.121 / Chapter A4 --- Conclusive remarks --- p.123 / Bibliography --- p.124

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326031
Date January 2007
ContributorsLau, Shong., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, 1, xv, 129 leaves : ill. ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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