The study aims to identify the strategic considerations of genetically engineered foods on the micro-, market-, and macro-environment of business and to make recommendations to the biotechnology industry on how to strategically manage the issues surrounding genetically engineered foods. Plants and animals have been selectively bred for centuries to create hybrid strains containing favourable traits of both plants and animals. Plant biotechnology is an extension of this traditional plant breeding. Plant biotechnology allows for the transfer of a greater variety of genetic information in a more precise, controlled manner. Genetic engineering allows for the manipulation of gene(s) to include novel and new traits or even to exclude bad or unwanted traits. Genetic cloning, a sub-discipline of genetic engineering creates the ability to clone a single gene, many genes or even complete organisms and live forms to ensure crops or herds of superior value and quality. These evolutionary steps of genetic engineering have created many new skills and abilities that could possibly revolutionise the business environment at all the levels. / Prof. N. Lessing

No description available.

A dissertation Submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Health Sciences at the University of the Witwatersrand, Johannesburg, South Africa. November 2014 / The genome is comprised of a simple quaternary code that serves as the “software” which governs all the complex processes of life. The successful manipulation of this code holds enormous potential for applications involving genome engineering. However, the tools needed to navigate the complex landscape of the genome and efficiently and precisely introduce engineered modifications are lacking. The clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system found in prokaryotes functions to recognise and silence foreign pathogenic DNA by double-stranded break (DSB) DNA digestion. The type II CRISPR system of Streptococcus pyogenes has recently been reconstituted to function in mammalian cells as a highly programmable and efficient gene-targeting nuclease platform. Through the heterologous expression of the CRISPR associated (Cas) 9 endonuclease and a small single-guide RNA (sgRNA) molecule, specific gene sequences can be targeted for a DNA DSB. The repair of this targeted DNA damage can be exploited to mutate gene sequences or reconstitute break sites according to a homologous repair template for precise gene modifications. In this study, the capabilities of the CRISPR/ Cas9 system for genome editing was tested by using it to precisely mutate the KRAS proto-oncogene. A panel of five KRAS targeting sgRNAs were designed around a common G>T mutation in codon 12 and characterised. The ability of the CRISPR/ Cas9 system to stimulate DSBs in this region was assessed using the Surveyor Assay, which is able to detect the non-homologous end joining repair of these breaks. A maximum cleavage activity of 6.01 and 2.74% was detected, upstream and downstream of the mutation site, respectively. Simultaneous cleavage by these two sgRNAs was able to successfully introduce a locus-specific micro-deletion. The homology directed repair of these DSBs according to a 90-mer single-stranded oligodeoxynucleotide repair template was shown with the RFLP assay. Analysis of these results implicated guide sequence composition and DSB repair pathway bias as potential factors which may affect the efficiency of desired gene-editing outcomes. These characterised sgRNAs were then applied to generate selectable G>T KRAS mutants. A “dual-cut” strategy, which was designed to overcome gene conversion limitations was employed, and the outcomes were measured with qPCR. The results show that 0.123% of transfected cells were successful recombinants, demonstrating that the use of a “dual-cut” strategy with the CRISPR/Cas9 system was functional and efficient for the generation of knock-in mutants. The CRISPR/Cas9 system has proven to be efficient and robust in modifying the human KRAS locus in various ways. With its modularity and simplicity, CRISPR/Cas9 is a powerful tool that will allow for the modification and interrogation of gene function.

DCUN1D1 is an E3 ligase of the neddylation pathway. It mediates the posttranslational modification of majority of the cullin family of proteins with NEDD8. This activity is known to enhance ubiquitination of the cullin RING E3 ligases, however, the extent of the impact of DCUN1D1's activity is underexplored. Studies performed previously in our lab demonstrated the role of DCUN1D1 in prostate cancer in vitro and in vivo. We also identified potential inhibitors of DCUN1D1 which inhibited the proliferation of prostate cancer cells in a DCUN1D1-specific manner. This study seeks to determine the mechanism of action of DCUN1D1 in prostate cancer and to identify DCUN1D1 inhibitors using a proteomics approach. Immunoprecipitation-coupled mass spectrometry was performed to identify DCUN1D1 binding partners and we identified some known substrates of DCUN1D1 in the form of cullin 3, cullin 4B and cullin 5. We also observed that the DCUN1D1 pulldown products implicated the ubiquitin proteasome pathway, transcription, lipid metabolism and inflammatory pathways. SILAC quantitative proteomics analysis was also performed to determine the proteins that were differentially expressed in DU145 DCUN1D1 knockdown cells relative to DU145 control cells. Interestingly, we did not identify the cullin proteins or classical components of the neddylation pathway but identified the ubiquitin activating enzyme, UBA1. We also found that dysregulation of DCUN1D1 in prostate cancer led to a dysregulation in translation-related and protein processing activities such as dysregulation of eukaryotic protein translation, and protein processing in the endoplasmic reticulum. We also observed the recurrence of the WNT signalling pathway across the proteomics approaches. This culminated in the exploration of the mechanism of action of DCUN1D1 in prostate cancer using changes in protein expression as measured by western blot analysis. Significantly, we determined that DCUN1D1 mediates its mechanism of action in prostate cancer, through the neddylation pathway and preferential neddylation of cullin proteins. We also observed that knockdown of DCUN1D1 in prostate cancer led to the dysregulation of the ubiquitination and WNT/β-catenin pathways. Furthermore, advanced connectivity map analysis was performed to identify potential inhibitors of DCUN1D1 based on a proteomics approach. The drugs found to strongly connect with the DCUN1D1 knockdown signature included kinase inhibitors and anti-inflammatory agents. The above observations could lead to improved understanding of DCUN1D1 and its potential for molecular target based treatment of prostate cancer.

The broad objective of this research was to develop transgenic poinsettia that express tryptophan decarboxylase (TDC) capable of protecting poinsettia against whitefly. An effective and efficient in vitro micro propagation and proliferation technique of poinsettia ‘Prestige Red’ was successfully developed in this study and this protocol can be used for potential development of transgenic poinsettia. Poinsettia ‘Prestige Red’ was successfully infected by Agrobacterium rhizogenes producing hairy roots at the site of infection. Investigations of more effective PGR concentrations are necessary in order to develop transgenic poinsettia through hairy roots. Stem disks of poinsettia ‘Eckespoint Pollys Pink’ developed into somatic embryos when they were transformed by A. tumefaciens harboring TDC. A. tumefaciens-mediated transformation of poinsettia through somatic embryogenesis is cultivar dependent. Additional research into more effective PGR combinations, antibiotic concentrations and antinecrosis chemicals is required in order to develop transgenic poinsettia harboring TDC through somatic embryogenesis using A. tumefaciens.

Dissertation (PhD(Agric))--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Considerable efforts have been made to improve strains of the wine yeast Saccharomyces cerevisiae through the use of genetic engineering. Although the process is well defined, globally there is much resistance towards the use of genetically modified organisms (GMOs), primarily because little is known about their environmental fate and their potential effect on naturally occurring organisms. The public concern is mainly focused on the uncertainty associated with the impact of the deliberate or accidental release of a GMO into the environment. As a consequence, thére is an urgent need to assess the potential risks involved with the use of this new technology. For the eventual global acceptance of any GMO, it is imperative that the consumer must be convinced that it is ultimately safe for human consumption and the environment. In order to achieve this, certain risk assessment procedures must be performed on each and every GMO that is planned to be released into the environment. Although some of the genetically modified (GM) yeasts that have been developed comply with the strict legislation of most countries and have been cleared by regulatory authorities for commercial use, GM yeasts have not, as yet, been used for the commercial production of GM bread, beer or wine. Nevertheless, the use of GM yeasts in the market appears imminent and there is an urgent need to assess and address the perceived health and environmental risks associated with GM foods. The overall objective of this research was to evaluate key environmental issues concerning the use of GM yeasts. The focus was on comparing the behaviour of specific parental and GM yeast strains in model systems in order to determine whether the GM strains may have any selective advantage, which could lead to their spreading. Specifically, it involved monitoring of the growth behaviour of selected GM yeasts within a vineyard microbial community and in fermentations, as well as the interaction of these yeasts with sand and glass surfaces in an aqueous environment. The GM yeasts under investigation were recombinant strains of a well-known, industrial strain of S. cerevisiae VIN13 expressing an a-amylase (designated GMY1); an endo-p-1,4-glucanase and endo-p-xylanase (designated GMY2); and a pectate lyase and polygalacturonase (designated GMY3). The GM yeasts were mist-inoculated onto individually-contained blocks consisting of one-year old grapevines in a secluded glasshouse environment. Specifically, the numbers and dynamics of GM yeast survival, as well as the effect of an introduced GM yeast on the yeast community dynamics and numbers, were investigated. Overall, it was found that the most prevalent wild yeasts isolated from the grapevines were Rhodo torula, Yarrowia lipolytica, Pichia and Candida spp. VIN13 and the GM yeasts did not affect the overall ecological balance of the microflora on the grapevines. Wild strains of S. cerevisiae were seldom isolated from the grapevines. With a few exceptions, the overall detection of GM yeasts was numerically limited. Co-inoculation of (VIN13+GMY1) and (GMY1+GMY2) revealed detection approximately in the same ratio at which they were inoculated, with small differences in the order of GMY2>GMY1 >GMY3. GM yeasts were rarely isolated from bark and soil samples. Spontaneous fermentation of the grapes harvested from the different treated blocks indicated that the GM yeasts survived on the berries, that the natural fermenting ability of VIN13 was conserved in the recombinant strains, and that the GM yeasts did not have any competitive advantage. The soil environment forms an important part of the biosphere and the transport and attenuation of a GM yeast in this matrix will to a large extent affect their ultimate fate in the environment. In soil, microorganisms either occur as suspended cells in pore water or as biofilms on soil surfaces. Although less extensive than a typical soil yeast, Cryptococcus, epifluorescent staining of biofilms confirmed that VIN13 and GMY1 were capable of existing in a biofilm mode on sand granules and glass. Data on effluent numbers detected in flow cells indicated that GMY1 had no advantage due to the genetic modification and had the same reproductive success as VIN13. These strains either had no difference in biofilm density or GMY1 was less dense than VIN13. When co-inoculated, GMY1 had no negative influence on the mobility of Cryptococcus through a sand column, as well as the ability of Cryptococcus to form biofilms. Furthermore, it was found that GMY1 did not incorporate well into a stable biofilm community on glass, but did not disrupt the biofilm community either. This is the first report of the assessment of the fate of GM strains of VIN13 that are suitable for the wine and baking industry. The investigation of the GM yeasts in this study under different scenarios is a good start to an extensive and necessary risk assessment procedure for the possible use of these GM yeasts in the industry. This study could lead to the provision of much-needed scientific and technical information to both industry and regulating bodies. The outcome of this research is also intended to serve as a basis for information sharing with public interest groups. / AFRIKAANSE OPSOMMING: Aansienlike pogings is reeds aangewend om rasse van die wyngis, Saccharomyces cerevisiae, deur middel van genetiese manipulering te verbeter. Alhoewel hierdie proses goed gedefinieerd is, is daar wêreldwyd heelwat teenkanting teen die gebruik van geneties gemanipuleerde organismes (GMO's). Dit is hoofsaaklik te wyte daaraan dat so min bekend is oor hul lot in die omgewing en hul potensiële effek op die organismes wat natuurlik voorkom. Die publiek is veral besorg oor die onsekerheid verbonde aan die bestemde of toevallige vrylating van 'n GMO in die omgewing. Gevolglik is daar 'n dringende behoefte om die potensiële risiko's in die gebruik van hierdie nuwe tegnologie te bepaal. Dit is van uiterste belang dat die verbruiker oortuig sal word van die veiligheid vir menslike gebruik en die omgewing voordat enige GMO uiteindelik wêreldwyd aanvaarbaar sal word. Om dit te kan bereik sal sekere risiko-bepalende prosedures toegepas moet word op ieder en elke GMO wat beplan word om vry gelaat te word in die omgewing. Alhoewel sommige van die geneties gemanipuleerde (GM) giste aan die streng wetgewing van die meeste lande voldoen en deur die owerhede vir kommersiële gebruik goedgekeur is, word GM-giste nog steeds nie vir die produksie van GM brood, bier of wyn gebruik nie. Ten spyte hiervan, blyk die gebruik van GM-giste onafwendbaar te wees en is daar dus 'n dringende behoefte om die voorspelde gesondheids- en omgewingsrisiko's wat met GM voedsel geassosieer word, aan te spreek. Die oorhoofse doel van hierdie navorsing was om belangrike omgewingskwessies aangaande die gebruik van GM-giste te evalueer. Die fokus was op die vergelyking van die gedrag van spesifieke oorspronklike gisrasse en GM-gisrasse in modelsisteme sodat daar bepaal kon word of die GM-gisrasse enige selektiewe voordele het wat moontlik tot hulonbeheerde verspreiding in die natuur sou kon lei. Dit het spesifiek die monitering van die groei van geselekteerde GMgiste binne 'n mikrobiese gemeenskap op wingerd en in fermentasies behels, asook die interaksie van hierdie giste met grond en glas oppervlaktes in 'n wateromgewing. Die GM-giste wat in hierdie studie gebruik is, was rekombinante rasse van 'n bekende industriële ras van S. cerevisiae, VIN13, wat geneties gemodifiseerd was om 'n a-amylase (aangedui as GMG1); 'n endo-p-1,4-glukanase en 'n endo-B-xilanase (aangedui as GMG2); en 'n pektaatliase en 'n poligalaktorinase (aangedui as GMG3) uit te druk. Die GM-giste is op afsonderlike blokke van eenjaaroue wingerdplante binne-in 'n beskutte kweekhuis gesproei-inokuleer. Daar was spesifiek na die selgetalle en dinamika van die oorlewende GM-giste gelet, asook wat die invloed was van die inokulasie van 'n GM gis op die selgetalle van die natuurlike gisgemeenskap. Daar is bevind dat die wildegiste Rhodotorula, Yarrowia Iipolytica, Pichia en Candida spp die gereeldste van die wingerd geïsoleer is. VIN13 en die GM-giste het nie die ekologiese balans van die natuurlike mikrobiese populasie op die wingerd versteur nie. Wilde rasse van S. cerevisiae is selde geïsoleer vanaf die wingerd. In die meeste gevalle is daar bevind dat wanneer GM-giste opgespoor is, hulle in lae selgetalle voorgekom het. Waar giste saam geïnokuleer was, was die opsporing van (VIN 13+GMY1) en (GMY1 +GMY2) ongeveer in dieselfde verhouding as waarin hul geïnokuleer was, terwyl klein verskille in die orde van GMY2>GMY1 >GMY3 opgemerk is. GM-giste is selde vanaf bas- en grond-monsters geïsoleer. Spontane fermentasies van druiwe wat geoes vanaf die verskillende behandelde blokke is, het daarop gedui dat die GM-giste wel op die druiwe oorleef, dat die natuurlike vermoë van VIN13 om te kan fermenteer in die gemodifiseerde gisrasse behoue gebly het en dat die GM-giste geensins deur die genetiese modifikasies bevoordeel was nie. Grond is 'n belangrike deel van die biosfeer en die verspreiding en aanhegting van 'n GM-gis in hierdie matriks sal sy algehele lot in die omgewing tot 'n groot mate beïnvloed. In die grond kom mikroorganismes as gesuspendeerde selle in poriewater of as biofilms op die oppervlaktes van grond voor. Alhoewel biofilmvorming van VIN13 en GMG1 swakker was as in die geval van 'n tipiese grondgis, Cryptococcus, het epifluoresserende kleuring van hierdie S. cerevisiaegiste bevestig dat VIN13 en GMG1 in staat was om as biofilms op sandkorrels en glas te oorleef. Gebaseer op seltellings in vloeiseluitlaat, kon daar afgelei word dat GMG1 geen selektiewe voordeel geniet het as gevolg van die genetiese modifikasie nie en dat die gis net so reproduktief was as VIN13. Hierdie gisrasse het geen verskil in biofilmdigtheid getoon nie of die biofilmvorming van GMG1 was minder dig as die van VIN13. Wanneer GMG1 saam met Cryptococcus geïnokuleer was, het GMG1 geen negatiewe invloed op die beweeglikheid van Cryptococcus deur 'n sandkolom gehad nie en die vermoë van Cryptococcus om biofilms te vorm is ook nie beïnvloed nie. Daar is verder ook bevind dat GMG1 nie goed binne-in 'n gestabiliseerde biofilmgemeenskap op glas geïnkorporeer het nie, maar dat die gis ook nie die biofilmgemeenskap versteur het nie. Hierdie studie verteenwoordig die eerste ondersoek ooit oor die lot, oorlewing en groeigedrag van GM-wyngiste in biologies-afgesonderde wingerd-, fermentasie-, modelgrond- en modelwater-ekosisteme. Die bestudering van hierdie GM-giste onder verskillende omgewingstoestande in afgeslote ekosisteme lê 'n stewige basis vir verdere ondersoeke en die ontwikkeling van omvattende en noodsaaklike risikobepalingsprosedures betreffende die moontlike toekomstige gebruik van GM-giste in die industrie. Hierdie studie baan die weg tot die verkryging van noodsaaklike wetenskaplike en tegniese inligting oor die veiligheidsaspekte rakende GM-wyngiste en dit kan van groot waarde vir die industrie, owerhede en verbruikerspubliek wees.

Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total transforming DNA concentration, transforming DNA conformation, and gene dosage on transformation efficiency are presented. Addition of Acinetobacter plasmid DNA sequences to the manipulated constructs did not have an effect on transformation rates. Results suggest that a broadly applicable and efficient method to carry out site-directed genetic manipulations of large plasmids has been identified. The ability to easily reintroduce the recombinant DNA molecules back into the original host organism was maintained.

Shen Dong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 97-104). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- High fructose corn syrup (HFCS) --- p.2 / Chapter 1.1.1 --- Status quo and prospect of HFCS --- p.2 / Chapter 1.1.2 --- Industrial process for HFCS production --- p.3 / Chapter 1.1.3 --- Glucose (xylose) isomerase in industrial application --- p.5 / Chapter 1.2 --- Glucose (xylose) isomerase --- p.9 / Chapter 1.2.1 --- Source organisms --- p.9 / Chapter 1.2.2 --- Functions of glucose (xylose) isomerase --- p.12 / Chapter 1.2.3 --- Structure of glucose isomerase --- p.13 / Chapter 1.2.4 --- Catalytic mechanism of glucose isomerase --- p.17 / Chapter 1.2.5 --- Biochemical properties of glucose isomerase --- p.18 / Chapter 1.2.6 --- Immobilization studies --- p.22 / Chapter 1.3 --- Aims of my study --- p.25 / Chapter Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Cloning of parental glucose isomerase gene --- p.27 / Chapter 2.1.1 --- Materials --- p.27 / Chapter 2.1.1.1 --- Bacterial strain --- p.27 / Chapter 2.1.1.2 --- Growth media --- p.29 / Chapter 2.1.1.3 --- Antibiotics --- p.29 / Chapter 2.1.1.4 --- Reagents for isolation of chromosomal DNA --- p.30 / Chapter 2.1.1.5 --- Reagents for PCR reaction --- p.30 / Chapter 2.1.1.6 --- Reagents for agarose gel electrophoresis --- p.30 / Chapter 2.1.1.7 --- Reagents for DNA recovery from agarose gel --- p.31 / Chapter 2.1.1.8 --- Vector and enzyme for ligation --- p.31 / Chapter 2.1.1.9 --- Reagents for preparation of competent cells --- p.32 / Chapter 2.1.1.10 --- Reagents for extraction of plasmid DNA --- p.32 / Chapter 2.1.1.11 --- Reagents for DNA sequencing --- p.32 / Chapter 2.1.2 --- Methods --- p.32 / Chapter 2.1.2.1 --- Isolation of chromosomal DNA --- p.32 / Chapter 2.1.2.2 --- Preparation of primers --- p.33 / Chapter 2.1.2.3 --- Amplification of parental glucose isomerase gene --- p.33 / Chapter 2.1.2.4 --- Agarose gel electrophoresis of DNA --- p.35 / Chapter 2.1.2.5 --- DNA recovery from agarose gel --- p.35 / Chapter 2.1.2.6 --- Ligation of purified DNA fragment into vector --- p.36 / Chapter 2.1.2.7 --- Making competent cells --- p.37 / Chapter 2.1.2.8 --- Transformation / Chapter 2.1.2.9 --- Plasmid DNA preparation --- p.38 / Chapter 2.1.2.10 --- DNA sequencing --- p.39 / Chapter 2.2 --- Mutagenesis of glucose isomerase --- p.40 / Chapter 2.2.1 --- Materials --- p.40 / Chapter 2.2.2 --- Methods --- p.40 / Chapter 2.2.2.1 --- Preparation of primers --- p.40 / Chapter 2.2.2.2 --- Introduction of point mutations --- p.42 / Chapter 2.2.2.3 --- Assembly of DNA fragments --- p.44 / Chapter 2.2.2.4 --- Amplification of full-length genes --- p.45 / Chapter 2.2.2.5 --- Agarose gel electrophoresis of DNA --- p.46 / Chapter 2.2.2.6 --- DNA recovery from agarose gel --- p.46 / Chapter 2.2.2.7 --- Ligation of purified DNA fragment into vector --- p.46 / Chapter 2.2.2.8 --- Transformation --- p.46 / Chapter 2.2.2.9 --- Plasmid DNA preparation --- p.46 / Chapter 2.2.2.10 --- DNA sequencing --- p.46 / Chapter 2.3 --- Expression and purification of glucose isomerase --- p.47 / Chapter 2.3.1 --- Materials --- p.47 / Chapter 2.3.1.1 --- Phosphate buffer preparation --- p.47 / Chapter 2.3.1.2 --- Reagents for SDS-PAGE --- p.48 / Chapter 2.3.2 --- Methods --- p.48 / Chapter 2.3.2.1 --- Incubation of bacteria --- p.48 / Chapter 2.3.2.2 --- Extraction of crude protein --- p.49 / Chapter 2.3.2.3 --- Partial purification of glucose isomerase --- p.49 / Chapter 2.3.2.4 --- Further purification of glucose isomerase --- p.50 / Chapter 2.3.2.5 --- SDS-PAGE --- p.51 / Chapter 2.4 --- Enzyme assays --- p.52 / Chapter 2.4.1 --- Materials --- p.52 / Chapter 2.4.1.1 --- Substrate for activity assay --- p.52 / Chapter 2.4.1.2 --- Buffer and bivalent metal cations --- p.52 / Chapter 2.4.1.3 --- Reagents for protein concentration determination --- p.53 / Chapter 2.4.1.4 --- Reagents for activity determination --- p.54 / Chapter 2.4.2 --- Methods --- p.54 / Chapter 2.4.2.1 --- Protein concentration determination --- p.54 / Chapter 2.4.2.2 --- Specific activity assay --- p.55 / Chapter 2.4.2.3 --- Thermostability assay --- p.57 / Chapter 2.4.2.4 --- Temperature curve of activity --- p.57 / Chapter 2.4.2.5 --- pH effects --- p.57 / Chapter 2.4.2.6 --- pH stability assay --- p.58 / Chapter 2.4.2.7 --- Bivalent metal cations --- p.58 / Chapter 2.4.2.8 --- Conversion rate of isomerization --- p.59 / Chapter Chapter 3 --- Results --- p.61 / Chapter 3.1 --- Cloning of parental glucose isomerase gene --- p.62 / Chapter 3.2 --- Mutagenesis of glucose isomerase --- p.64 / Chapter 3.3 --- Expression and purification of glucose isomerase --- p.65 / Chapter 3.4 --- Enzyme assays of glucose isomerase --- p.70 / Chapter 3.4.1 --- Specific activity --- p.70 / Chapter 3.4.2 --- Thermostability --- p.72 / Chapter 3.4.3 --- Activity at different temperatures --- p.76 / Chapter 3.4.4 --- pH effects --- p.77 / Chapter 3.4.5 --- pH stability --- p.78 / Chapter 3.4.6 --- Bivalent metal cations --- p.79 / Chapter 3.4.7 --- Conversion rate of isomerization --- p.84 / Chapter Chapter 4 --- Discussions --- p.87 / Chapter 4.1 --- Different glucose isomerase mutants --- p.88 / Chapter 4.2 --- Enzymatic physicochemical and catalytic properties --- p.94 / Chapter 4.3 --- Future work --- p.95 / References --- p.97

Wong Wai Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 175-192). / Abstracts in English and Chinese. / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract --- p.iv / Abbreviation --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Section I --- The Making of Genetically Modified Organisms --- p.2 / Chapter 1.1 --- Conventional breeding in agriculture --- p.2 / Chapter 1.2 --- What is genetic engineering? --- p.4 / Chapter 1.3 --- Plant transformation --- p.5 / Chapter 1.3.1 --- Agrobacterium-mediated --- p.6 / Chapter 1.3.2 --- Direct gene transfer --- p.8 / Chapter 1.3.2.1 --- Microparticle bombardment --- p.8 / Chapter 1.3.2.2 --- Protoplasts --- p.9 / Chapter 1.3.3 --- Gene silencing --- p.10 / Chapter 1.4 --- Examples of genetically modified crops --- p.13 / Chapter 1.5 --- Foreign genes commonly found in transgenic plants --- p.14 / Chapter Section II --- Benefits and Environmental Concern of GMOs --- p.17 / Chapter 2.1 --- Mechanism of GMO --- p.17 / Chapter 2.1.1 --- Herbicide tolerant crops --- p.18 / Chapter 2.1.2 --- Insect resistant crops --- p.19 / Chapter 2.1.3 --- Delayed ripening crops --- p.20 / Chapter 2.1.4 --- Virus resistant crops --- p.20 / Chapter 2.2 --- Benefits of GMOs --- p.21 / Chapter 2.3 --- Impact of GM foods to human health and the environment --- p.22 / Chapter 2.3.1 --- Human health --- p.22 / Chapter 2.3.1.1 --- GM potatoes --- p.23 / Chapter 2.3.1.2 --- CaMV risks? --- p.24 / Chapter 2.3.1.3 --- Food allergy --- p.25 / Chapter 2.3.2 --- Environmental concerns --- p.26 / Chapter 2.3.2.1 --- Horizontal gene transfer --- p.27 / Chapter 2.3.2.1.1 --- Selectable marker genes --- p.27 / Chapter 2.3.2.1.2 --- Herbicide resistant genes --- p.29 / Chapter 2.3.2.1.3 --- Insect resistant genes --- p.29 / Chapter 2.3.2.2 --- Ecology --- p.30 / Chapter 2.3.2.2.1 --- Monarch butterfly --- p.30 / Chapter Section III --- Future developments of GMO --- p.32 / Chapter 3.1 --- Designer Food and engineered plants --- p.32 / Chapter 3.1.1 --- Insect resistance --- p.33 / Chapter 3.1.2 --- Viral resistance --- p.33 / Chapter 3.1.3 --- Fungal resistance --- p.34 / Chapter 3.1.4 --- Nutritional quality --- p.34 / Chapter 3.1.5 --- Modifications of oil composition --- p.35 / Chapter 3.1.6 --- Medical applications --- p.37 / Chapter 3.1.7 --- Environmental applications --- p.40 / Chapter 3.1.7.1 --- Tolerance to high salinity and drought --- p.40 / Chapter 3.1.7.2 --- Tolerance to frost --- p.41 / Chapter 3.1.7.3 --- Bioremediation --- p.42 / Chapter 3.1.7.4 --- Biodegradable products --- p.43 / Chapter Section IV --- Regulation of GMO --- p.44 / Chapter 4.1 --- The question of labeling --- p.44 / Chapter 4.1.1 --- Moral and ethical issues --- p.44 / Chapter 4.1.2 --- Animal welfare --- p.45 / Chapter 4.2 --- International practice in GMO labeling --- p.46 / Chapter 4.2.1 --- United States of America --- p.46 / Chapter 4.2.2 --- Canada --- p.48 / Chapter 4.2.3 --- European Union --- p.49 / Chapter 4.2.4 --- Australia and New Zealand --- p.50 / Chapter 4.2.5 --- Japan --- p.51 / Chapter 4.2.6 --- Republic of Korea --- p.52 / Chapter 4.2.7 --- China --- p.53 / Chapter 4.2.8 --- Taiwan --- p.53 / Chapter 4.2.9 --- Hong Kong --- p.54 / Chapter Section V --- Uses of crops --- p.56 / Chapter 5.1 --- Uses of crops --- p.56 / Chapter 5.1.1 --- Soybean --- p.56 / Chapter 5.1.2 --- Corn --- p.57 / Chapter 5.1.3 --- Tomato --- p.58 / Chapter 5.1.4 --- Potato --- p.59 / Chapter 5.1.5 --- Rice --- p.60 / Chapter 5.1.6 --- Rapeseed --- p.61 / Chapter 5.1.7 --- Oil --- p.62 / Chapter 5.2 --- "Food additives, hormones and flavourings" --- p.63 / Chapter Chapter 2 --- Materials & Methods --- p.65 / Chapter 2.1 --- Materials --- p.66 / Chapter 2.1.1 --- Growth media & agar --- p.66 / Chapter 2.1.2 --- Reagents for agarose gel electrophoresis --- p.67 / Chapter 2.1.3 --- Reagents for preparation of competent cells --- p.67 / Chapter 2.1.4 --- Reagents for measurement of DNA concentration --- p.68 / Chapter 2.1.4.1 --- Measurement of DNA concentration by PicoGreen --- p.68 / Chapter 2.1.5 --- Reagents for Southern hybridization --- p.68 / Chapter 2.2 --- Methods --- p.70 / Chapter 2.2.1 --- Restriction endonuclease digestion --- p.70 / Chapter 2.2.2 --- Agarose gel electrophoresis of DNA --- p.70 / Chapter 2.2.3 --- DNA recovery from agarose gel --- p.71 / Chapter 2.2.3.1 --- QIAquick® gel extraction --- p.71 / Chapter 2.2.4 --- Ligation of purified DNA fragment into vector --- p.72 / Chapter 2.2.5 --- Transformation --- p.72 / Chapter 2.2.6 --- Rubidium chloride method for making competent cells --- p.12 / Chapter 2.2.7 --- Plasmid DNA preparation --- p.73 / Chapter 2.2.7.1 --- Concert Rapid Mini Prep --- p.73 / Chapter 2.2.7.2 --- QIAprep® Miniprep --- p.74 / Chapter 2.2.8 --- Extraction of plant genomic DNA --- p.75 / Chapter 2.2.8.1 --- Qiagen DNeasy´ёØ Plant Mini Kit --- p.75 / Chapter 2.2.9 --- Southern Hybridization --- p.75 / Chapter 2.2.9.1 --- Denaturation --- p.76 / Chapter 2.2.9.2 --- Blot transfer --- p.76 / Chapter 2.2.9.3 --- Pre-hybridization --- p.77 / Chapter 2.2.9.4 --- Synthesis of radiolabelled probe --- p.77 / Chapter 2.2.9.5 --- Hybridization of radiolabelled probe on filter --- p.77 / Chapter 2.2.9.6. --- Detection of hybridized probes --- p.78 / Chapter 2.2.10 --- Measurement of DNA concentration --- p.78 / Chapter 2.2.10.1 --- Determination of DNA on EtBr stained gel --- p.78 / Chapter 2.2.10.2 --- Determination of DNA by UV spectrophotometer --- p.78 / Chapter 2.2.10.3 --- Determination of DNA by PicoGreen --- p.79 / Chapter 2.2.11 --- DNA sequencing --- p.80 / Chapter 2.2.11.1 --- Automated sequencing by ABI Prism 377 --- p.80 / Chapter Chapter 3 --- PCR Diagnostics --- p.81 / Chapter 3.1 --- Applications of PCR to processed foods --- p.82 / Chapter 3.1.1 --- DNA quality --- p.82 / Chapter 3.1.2 --- PCR & Multiplex PCR --- p.83 / Chapter 3.1.3 --- Choice of primers --- p.84 / Chapter 3.1.4 --- Inhibitors --- p.84 / Chapter 3.2 --- Materials & Methods --- p.85 / Chapter 3.2.1 --- Selection of primers --- p.85 / Chapter 3.2.2 --- Amplification of target sequences --- p.86 / Chapter 3.2.3 --- Multiple amplification of target sequences --- p.87 / Chapter 3.3 --- Results --- p.88 / Chapter 3.4 --- Discussion --- p.93 / Chapter Chapter 4 --- Quality Control in GMO detection --- p.95 / Chapter 4.1 --- Standardization of pre- and post- PCR analysis --- p.96 / Chapter 4.1.1 --- General guidelines --- p.96 / Chapter 4.1.2 --- UV irradiation --- p.97 / Chapter 4.1.3 --- Inactivation protocols --- p.93 / Chapter 4.1.4 --- Positive and negative controls --- p.99 / Chapter 4.1.5 --- PCR verification --- p.99 / Chapter 4.1.6 --- Equipment decontamination --- p.100 / Chapter 4.2 --- Materials & Methods --- p.101 / Chapter 4.2.1 --- Selection of primers for external control --- p.101 / Chapter 4.2.2 --- Development of the external control --- p.101 / Chapter 4.2.3 --- Selection of primers for internal control --- p.103 / Chapter 4.3 --- Results --- p.104 / Chapter 4.4 --- Discussion --- p.107 / Chapter Chapter 5 --- DNA extraction from food samples --- p.110 / Chapter 5.1 --- Introduction --- p.111 / Chapter 5.2 --- Reagents and Buffers for DNA extraction from food samples --- p.112 / Chapter 5.2.1 --- Cetyltrimethylammonium bromide (CTAB) extraction method --- p.112 / Chapter 5.2.2 --- Organic-based extraction method --- p.113 / Chapter 5.2.3 --- Potassium acetate/sodium dodecyl sulphate precipitation method --- p.113 / Chapter 5.2.4 --- Hexane-based extraction method --- p.114 / Chapter 5.3 --- Weight and names of samples --- p.115 / Chapter 5.4 --- DNA extraction methods --- p.115 / Chapter 5.4.1 --- CTAB extraction method --- p.115 / Chapter 5.4.2 --- Qiagen DNeasy´ёØ plant mini kit --- p.116 / Chapter 5.4.3 --- Promega Wizard® genomic DNA purification --- p.116 / Chapter 5.4.4 --- Promega Wizard® Magnetic DNA purification system --- p.117 / Chapter 5.4.5 --- Promega Wizard® DNA Clean-Up system --- p.118 / Chapter 5.4.6 --- Qiagen QIAshreddrer´ёØ and QIAamp spin column --- p.119 / Chapter 5.4.7 --- Chelex-based extraction method --- p.119 / Chapter 5.4.8 --- Organic-based extraction method --- p.120 / Chapter 5.4.9 --- Nucleon PhytoPure extraction and purification method --- p.120 / Chapter 5.4.10 --- Potassium acetate/SDS precipitation method --- p.121 / Chapter 5.4.11 --- Hexane-based extraction method --- p.122 / Chapter 5.5 --- Results --- p.123 / Chapter 5.5.1 --- Comparison of eleven extraction methods --- p.123 / Chapter 5.5.2 --- Comparison of DNA extraction on selected methods --- p.125 / Chapter 5.6 --- Discussion --- p.132 / Chapter Chapter 6 --- Quantitative Analysis --- p.136 / Chapter 6.1 --- Introduction --- p.137 / Chapter 6.1.1 --- Chemistry of quantitative PCR --- p.138 / Chapter 6.1.2 --- PCR system --- p.140 / Chapter 6.2 --- Materials & Methods --- p.142 / Chapter 6.2.1 --- Design of primers and probes --- p.142 / Chapter 6.2.2 --- Methods --- p.145 / Chapter 6.3 --- Results --- p.146 / Chapter 6.3.1 --- Selection of primer/probe --- p.146 / Chapter 6.3.2 --- Primer optimization --- p.149 / Chapter 6.3.3 --- Quantitative analysis of real samples --- p.158 / Chapter 6.4 --- Discussion --- p.152 / Chapter Chapter 7 --- Conclusion --- p.168 / References --- p.175 / Appendix --- p.193

Bibliography: leaves 281-330.