No description available.

No description available.

No description available.

Two cationic antimicrobial peptides, ESF I-GR7, and pleurocidin, were assessed for their stability in plant intercellular fluid, the targeted locale for their expression in transgenic plants. Incubation of ESFI-GR7 and pleurocidin with intercellular fluid (ICF) extracted from sugarcane, tomato and tobacco leaves reduced their biotoxicity towards various pathogens, namely Camobacterium mobile DMSO and Xanthomollas campestris. It was concluded that it may be necessary to modify the aminoacid structures of the peptides in order to ensure that endogenous proteases would not degrade the peptides once expressed in a transgenic environment. The presence of pleurocidin was detected in transgenic sugarcane transformed (in a previous study) with pleurocidin gene cloned into the pUBI 510 plasmid. ICF was extracted from four month old transgenic Saccharum hybrid species (sugarcane). Western blotting verified the presence of the transgenic protein in crude protein extracts. Immunogold labelling and transmission electron microscopy were performed to investigate the localisation of transgenic pleurocidin. The peptide was localized predominantly in the intercellular spaces and cell wall sugarcane leaves. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

This thesis examines the development and changing practices of governance in China by example of the evolution of policy development in agricultural biotechnology (especially in the case of genetically modified rice). In particular, the process of negotiation between the central government, scientific community, NGOs and the media are brought up to investigate the paradigmatic change in China's development that has been taking place over the last three decades of reform. By drawing on an STS perspective in tandem with social theories, it is argued that the governance of agri-biotech in China could be seen as a process of defining and redefining collective action problems by a widening range of policy actors. This approach is of special pertinence in studying China since its 'techno-nationalist' milieu and complicated and often inconsistent policy process seem to defy the concept of governance. The thesis traces the historical policy development over the governance of agri-biotech in China, and provides a panoramic view on how an increasing number of agents have participated in the process and thereby shaped the collective policy problem. National agri-biotech policy has developed in four distinct phases: an initial phase, marked by technological optimism and lack of regulation; a second, 'the millennium policy‘, distinguished by international pressure from WTO and the Cartagena Protocol; the third phase when scandals of GM rice leakage mobilised widespread public opposition to challenge the current expert policy system; the last stage of intensive policy development occurred since 2008 when global food crises led to the re-evaluation of the collective problem in terms of food security. The China story is not a mere repetition of the European experience in regards to GMO regulation, in that China is still a developing country caught between international forces of trade liberalisation and global biosafety governance, a conflict that is currently complicated in the transatlantic disputes over GMOs. The current policy ambivalence of China is under great pressure to solidify into solutions that can both protect local biodiversity and stand the challenge from international GMO trade. Finally, the opaque nature of China's political culture compromises the efficacy of policy intervention from below, making the policy negotiations an interesting test ground for the possibility of governance from below in the budding prospect of democratisation in China.

This research is concerned with analyzing a set of viral genomes to elucidate the underlying characteristics and determine the information-theoretic aspects of the genomic signatures. The goal of this study thereof, is tailored to address the following: (i) Reviewing various methods available to deduce the features and characteristics of genomic sequences of organisms in general, and particularly focusing on the genomes pertinent to viruses; (ii) applying the concepts of information-theoretics (entropy principles) to analyze genomic sequences; (iii) envisaging various aspects of biothermodynamic energetics so as to determine the framework and architecture that decide the stability and patterns of the subsequences in a genome; (iv) evaluating the genomic details using spectral-domain techniques; (v) studying fuzzy considerations to ascertain the overlapping details in genomic sequences; (vi) determining the common subsequences among various strains of a virus by logistically regressing the data obtained via entropic, energetics and spectral-domain exercises; (vii) differentiating informational profiles of coding and non-coding regions in a DNA sequence to locate aberrant (cryptic) attributes evolved as a result of mutational changes and (viii) finding the signatures of CDS of genomes of viral strains toward rationally conceiving plausible designs of vaccines. Commensurate with the topics indicated above, necessary simulations are proposed and computational exercises are performed (with MatLabTM R2009b and other software as needed). Extensive data gathered from open-literature are used thereof and, simulation results are verified. Lastly, results are discussed, inferences are made and open-questions are identified for future research. / by Sharmistha P. Chatterjee. / Thesis (Ph.D.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Leung Chun-wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 113-122). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgment --- p.iii / Abstract --- p.v / Table of contents --- p.ix / List of abbreviations --- p.xiv / List of figures --- p.xvii / List of tables --- p.xix / Chapter 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Artificial sweeteners --- p.3 / Chapter 1.2.1 --- SACCHARIN --- p.3 / Chapter 1.2.2 --- cyclamate --- p.4 / Chapter 1.2.3 --- Aspartame --- p.4 / Chapter 1.2.4 --- acesulfame-k --- p.5 / Chapter 1.2.5 --- SUCRALOSE --- p.5 / Chapter 1.3 --- natural sweet plant proteins --- p.7 / Chapter 1.3.1 --- THAUMATIN --- p.7 / Chapter 1.3.2 --- MONELLIN --- p.10 / Chapter 1.3.3 --- CURCULIN --- p.11 / Chapter 1.3.4 --- PENTADIN AND BRAZZEIN --- p.11 / Chapter 1.3.5 --- MIRACULIN --- p.12 / Chapter 1.3.6 --- MABINLIN --- p.12 / Chapter 1.4 --- Genetic Engineering of Sweet Plant Protein --- p.19 / Chapter 1.4.1 --- biotechnological studies on thaumatin --- p.20 / Chapter 1.4.1.1 --- Protein modification and sweetness --- p.20 / Chapter 1.4.1.2 --- Transgenic expression in microbes --- p.21 / Chapter 1.4.1.3 --- Transgenic expression in higher plants --- p.23 / Chapter 1.4.2 --- BIOTECHNOLOGICAL STUDIES ON MONELLIN --- p.24 / Chapter 1.4.2.1 --- Gene modification and transgenic expression in microbes --- p.24 / Chapter 1.4.2.2 --- Transgenic expression in plants --- p.25 / Chapter 1.4.3 --- TRANSGENIC EXPRESSION OF MABINLIN IN PLANTS --- p.26 / Chapter 1.5 --- phaseolin and its regulatory sequences --- p.27 / Chapter 1.6 --- ARABIDOPSIS --- p.29 / Chapter 1.6.1 --- ARABIDOPSIS THALIANA as a model plant --- p.29 / Chapter 1.6.2 --- Transformation methods --- p.29 / Chapter 1.6.2.1 --- Direct DNA uptake --- p.30 / Chapter 1.6.2.2 --- Agrobacterium-mediated transformation --- p.31 / Chapter 1.6.2.3 --- In planta transformation --- p.31 / Chapter 2 --- GENKRAL INTRODUTION AND HYPOTHESIS --- p.22 / Chapter 2.1 --- General Introduction --- p.33 / Chapter 2.2 --- Hypothesis --- p.34 / Chapter 3 --- MOLECULAR STUDIES ON SWEET PROTEIN MARINLIN : THERMAL STABILITY --- p.28 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Materials --- p.40 / Chapter 3.2.1 --- laboratory wares --- p.40 / Chapter 3.2.2 --- Equipments --- p.40 / Chapter 3.2.3 --- Chemicals --- p.40 / Chapter 3.2.4 --- commerical kits --- p.41 / Chapter 3.2.5 --- DNA primers --- p.42 / Chapter 3.2.6 --- DNA plasmids --- p.43 / Chapter 3.2.7 --- bacterial strains --- p.43 / Chapter 3.2.8 --- Plant materials --- p.44 / Chapter 3.2.9 --- Protein and Antibody --- p.44 / Chapter 3.3 --- Methods --- p.45 / Chapter 3.3.1 --- Transformation of Arabidopsis with mbliii and mbli genes --- p.45 / Chapter 3.3.1.1 --- Construction of mutant MBLIII and MBLI genes containing single codon mutation by megaprimer PCR --- p.45 / Chapter 3.3.1.2 --- Cloning of PCR-amplified MBLIII and MBLI cDNAs into vector pD3-8 --- p.48 / Chapter 3.3.1.3 --- In vitro site-directed mutagensis (for the construction of MBLIII and MBLI cDNAs containing single codon mutation) --- p.49 / Chapter 3.3.1.4 --- Cloning of the wild-type and mutated MBLIII and MBLI cDNA into vector pTZ / phas --- p.53 / Chapter 3.3.1.5 --- Confirmation of sequence fidelity and mutated codon in MBLIII and MBLI cDNA by DNA sequencing --- p.53 / Chapter 3.3.1.6 --- Transfer of wild-type MBLIII and MBLI cDNA flanked by phaseolin regulatory sequence into Agrobacterium binary vector --- p.55 / Chapter 3.3.1.7 --- Transformation of Agrobacterium with pBI / phas / MBLIII and pBI / phas / MBLI chimeric gene constructs --- p.57 / Chapter 3.3.1.8 --- Vacuum infiltration transformation of A rabidopsis --- p.58 / Chapter 3.3.1.9 --- Screening of homozygous transgenic Arabidopsis --- p.59 / Chapter 3.3.2 --- Expression analysis of MBLIII transgene --- p.61 / Chapter 3.3.2.1 --- GUS assay of transgenic plants --- p.61 / Chapter 3.3.2.2 --- Genomic DNA isolation from transgenic plants --- p.61 / Chapter 3.3.2.3 --- PCR amplification of transgene --- p.62 / Chapter 3.3.2.4 --- Total RNA isolation from transgenic Arabidopsis --- p.63 / Chapter 3.3.2.5 --- RT-PCR of total RNA from transgenic Arabidopsis --- p.64 / Chapter 3.3.2.6 --- Verification of the presence of mutagenic site and the fidelity of RNA transcript from transgenic Arabidopsis --- p.65 / Chapter 3.3.2.7 --- Protein extraction and tricine SDS-PAGE of putative transgenic protein from Arabidopsis --- p.65 / Chapter 3.3.2.8 --- N-terminal amino acid sequencing --- p.66 / Chapter 3.3.2.9 --- Isoelectric precipitation of MBL --- p.67 / Chapter 3.3.2.10 --- Production of polyclonal antibody against purified MBL --- p.67 / Chapter 3.3.2.11 --- Western-blotting and immunodectection of Arabidopsis protein by anti-MBL polyclonal antibody --- p.69 / Chapter 3.4 --- results & discussion --- p.71 / Chapter 3.4.1 --- Site-specific mutations of Arginine residue in mbliii cdna and glutamine in mbli cdna --- p.71 / Chapter 3.4.1.1 --- Megaprimer PCR --- p.71 / Chapter 3.4.1.2 --- Cloning into the seed-specific expression vector pD38 --- p.74 / Chapter 3.4.1.3 --- In vitro site-directed mutagenesis --- p.76 / Chapter 3.4.2 --- Construction of plant expression vectors containing chimeric MBLIII and MBLI --- p.80 / Chapter 3.4.2.1 --- Cloning of MBLIII and MBLI cDNAs into the seed-specific expression vector pTZ / phas --- p.80 / Chapter 3.4.2.2 --- Cloning into the plant expression vector pBI121 --- p.83 / Chapter 3.4.3 --- Generation of homozygous transgenic Arabidopsis --- p.84 / Chapter 3.4.3.1 --- Screening of transgenic R1 Arabidopsis --- p.84 / Chapter 3.4.3.2 --- Screening of transgenic R2 plants --- p.86 / Chapter 3.4.3.3 --- Screening of homozygous R3 transgenic plants --- p.88 / Chapter 3.4.4 --- Detection of MBLIII transgene in Arabidopsis --- p.89 / Chapter 3.4.4.1 --- Gus Assay --- p.89 / Chapter 3.4.4.2 --- Detection of transgene integration --- p.90 / Chapter 3.4.5 --- DETECTION of MBLIII TRANSCRIPT IN TRANSGENIC arabidopsis --- p.92 / Chapter 3.4.5.1 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.92 / Chapter 3.4.5.2 --- Verification of the presence of the mutant codon and sequence fidelity of the RT-PCR product --- p.94 / Chapter 3.4.6 --- DETECTION OF MBL III PROTEIN IN TRANSGENIC arabidopsis --- p.97 / Chapter 3.4.6.1 --- Expression of MBL protein --- p.97 / Chapter 3.4.6.2 --- Isoelectric precipitation --- p.101 / Chapter 3.4.6.3 --- Assay of titers and quality of primary polyclonal antibody against purified MBL protein --- p.103 / Chapter 3.4.6.4 --- Western blot / Immunodetection --- p.106 / Chapter 4 --- GENERAL DISCUSSION --- p.109 / Conclusion --- p.112 / References --- p.113

Chan Man Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 102-114). / Abstracts in English and Chinese. / Table of Contents / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.iv / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.xi / LIST OF TABLES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.4 / Chapter 2.1 --- The importance of rice --- p.4 / Chapter 2.2 --- The prevalence of hunger and malnutrition --- p.4 / Chapter 2.3 --- Limitation of essential amino acids in crop plants --- p.5 / Chapter 2.4 --- Lysine biosynthesis and catabolism --- p.7 / Chapter 2.5 --- Lysine biosynthetic pathway --- p.7 / Chapter 2.5.1 --- The biosynthesis of aspartate --- p.7 / Chapter 2.5.2 --- The aspartate family pathway --- p.9 / Chapter 2.5.2.1 --- Aspartate kinase (AK) --- p.11 / Chapter 2.5.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.13 / Chapter 2.5.2.3 --- Threonine Synthase (TS) and other enzymes --- p.15 / Chapter 2.6 --- The lysine catabolic pathway --- p.16 / Chapter 2.6.1 --- "LKR-SDHproteins, mRNAs and genes" --- p.18 / Chapter 2.6.2 --- Regulation of lysine catabolic pathway --- p.21 / Chapter 2.6.2.1 --- Regulation at biochemical level --- p.21 / Chapter 2.6.2.2 --- Regulation through linkage between LKR and SDH --- p.22 / Chapter 2.6.2.3 --- Regulation through LKR/SDH gene expression --- p.24 / Chapter 2.6.2.4 --- Implication of regulatory mechanism of saccharopine pathway --- p.26 / Chapter 2.7 --- Overall regulation of lysine content in plants --- p.27 / Chapter 2.8 --- Increasing lysine content in plants --- p.28 / Chapter 2.8.1 --- "Breeding, selection and naturally occurring mutants" --- p.28 / Chapter 2.8.2 --- Induced biochemical mutants --- p.29 / Chapter 2.8.3 --- Transgenic plants --- p.31 / Chapter 2.8.4 --- Insight into the way of lysine accumulation --- p.35 / Chapter 2.9 --- Gene silencing in plant --- p.36 / Chapter 2.9.1 --- Mechanism of antisense RNA and RNAi --- p.36 / Chapter 2.9.2 --- Application of antisense technology to produce transgenic plants --- p.39 / Chapter 2.10 --- Hypothesis --- p.41 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.43 / Chapter 3.1 --- Chemicals --- p.43 / Chapter 3.2 --- Bacterial strains --- p.43 / Chapter 3.3 --- Chimeric gene construction for rice transformation --- p.43 / Chapter 3.3.1 --- Plasmids and genetic materials --- p.43 / Chapter 3.3.2 --- Construction of chimeric genes with seed-specific promoters --- p.46 / Chapter 3.3.3 --- Construction of chimeric gene with 35S promoter --- p.51 / Chapter 3.3.4 --- Construction of antisense and RNAi constructs --- p.53 / Chapter 3.3.5 --- "Construction of chimeric genes expressing AK, DHPS and RNAi synchronously" --- p.58 / Chapter 3.3.6 --- Confirmation of sequence fidelity of chimeric genes --- p.59 / Chapter 3.4 --- Rice transformation --- p.59 / Chapter 3.4.1 --- Plant materials --- p.59 / Chapter 3.4.2 --- Preparation of Agrobacterium --- p.59 / Chapter 3.4.3 --- Agrobacterium-mediated rice transformation --- p.60 / Chapter 3.4.3.1 --- Callus induction from mature seed embryos --- p.60 / Chapter 3.4.3.2 --- Callus induction from immature seed embryos --- p.60 / Chapter 3.4.3.3 --- "Co-cultivation, selection and regeneration of transgenic rice" --- p.60 / Chapter 3.5 --- Analysis of transgenic expression --- p.62 / Chapter 3.5.1 --- Genomic DNA extraction --- p.62 / Chapter 3.5.2 --- Total RNA extraction --- p.62 / Chapter 3.5.3 --- Synthesis of DIG-labeled DNA probe / Chapter 3.5.4 --- Southern blot analysis --- p.65 / Chapter 3.5.5 --- Northern blot analysis --- p.65 / Chapter 3.5.6 --- Extraction of immature seed protein --- p.65 / Chapter 3.5.7 --- Tricine SDS-PAGE --- p.66 / Chapter 3.5.8 --- Western blot analysis --- p.66 / Chapter 3.6 --- Free amino acid analysis --- p.67 / Chapter CHAPTER 4. --- RESULTS --- p.68 / Chapter 4.1 --- Construction of chimeric genes --- p.68 / Chapter 4.2 --- Rice transformation --- p.70 / Chapter 4.3 --- Detection of target genes in transgenic rice lines --- p.72 / Chapter 4.3.1 --- PCR of Genomic DNA --- p.72 / Chapter 4.3.2 --- Southern blot analysis --- p.75 / Chapter 4.4 --- Northern blot analysis --- p.77 / Chapter 4.5 --- "Western blot analysis ofAK, DHPS and LKR protein" --- p.80 / Chapter 4.6 --- Free amino acid analysis --- p.82 / Chapter 4.6.1 --- Free lysine content --- p.82 / Chapter 4.6.2 --- Changes of other amino acids --- p.84 / Chapter CHAPTER 5. --- DISCUSSION --- p.93 / Chapter 5.1 --- Rice transformation and transgene expression --- p.93 / Chapter 5.2 --- Co-expression of E. coli feedback-insensitive AK and DHPS --- p.94 / Chapter 5.3 --- Enhancing free Lys through down-regulation of LKR --- p.95 / Chapter 5.4 --- Co-expression of AK and DHPS together with down-regulation of LKR --- p.96 / Chapter 5.5 --- Free amino acid changes in different genotypes --- p.97 / Chapter 5.6 --- Future perspectives --- p.98 / Chapter CHAPTER 6. --- CONCLUSION --- p.100 / REFERENCES --- p.102

Current research efforts are focused on 'second generation biofuels', which includes biofuels produced from lignocellulosic material. Lignocellulosic material is primarily composed of cellulose, a glucose polymer, xylose rich hemicellulose and non-fermentable lignin. Saccharomyces cerevisiae is widely used on an industrial scale for the production of ethanol from glucose; however, native S. cerevisiae does not contain the genes required for fermentation of xylose into ethanol. Others have sequentially expressed trans-genes from xylose fermenting organisms to engineer strains of S. cerevisiae capable of fermenting this pentose. The goal of this thesis was to generate a single cassette of 9 genes which have been shown to ferment xylose and arabinose. The 17 kb DNA fragment harboring all the genes necessary was introduced into the yeast genome using one-step homologous recombination based transformation. Expression of this cassette was verified by demonstrating that the first and last genes on this cassette were transcribed. The modified strain exhibited xylose utilization under microaerobic fermentation conditions. Further genetic and process engineering methods may be employed to improve the yield. The experiments described here demonstrate that generating a functional cassette of pentose fermenting genes is still achievable.

The gut microbiome is an integral component of the human body that plays a role in many physiological processes. Dysbiosis, an imbalance of the microbiome, has been associated with disease states including inflammatory bowel disease, type II diabetes, and obesity, and moreover, contributes to the pathogenesis of these states. Understanding the functional mechanisms governing microbial ecology and microbe-host interactions is essential to understanding the microbiome’s role in health and disease. However, at present, functional genetic studies of diverse natural mammalian gut microbiomes remain challenging, due to a lack of genetic tools for bacteria outside of a handful of well-studied model organisms. Altering the metagenome of a complex microbial community requires novel platform technologies for genetic engineering which can operate in a generalized fashion across many different host organisms. In this thesis, I present two novel genetic tools designed for genetic modification of bacterial communities. The first, the Cas-Transposon platform, is a host-independent targeted genome editing tool that utilizes programmable, targeted transposases to mediate site-specific gene insertions into user-defined loci. The Himar1 transposase naturally inserts transposases into random TA dinucleotides in a genome, but when fused to the dCas9 RNA-guided, DNA-binding protein, the fusion protein Himar1-dCas9 targets transposon insertions to a single TA site. The activity of Himar1-dCas9 was characterized using in vitro experiments, demonstrating that site-specific transposition is dependent on guide RNA (gRNA) orientation relative to the target site and the sequence surrounding the target site, but robust to variations in DNA and protein concentration, presence of background DNA, and temperature. We additionally showed that the Cas-Transposon platform is capable of performing site-specific transposition into a plasmid in vivo in E. coli, although further optimization of the system may be necessary to effect site-specific transposition into a genomic locus. The Himar1-dCas9 protein is the first example of a transposase that inserts transposons into locations programmable by an RNA, making it a novel tool for gene insertion and knockout in potentially any organism, without relying on DNA repair by a host cell. Metagenomic Alteration of Gut microbiome by In situ Conjugation (MAGIC) is an approach to directly modify gut bacteria in their native habitat by harnessing naturally occurring horizontal gene transfer activity to deliver engineered DNA. Because many gut bacteria are difficult to cultivate and thus difficult to genetically manipulate in the laboratory, MAGIC uses donor bacteria, delivered directly into the gut environment, to conjugate mobile vectors bearing engineered genetic payloads. Using payloads with selectable markers, we identified organisms across 4 major phyla of gut bacteria that were amenable to genetic modification with libraries of conjugative vectors we created. Using a lab-adapted E. coli strain as a donor, we achieved transient expression of the engineered payload in the microbiome. We also demonstrated that engineered native gut bacteria containing conjugative vectors could be deployed back into the gut to stably recolonize and mediate secondary transfer of the payload into other microbes, potentially enabling long-term infiltration of the payload into the metagenome. The results from this study suggest that both short-term and long-term genetic alteration of the metagenome are possible by choosing different donors, and that the MAGIC platform could enable development of more diverse microbial chasses for synthetic biology applications. MAGIC could also be used to create personalized engineered probiotics for diagnostic or therapeutic applications. In Chapter 4 of this thesis, we explored the targeted use of MAGIC to genetically modify Segmented Filamentous Bacteria, a gut commensal that is important for immune regulation but recalcitrant to in vitro cultivation. The Cas-Transposon and MAGIC technologies expand our capabilities in the areas of targeted genome editing and gene delivery into bacteria, respectively. Together, they form a suite of complementary approaches to genetically engineer undomesticated gut commensal bacteria and probe the functional genetic networks in the gut microbiome, which will enhance our understanding of microbiome ecology and host-microbiome interactions. In addition, the expanded range of genetic manipulations made possible by these tools may enable production of more diverse, perhaps personalized, probiotics containing engineered functions, such as sensing disease markers or drug delivery.