Single biomolecule studies using optical tweezers

Single biological molecule studies enable to probe and visualize exciting details of the events in physiological in vivo processes. The basic underlying question of this dissertation is to understand biological processes at a single molecule level. In contrast to ensemble techniques, advances in single molecule manipulation (e.g. optical and magnetic tweezers, atomic force microscopy) and / or fluorescence techniques allow to investigate the properties of individual molecules in real time with a possibility to change external conditions (buffers) in situ and modulate inter- and intra-molecular interactions. This thesis reports the application of a single molecule technique, dual beam optical tweezers, for the study of single biomolecules. A range of single molecule systems was investigated such as i)VirE2 protein DNA machinery, ii) DNA-surfactant, EtBr (ethidium bromide), SYBR® Green-DNA interactions and iii) dsDNA denaturation studies. In addition the development of the present experimental setup is described to enable combined force measurement as well as single molecule fluorescence studies. The presented biomolecular results provide new and complementary information on the different biological systems demonstrating the diversity of experiments that can be performed on single DNA molecules using optical tweezers. Chapter one gives a brief introduction to optical tweezers, describes how optical tweezers work, the physics behind it, details of the experimental setup and the method of force calibration required in micromanipulation. Optical tweezers have opened exciting avenues of research, especially in biology. Biologists will be able to investigate the nature of molecular machines one by one, and infer from their behavior those properties common to the population. In chapter 2, we show how optical tweezers were employed to study the change in the mechanical properties of single DNA molecules upon binding of small agents. The first part of this chapter reports on the changes in mechanics of single dsDNA in the presence of cationic and anionic surfactants (used as non-viral vectors in gene therapy). The second part describes the interaction of DNA binding ligands (SYBR® Green, EtBr) with individual DNA strands. Agrobacterium tumefaciens (AT), a Gram-negative bacterium, evolved a complex and unique mechanism to transfer a long single stranded DNA (ssDNA) molecule from its cytoplasm to the eukaryotic host plant cell nucleus. Central to this mechanism, chapter 3 discusses the results of the measurements on VirE2 protein interacting with single stranded DNA (ssDNA). VirE2 protein is a multifunctional protein from AT that coat the transferred-ssDNA (T-DNA), interacts with host factors assisting nuclear import of the complex, forms channels in lipid bilayers and displays a highly cooperative binding to ssDNA. The biological findings are presented in a new generic model which can be used to explain how generation of forces helps bacterial DNA to enter the plant cell based on our single molecule data. Single molecule dsDNA denaturation, relevant in many molecular biological experiments, induced by NaOH and mechanical pulling are studied in chapter 4. Here optical tweezers experiments give access to the ‘melting’ of hydrogen bonds by mechanical forces or alkali denaturation (NaOH) of dsDNA in real time. The mechanical stability and the transition of dsDNA to ssDNA is investigated at different ionic strength as well as in buffers. Fluorescent images of single λ DNA labeled with SYBR® Green were observed up to forces ≥ 65 pN and indicate a B-DNA to S −DNA transition. Chapter 5 describes the implementation of single-molecule fluorescence detection (SMF) in optical tweezers. The design and instrumental capabilities of optical tweezers combined with SMF are discussed in detail. The development of this instrument provides a worldwide unique experimental setup and opens up new possibilities in the studies of complex biological systems. Finally chapter 6 summarizes the results of this thesis and discusses future experimental applications. The appendices provide further details for DNA sample preparation, molecular biology and chemical surface activation recipes, an instruction manual for the setup and the list of currently published papers

Characterisation of agrobacterium VirD2 interacting protein DIP and its homologues

Ph.DDOCTOR OF PHILOSOPH

Identification and characterization of agrobacterium tumefaciens virD2-binding protein

Ph.DDOCTOR OF PHILOSOPH

Secreted Agrobacterium rhizogenes Effector Protein GALLS Interacts with Arabidopsis Host Proteins and Stimulates Agrobacterium-mediated Transformation

Agrobacterium species transform plant cells by targeting a portion of plasmid-encoded bacterial DNA to the host nucleus. Genetic transformation by A. tumefaciens and A. rhizogenes requires secreted effector proteins. The tumor-inducing (Ti) plasmid from virulent A. tumefaciens encodes VirE2, a secreted single-stranded DNA-binding protein required for efficient transformation of hosts. Some strains of A. rhizogenes harbor root-inducing (Ri) plasmids that lack the virE2 gene, and contain the GALLS gene instead. GALLS can fully substitute for the lack of virE2. The GALLS gene encodes two independently translated proteins: full-length (GALLS-FL) and a more abundant C-terminal domain (GALLS-CT) protein (collectively called GALLS). Both proteins are secreted from the bacterium into plant cells during transformation. GALLS-FL is a putative strand-transferase that is essential for GALLS-mediated transformation of all hosts. GALLS-CT is not required for transformation, but can significantly stimulate gene transfer to many plant species, including Arabidopsis thaliana. Host responses to Agrobacterium effectors are not well-understood. VirE2 interacts with host proteins in planta, however the exact roles and importance of these host proteins during transformation remain unclear. Host responses to GALLS-FL and GALLS-CT effectors have not been investigated before. We hypothesized that GALLS interacts with A. thaliana proteins that are important for Agrobacterium-mediated transformation. We also hypothesized that GALLS-CT, which is important for transformation of some hosts, may interact with host-specific proteins to alter host gene expression in favor of Agrobacterium infection. Using yeast two-hybrid screens, we identified LHS10, a putative host transcription factor that binds both GALLS proteins in planta. Overexpression of LSH10 in transgenic A. thaliana stimulated both GALLS- and VirE2-mediated transformation (transient and stable). RNA-Seq analysis of hypersusceptible plant lines overexpressing LSH10 revealed differential expression of many defense related genes, including up-regulation of WRKY38. WRKY38 is a transcription factor that suppresses expression of plant defense genes. Expression of WRKY38 from the CaMV 35S constitutive promoter in transgenic A. thaliana increased host susceptibility to Agrobacterium-mediated transformation. LSH10 may stimulate Agrobacterium-mediated transformation by lowering plant defense responses, leading to improved transformation efficiency. To identify the effects of GALLS-CT on host responses, we created transgenic A. thaliana that expressed GALLS-CT from an inducible promoter. GALLS-CT expressed in planta for six hours prior to Agrobacterium infection stimulated transient transformation. RNA-Seq analysis of transgenic plants producing GALLS-CT showed down regulation of several genes encoding heat shock and small heat shock molecular chaperones important for protein processing. Several of these heat shock proteins are involved in stabilizing plant disease resistance (R) proteins. Down regulation of molecular chaperone genes may lead to destabilization of defense response proteins, which may dampen host defense responses and improve Agrobacterium-mediated transformation frequencies in A. thaliana. Despite the importance of Agrobacterium-mediated transformation in agriculture, biotechnology, and plant science, plant responses to Agrobacterium are not well-understood. Genetic transformation of plants by Agrobacterium tumefaciens and A. rhizogenes requires secreted effector proteins VirE2 or GALLS. No studies prior to our work have investigated whether any host proteins interact with GALLS-FL or GALLS-CT, or how GALLS-CT may alter host-specific responses to stimulate transformation. We discovered that GALLS interacts with host protein LSH10. Overexpression of LSH10 made plants hypersusceptible to GALLS- and VirE2-mediated transformation, possibly by dampening host defense responses via up-regulation of WRKY38. We also observed that GALLS-CT produced in transgenic A. thaliana stimulated GALLS-mediated transient transformation. Transgenic plants that produced GALLS-CT showed decreased expression of genes encoding heat shock molecular chaperones involved in stabilizing disease resistance proteins. Our studies suggest that LSH10 and GALLS-CT may stimulate transformation by dampening host defense responses against Agrobacterium. These results shed light on a potentially new defense response pathway in A. thaliana, as well as how bacterial effector proteins may alter host responses to promote infection. Our findings may also prove useful in improving transformation frequencies in recalcitrant hosts

LIVE-TRACKING VIRE2 PROTEIN AND MOLECULAR ANALYSIS OF YEAST FACTOR PMP3P DURING AGROBACTERIUM-MEDIATED TRANSFORMATION

Ph.DDOCTOR OF PHILOSOPH

Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

Biotechnology and Crop Improvement in Asia

This book results from a workshop held at ICRISAT 3-7 Dec 1990 by the Asian Development Bank (ADB). The participants were representatives of Asian countries, and scientific leaders in [he various fields of biotechnology The introductory chapter outlines the role of the ADS in strengthening biotechnology research in Asia; this is followed by a presentation of the Indian biotechnology program and the problems thaI are being addressed. A chapter on Industry and Public Sector the and of private companics becoming involved in biotechnology research. The country representatives presented papers on the slatus of biotechnology in ,heir country, and these chaplers give an indication of the wide range of achievement in the area. Participants also outlined those crops and areas of crop improvement to which biotechnology could he applied. The remaining are reviews of the major disciplines in biotechnology and reports by Asian scientists and others on research relevant to Asian agriculture. These reviews cover cell lind tissue culture. with emphasis on cereals. and haploids. There are four reviews on transformation, covering Agrobacterium-medialed transformation, physical methods gene transfer the use of viruses as vectors and gene action in transformed plants. The section on genome characterization and diagnostics covers the genomes of plants-nuclear mitochondrial, and plastid and of viruses, and the relevance of genome research to plant improvement, through the use of markers-restriction fragment length polymorph isms (RFLP), polymerase chain reaclion (PCR), and random amplified polymorphic DNA (RAPD). The recommendations of the workshop give a useful insight into how a representative cross section view the future of biotechnology. Title book contains a glossary and index