Nano and Biotechnology
Morning Session 1: Nano- and Bio-technology (0945-1055)
Chair: Mark Hildebrand
0945-1015 (Invited Speaker)
ENGINEERING MICROALGAE FOR BIOTECHNOLOGY
Alison G Smith
Department of Plant Sciences, University of Cambridge, Cambridge, UK
There is enormous potential to use microalgae as feedstocks for everything from recombinant proteins and high value chemicals to biofuels, but to implement this technology in a sustainable and economic manner, it will be necessary to optimize many parameters, and metabolic engineering strategies will be essential. However, in comparison with the well-developed molecular biology approaches available for manipulation of bacteria, yeast, and even land plants, those for algae are limited, even for the well-studied Chlamydomonas reinhardtii. We have established a synthetic biology workflow to enable rapid assembly of different genetic elements (eg coding region, regulatory elements, targeting and epitope tags), allowing high throughput testing of different components, and ultimately orthogonality – where standard parts can be used in any system to generate predictable outcomes. Using this approach we have investigated the activity of the untranslated regions (UTRs) in transgenes introduced into C. reinhardtii. For 3’ UTRs, a 10-fold variation in transgene expression and stability is observed with different sequences. The presences of a thiamine-pyrophosphate riboswitch in the 5’ UTR can be used to regulate transgene expression with nM concentrations of thiamine added to the cultures. We are actively exploring whether the same responses are seen in Phaeodactylum tricornutum.
DEVELOPMENT OF REPLICATING PLASMIDS DELIVERED TO DIATOMS VIA BACTERIAL CONJUGATION
Rachel E. Diner1,2, Bogumil J. Karas3, Stephane C. Lefebvre2, Jeff McQuaid2, Andrew E. Allen1, 2, Christopher L. Dupont2, Philip D. Weyman3
1 Integrative Oceanography Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, USA
2 Microbial and Environmental Genomics Group. J. Craig Venter Institute. La Jolla, CA, USA
3 Synthetic Biology and Bioenergy Group. J. Craig Venter Institute. La Jolla, CA, USA
The importance of diatoms in both nature and biotechnology necessitates the development of molecular tools to study them. We have engineered plasmids capable of replication and stable maintenance in the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Replication of the plasmids in diatoms was enabled by the addition of the yeast replication elements CEN (centromere), ARS (origin or replication), and HIS (histidine marker).Plasmids were delivered to the diatoms via an optimized method of conjugation from the bacteria Escherichia coli, with an efficiency of 4 x 10-4 diatom cells. The low cost and high efficiency of this transformation protocol makes it an excellent alternative to current methods (e.g. biolistic particle delivery). Plasmids of up to 75kb were shown to be delivered and stably maintained, demonstrating potential for the introduction of entire biological pathways and synthetic chromosomes into diatoms. Furthermore, the demonstrated delivery of bacterial DNA by conjugation into diatoms (the first example discovered in the Stramenopile lineage) raises interesting questions regarding inter-kingdom horizontal gene transfer, which is a topic of ongoing research.
SURFACE CHARACTERIZATION AND CATALYTIC APPLICATION OF DIATOM BIOSILICA
Cathleen Fischer1, Martin Oschatz2, Stefan Kaskel2, Nils Kröger3, Eike Brunner1
1 Institute for Bioananlytical Chemistry TU Dresden, Germany
2 Institute for Inorganic Chemistry TU Dresden, Germany
3 Center of Molecular Bioengineering TU Dresden, Germany
Diatom biosilica offers enormous perspectives for technological applications due to its special, outstanding properties. Diatom biosilica exhibits a species-specific nano- to microporous patterning. It is highly porous, biocompatible, thermally stable, and chemically inert. The potential of diatom biosilica for different applications, e.g. in catalysis, for drug-delivery-systems, and biophotonics is a subject of ongoing research.1
The catalytic behavior of different silica nanoparticle composite materials was investigated for the redox reaction between hexacyanoferrate(III) and thiosulfate in flow through and batch mode catalysis. The catalytic performance of biosilica of different diatom species with different structures was compared to synthetic silica materials. Platinum nanoparticles were attached to the surface via a covalent coupling method. The results of these studies show interesting differences between the materials, which might be due to structure, porosity, and/or surface chemistry. Hence the comprehensive characterization of the biosilica surface with different analytical methods is essential in this context. Various methods for characterizing the porosity and surface chemistry were applied to the biosilica and synthetic silica materials. With respect to the catalytic activity, the diatom biosilica from Eucampia zodiacus shows the most promising results.
Morning Session 2: Nano- and Bio-technology (1130-1240)
Chair: Nicole Poulsen
1130-1200 (Invited Speaker)
HIERARCHICAL FUNCTIONALISATION OF LIVING SUBSTRATES
CÚRAM-Centre for Research in Medical Devices, National University of Ireland, Galway.
Biomaterials are no longer considered innate structures and using functionalisation strategies to modulate a desired response whether it is a host or implant is currently an important focus in current research paradigms. Using functionalisation strategies such as enzymatic and hyperbranched linking systems, we have been able to link biomolecules to different structural moieties. The programmed assembly of biomolecules into higher-order self-organized systems is central to innumerable biological processes and development of the next generation of functionalized scaffolds. Recent design efforts have utilized a bottom-up approach toward both understanding and engineering supramolecular protein assemblies. These include functionalisation of micro and nanoparticles with biomolecules that include designed peptide motifs, growth factors and a multitude of gene vector systems. Structural moieties have taken a variety of different forms such as nanofibers and nanoparticulate.
In addition; development of nature inspired complex hierarchical structures and quantification of these geometries that have aided these investigations along with examples of how unicellular algae (diatoms) with a hallmark intricate siliceous cell wall, have provided such a stimulus. The ability to modify the chemistry of diatoms in vivo opens the possibility to tailor their structures to the end application. We have chemically modified the living diatom using several precursor systems (e.g. titanium (IV) bis-(ammonium lactato)-dihydroxide (TiBALDH)). Incorporation of these precursors into the diatom is achieved via a optimisation process that is dependent on the entity of the precursor. The characteristic architectural features of the diatom can be altered and are precursor dependent (1) Transformation of the living diatom provides opportunity to confer unique structural, chemical or functional properties upon the diatom.(2,3)
1. Lang Y. et. al. Integration of TiO2 into the diatom Thalassiosira weissflogii during frustule synthesis. Sci Rep. 2013 Nov 13;3:3205
2. Lang Y et. al. Functionalization of the living diatom Thalassiosira weissflogii with thiol moieties. Nat Commun. 2013;4:2683.
3. O'Connor J et. al. Nano-structured polymer-silica composite derived from a marine diatom via deactivation enhanced atom transfer radical polymerization grafting. Small. 2014 Feb 12;10(3):469-73.
MOLECULAR DYNAMICS SIMULATION OF THE INTERACTION OF POLYAMINES WITH SILICA SUBSTRATES
Sergii Donets, Arezoo Dianat, Rafael Gutierrez, Manfred Bobeth and Gianaurelio Cuniberti
Institute for Materials Science and Max Bergmann Center of Biomaterials,Dresden University of Technology, Germany
The role played by organic components in the process of biosilicification, resulting in well-defined three-dimensional silica structures in biosystems, has not been completely clarified. Especially in the case of diatoms several organic components have been meanwhile identified, including silaffins, silacidins, and polyamines. In this study we perform classical and quantum molecular dynamics simulations addressing two major issues related to polyamines: 1) the possible protonation states and protonation sites in short polyamines, and 2) the interaction of differently protonated polyamines with silica surfaces in order to identify the possible molecular conformations in dependence of the degree of protonation. Our results are in good quantitative agreement with results based on NMR experiments.
CYTOTOXICITY EVALUATION OF SI-DIATOM FRUSTULES IN A MOUSE MODEL
Pamela J Walsh1,2, Phillip B Messersmith2, Matthew Julius3, Fraser Buchanan4
Susan A Clarke5
1 School of Chemistry & Chemical Engineering, Queen’s University, Belfast, UK
2 Engineering Department, Northwestern University, Evanston, Illinois, USA
3 Department of Biological Sciences, St. Cloud State University Minnesota, MN, USA
4 School of Mechanical & Aerospace Engineering, Queen’s University, Belfast, UK
5 School of Nursing & Midwifery, Queen’s University, Belfast, UK
Silica additives in bone substitute materials are topical, clinically interesting and have significant support in the Orthopaedic field. Biosilica, e.g isolated from diatoms, has many advantages over its synthetic counterparts, e.g. it is amorphous, thus will be absorbed by the body, however, issues such as purity, presence of endotoxins and cytotoxicity need to be addressed before it can be further exploited. Biosilica isolated from Cyclotella Meneghiniana was then tested in a mouse model, to test the immunological response, organ toxicity (kidney, spleen, liver) and route of metabolism/excretion of silica. Five-week-old Balb-c mice were injected subcutaneously with a single high dose (50mg/ml) of Si-frustules, Si-frustules + organic linker and vehicle only control. Animals were sacrificed at 1d and 28d. The animal studies were conducted under an ethically approved protocol at Queen’s University, Belfast. The animals showed no adverse stress during the experiment and remained healthy until sacrifice. Blood results using ICP-OES analysis suggest the frustules were metabolized between comparator groups at different rates, and clearly showed elevated levels of silicon in groups injected with frustules relative to control. The histology of organs showed no variation in morphology of mice injected frustules relative compared to the control group.
Acknowledgements: The authors would like to thank Marie Curie International Outgoing Fellowships from the EU and Beaufort Marine Biodiscovery Award as part of the Marine Biotechnology Ireland Programme for providing financial support to this project.