Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.Proteiinien muokkauksella tähdätään entsyymien ja sitojareagenssien ominaisuuksien parantamiseen geneettisten muutosten avulla. Tyypillisiä parannettavia ominaisuuksia ovat sitomisvoimakkuus, spesifisyys, kestävyys, tuotto-ominaisuudet ja liukoisuus. Koska proteiinit ovat monimutkaisia biomolekyylejä, geneettisten muutosten vaikutukset ovat vain harvoin tarkkaan ennustettavissa. Siksi suosittu proteiinien muokkausstrategia on luoda kohdemolekyylistä lukuisia geenivariantteja ja asettaa luotu joukko valintakokeeseen, jonka perusteella variantit erottuvat toisistaan tavoitellun ominaisuuden perusteella. Tämä suunnattuna evoluutiona tunnettu tekniikka on keskeinen työkalu kehitettäessä proteiinituotteita, joita käytetään monenlaisissa sovelluksissa vaatteiden pesuaineista syöpälääkkeisiin. Proteiinituotteiden kehitystyön nopeuttaminen edellyttää jatkuvasti uusia menetelmiä, joilla voidaan rakentaa aiempaa laajempia geenikirjastoja ja niille yhteensopivia valintatyökaluja. Tässä työssä tutkittiin alukepidennysmutageneesitekniikan mahdollisuuksia korkealaatuisempien geenivarianttikirjastojen rakentamiseksi Escherichia coli-bakteerin soluihin. Toisessa osajulkaisussa tutkittiin rekombinaatiota menetelmänä, jolla voitaisiin lisätä seulottavissa olevien entsyymivarianttien lukumäärää. Kolmannessa osajulkaisussa kehitettiin valintaprosessi, jonka tarkoituksena oli parantaa vastaainefragmenttien ilmentymistä filamenttifaagin pinnalla näyttötekniikkaa varten. Neljännessä osajulkaisussa testattiin geenikirjaston suunnittelustrategioita käytännössä kahdella eri periaatteiden mukaan monimuotoistetulla vasta-ainekirjastolla. Viimeisessä osajulkaisussa vertailtiin faagin eri pintaproteiineihin fuusioidun vasta-ainekirjaston toimintaa valintakokeiden avulla. Samalla tutkittiin vasta-ainefragmenttien Fab (engl. antigen binding fragment) ja ScFv (engl. single-chain fragment of antibody variable domains) vaikutusta valintakokeen onnistumiseen, jotta selviäisi, mikä on käyttökelpoisin näyttötekniikka käytössä olevan kirjaston hyödyntämiseksi. Tutkimuksen tuloksena kehitettiin uusi geenivarianttikirjaston rakennusmenetelmä nimeltään sRCA (engl. selective rolling circle amplification), joka lisää merkittävästi mutageneesitehokkuutta ja jopa yli satakertaistaa kirjaston muodostavien transformanttien määrän verrattuna tavanomaiseen alukepidennysmutageneesiin. Toisessa osajulkaisussa havaittiin Cre-loxP-rekombinaation olevan sovelias työkalu RCA-satunnaismutageneesin tuloksena syntyvän DNA-vyyhdin pilkkomiseen kehämäisiksi plasmidiyksiköiksi. Uusi menetelmä lisäsikin DNA:n transformaatiotehokkuutta E. coli-bakteeriin. Kirjastoseulontojen avulla osoitettiin, että kirjastosta, jossa aminohappojen vaihtelua esiintyi lähempänä vasta-aineen sitomiskohdan keskustaa, löytyi enemmän pienmolekyylejä ja peptidejä tunnistavia vasta-aineita. Ulompana keskustasta sijaitsevien aminohappojen vaihtelu puolestaan tuki proteiineja tunnistavien vasta-aineiden kehitystyötä. Vasta-ainekirjaston faaginäyttötekniikkavertailun johtopäätökset olivat, että käyttämällä filamenttifaagin vasta-aineiden fuusiokumppanina lyhennettyä proteiini kolmea (p3Δ) pystyttiin eristämään runsaammin erilaisia kohdetta tunnistavia vasta-ainemolekyylejä kuin käyttämällä kokopitkää proteiini kolmea tai proteiini yhdeksää (p9). Erityisesti ScFvp3Δ- formaatissa oleva kirjasto tarjosi sitomisvoimakkuudeltaan parhaita vasta-aineita molekyylikooltaan pienen digoksigeniinin tunnistukseen. Tutkimus geenivarianttikirjastojen suunnittelusta, rakentamisesta ja seulontaan soveltuvista valintatyökaluista lisää käytännön tietoa suunnattujen evoluutiokokeiden toteuttamiseksi ja on arvokasta tietoa alan tutkijoille heidän tulevissa hankkeissaan.Siirretty Doriast

Fragility as Resilience: Designing the Balance of the Natural and Built on the example of an Open Competition for the Wider Area of the Hippodrome in Belgrade

In a global and local context today, it is difficult to recognize let alone to design a sensitive balance between natural and built, which will enable further development of human activities while developing awareness of the importance of preserving nature, its processes and balance. We must regulate the indisputable pressure to build new capacities of urban tissue with strategies that intertwine built and unbuilt, manmade and natural, into a single mechanism of interaction. The design concept, which puts the fragility of the named balance in the foreground, enables the spatial-program scenarios of life in cities to be placed in an interdependent relationship with the nature so that the concept of built structure does not rest on the strength of architectural form or technological solution. As a testing ground for studying these issues, we take the winning competition proposal of the urban-architectural competition for the area of the Hippodrome. The competition site belongs to the wider spatial area "Topčider", an especially significant and authentic space since it unites the city's natural, historical and cultural heritage. The competition proposal examines the possibilities for the development of the interconnectedness of nature and the city through the concept of fragility

Time-Based Design Paradigms

Associate Professor in Interior and Spatial Design at Politecnico di Milano. She has been visiting professor at Tsinghua University, Beijing (China); Kookmin University, Seoul (South Korea); Hosei University, Tokyo (Japan) and many others. She designed professional projects in China, Japan, USA, Europe, UK and UAE, as founder of Senselab, most of them awarded and selected by international juries. Some of her researches and products have been selected by ADI-Index 2019, Italian Design Ambassador 2020, 2021; awarded Eccellenze della Lombardia. She exhibited her works at Biennale di Venezia 2010, 2011, 2021; Triennale di Milano 2018. The relationships between senses, time, spaces and design are developed in education, conferences, publications and professional works. She is the author of Storie di Architettura attraverso i sensi (Stories of architecture through the senses, Bruno Mondadori, 2000), Invisible Architectures. Experiencing places throught the senses of smell (Skira, 2006), Sensi, tempo e architettura (Senses, time and architecture, Postmedia Books, 2012), Sensefulness, new paradigms for Spatial Design (Postmedia Books, 2019), and the book Extended Store. How digitalization effects the retail space design, written in collaboration with the author Yuemei Ma (FrancoAngeli, 2021), as well as many other international publications

A Smoothed Particle Hydrodynamics Method for the Simulation of Centralized Sloshing Experiments

The Smoothed Particle Hydrodynamics (SPH) method is proposed for studying hydrodynamic processes related to nuclear engineering problems. A problem of possible recriticality due to the sloshing motions of the molten reactor core is studied with SPH method. The accuracy of the numerical solution obtained in this study with the SPH method is significantly higher than that obtained with the SIMMER-III/IV reactor safety analysis code

Functional genomics of the unicellular cyanobacterium Synechococcus elongatus PCC 7942

Unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942 is the model organism for studying the circadian clock in cyanobacteria. Despite tremendous work over the last decade in identification of clock-related loci and elucidation of molecular mechanisms of the central oscillator, many details of the basic steps in generating circadian rhythms of biological processes remain unsolved and many components are still missing. A transposon-mediated mutagenesis and sequencing strategy has been adopted to disrupt essentially every locus in the genome so as to identify all of the loci that are involved in clock function. The complete genome sequence has been determined by a combination of shotgun sequences and transposon-mediated sequences. The S. elongatus PCC 7942 genome is 2,695,903 bp in length, and has a 55.5% GC content. Automated annotation identified 2,856 protein-coding genes and 51 RNA coding loci. A system for community refinement of the annotation was established. Organization and characteristic features of the genome are discussed in this dissertation. More than 95% of the PCC 7942 genome has been mutagenized and mutants affected in approximately 30% of loci have been screened for defects in circadian function. Approximately 70 new clock loci that belong to different functional categories have been discovered through a team effort. Additionally, functional analysis of insertion mutants revealed that the Type-IV pilus assembly protein PilN and the RNA chaperon Hfq are involved in transformation competence of S. elongatus cells. Functional analysis of an atypical short period kaiA insertional mutant showed that the short period phenotype is caused mainly by the truncation of KaiA by three amino acid residues. The interaction between KaiC and the truncated KaiA is weakened as shown by fluorescence anisotropy analysis. Deletion analysis of pANL, the large endogenous plasmid, implies that two toxin-antitoxin cassettes were responsible for inability to cure cells of this plasmid. In summary, the results indicate that this functional genomics project is very promising toward fulfilling our goal to assemble a comprehensive view of the cyanobacterial circadian clock. The mutagenesis reagents and dataset generated in this project will also benefit the greater scientific community

NASA Tech Briefs, October 2003

Topics covered include: Cryogenic Temperature-Gradient Foam/Substrate Tensile Tester; Flight Test of an Intelligent Flight-Control System; Slat Heater Boxes for Thermal Vacuum Testing; System for Testing Thermal Insulation of Pipes; Electrical-Impedance-Based Ice-Thickness Gauges; Simulation System for Training in Laparoscopic Surgery; Flasher Powered by Photovoltaic Cells and Ultracapacitors; Improved Autoassociative Neural Networks; Toroidal-Core Microinductors Biased by Permanent Magnets; Using Correlated Photons to Suppress Background Noise; Atmospheric-Fade-Tolerant Tracking and Pointing in Wireless Optical Communication; Curved Focal-Plane Arrays Using Back-Illuminated High-Purity Photodetectors; Software for Displaying Data from Planetary Rovers; Software for Refining or Coarsening Computational Grids; Software for Diagnosis of Multiple Coordinated Spacecraft; Software Helps Retrieve Information Relevant to the User; Software for Simulating a Complex Robot; Software for Planning Scientific Activities on Mars; Software for Training in Pre-College Mathematics; Switching and Rectification in Carbon-Nanotube Junctions; Scandia-and-Yttria-Stabilized Zirconia for Thermal Barriers; Environmentally Safer, Less Toxic Fire-Extinguishing Agents; Multiaxial Temperature- and Time-Dependent Failure Model; Cloverleaf Vibratory Microgyroscope with Integrated Post; Single-Vector Calibration of Wind-Tunnel Force Balances; Microgyroscope with Vibrating Post as Rotation Transducer; Continuous Tuning and Calibration of Vibratory Gyroscopes; Compact, Pneumatically Actuated Filter Shuttle; Improved Bearingless Switched-Reluctance Motor; Fluorescent Quantum Dots for Biological Labeling; Growing Three-Dimensional Corneal Tissue in a Bioreactor; Scanning Tunneling Optical Resonance Microscopy; The Micro-Arcsecond Metrology Testbed; Detecting Moving Targets by Use of Soliton Resonances; and Finite-Element Methods for Real-Time Simulation of Surgery

An exploration to integrate pliable textile and rigid metal properties within hybrid self-supporting woven forms using selective finishing

This practice-based research aims to integrate and control pliable anisotropic textile properties with rigid isotropic metal properties in self-supporting three-dimensional woven forms. When constructing self-supporting form using textiles, the drape and pliability can become compromised, for example, when placed under high tensile force, or a rigid finishing process is applied. This inquiry aims to improve the integration and control of the pliability and rigidity within metallised woven hybrid self-supporting forms. The methodology uses woven textile design methods and thinking, combined with industrial textile production and engineering techniques, to form integrated cognitive problem-solving spaces during practice-based experimentation and reflection. The design and making of the woven textiles are inextricably linked with the finishing process. This extends Seitamaa-Hakkarainen and Hakkarainen's (2001) dual-space parallel processing to incorporate a third specific thinking space: finishing. This is described as a Design-make Tri-space that is used as a research framework when problem-solving during this material investigation. My research question explores my hypothesis that using an experienced weaver’s parallel processing method could offer an alternative finishing technique to previous metallisation of textiles. This approach simultaneously considers the composition and construction of a woven textile with the finishing process. In my collaboration with industry a second research framework was used: Tri-space Roles. The roles of academic researcher, designer collaborating with industry and apprentice were integrated to become one interconnected role. Three case studies demonstrate how using different making and finishing sequences control and refine the properties of the hybrid forms. Qualitative haptic interaction was used to evaluate the relationship between the pliable fabric and the rigidity created by the finishing process. This research contributes new knowledge to the metallisation of textiles by establishing a new making process that enables the control of selective finishing on anisotropic woven textiles. It also proposes that the Design-make Tri-space and the Tri-space Roles problem-solving approaches are frameworks that facilitate parallel processing. These method frameworks have the potential to be modified and used by other design researchers using alternative textile processes, such as knit or embroidery, or other materials focused disciplines, such ceramics or glass

Sound Aesthetic: A Form of Narrative

This research presents an exploration into a novel design methodology that incorporates architecture, multimedia, and interactive digital technologies to create an immersive experience that encourages a spatial and sensorial discourse between user and their built environment. This immersive design method creates a continuous narrative that allows a multi-directional interaction between the two. This interaction creates a “sound” architectural aesthetic that changes the experience of space. The target of the interaction between user and space is the five human senses resulting in an immersive aesthetic. In order to illustrate this immersive aesthetic, five architectural prototypes were created using an assorted design workflow of parametric programming environment and interactive prototyping platform. This workflow is employed for the creation of five prototypes used for the simulation that has user interaction as an input and formal geometries as an output. These five prototypes target various human senses in order to enhance the immersive aesthetic. Each protoype is evaluated according to individual prototype’s ability to stimulate user’s senses. Finally, future research based on the outcomes of this research is suggested