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

NASA Tech Briefs, February 1997

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The benefits of an additional practice in descriptive geomerty course: non obligatory workshop at the Faculty of Civil Engineering in Belgrade

At the Faculty of Civil Engineering in Belgrade, in the Descriptive geometry (DG) course, non-obligatory workshops named “facultative task” are held for the three generations of freshman students with the aim to give students the opportunity to get higher final grade on the exam. The content of this workshop was a creative task, performed by a group of three students, offering free choice of a topic, i.e. the geometric structure associated with some real or imagery architectural/art-work object. After the workshops a questionnaire (composed by the professors at the course) is given to the students, in order to get their response on teaching/learning materials for the DG course and the workshop. During the workshop students performed one of the common tests for testing spatial abilities, named “paper folding". Based on the results of the questionnairethe investigation of the linkages between:students’ final achievements and spatial abilities, as well as students’ expectations of their performance on the exam, and how the students’ capacity to correctly estimate their grades were associated with expected and final grades, is provided. The goal was to give an evidence that a creative work, performed by a small group of students and self-assessment of their performances are a good way of helping students to maintain motivation and to accomplish their achievement. The final conclusion is addressed to the benefits of additional workshops employment in the course, which confirmhigherfinal scores-grades, achievement of creative results (facultative tasks) and confirmation of DG knowledge adaption

The contemporary visualization and modelling technologies and the techniques for the design of the green roofs

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Европейский и национальный контексты в научных исследованиях

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Тhe benefits of an additional practice in descriptive geometry course: non obligatory workshop at the Faculty of civil engineering in Belgrade

At the Faculty of Civil Engineering in Belgrade, in the Descriptive geometry (DG) course, non-obligatory workshops named “facultative task” are held for the three generations of freshman students with the aim to give students the opportunity to get higher final grade on the exam. The content of this workshop was a creative task, performed by a group of three students, offering free choice of a topic, i.e. the geometric structure associated with some real or imagery architectural/art-work object. After the workshops a questionnaire (composed by the professors at the course) is given to the students, in order to get their response on teaching/learning materials for the DG course and the workshop. During the workshop students performed one of the common tests for testing spatial abilities, named “paper folding". Based on the results of the questionnairethe investigation of the linkages between:students’ final achievements and spatial abilities, as well as students’ expectations of their performance on the exam, and how the students’ capacity to correctly estimate their grades were associated with expected and final grades, is provided. The goal was to give an evidence that a creative work, performed by a small group of students and self-assessment of their performances are a good way of helping students to maintain motivation and to accomplish their achievement. The final conclusion is addressed to the benefits of additional workshops employment in the course, which confirmhigherfinal scores-grades, achievement of creative results (facultative tasks) and confirmation of DG knowledge adaption

The contemporary visualization and modelling technologies and techniques for the design of the green roofs

The contemporary design solutions are merging the boundaries between real and virtual world. The Landscape architecture like the other interdisciplinary field stepped in a contemporary technologies area focused on that, beside the good execution of works, designer solutions has to be more realistic and “touchable”. The opportunities provided by Virtual Reality are certainly not negligible, it is common knowledge that the designs in the world are already presented in this way so the Virtual Reality increasingly used. Following the example of the application of virtual reality in landscape architecture, this paper deals with proposals for the use of virtual reality in landscape architecture so that designers, clients and users would have a virtual sense of scope e.g. rooftop garden, urban areas, parks, roads, etc. It is a programming language that creates a series of images creating a whole, so certain parts can be controlled or even modified in VR. Virtual reality today requires a specific gadget, such as Occulus, HTC Vive, Samsung Gear VR and similar. The aim of this paper is to acquire new theoretical and practical knowledge in the interdisciplinary field of virtual reality, the ability to display using virtual reality methods, and to present through a brief overview the plant species used in the design and construction of an intensive roof garden in a Mediterranean climate, the basic characteristics of roofing gardens as well as the benefits they carry. Virtual and augmented reality as technology is a very powerful tool for landscape architects, when modeling roof gardens, parks, and urban areas. One of the most popular technologies used by landscape architects is Google Tilt Brush, which enables fast modeling. The Google Tilt Brush VR app allows modeling in three-dimensional virtual space using a palette to work with the use of a three-dimensional brush. The terms of two "programmed" realities - virtual reality and augmented reality - are often confused. One thing they have in common, though, is VRML - Virtual Reality Modeling Language. In this paper are shown the ways on which this issue can be solved and by the way, get closer the term of Virtual Reality (VR), also all the opportunities which the Virtual reality offered us. As well, in this paper are shown the conditions of Mediterranean climate, the conceptual solution and the plant species which will be used by execution of intensive green roof on the motel “Marković”