Clamping jig for clamping of connecting rod forgings

V diplomskem delu smo s programom Solidworks zasnovali 3D-vpenjalne priprave za vpenjanje ojnic. Od odkovka ojnice do končnega izdelka smo si zamislili dve vmesni CNC-obdelavi. Za prvo CNC-obdelavo smo izdelali dva različna 3D-modela vpenjalnih priprav in jih med seboj primerjali. Za drugo CNC-obdelavo smo izdelali en 3D-model vpenjalne priprave. Prednostne naloge vpenjalne priprave so bile, da obdelovanec med obdelavo povsem fiksira, cenovna ugodnost, obratovalna zanesljivost, enostavna zamenjava deformiranih oziroma obrabljenih delov. Standardne dele vpenjalnih priprav smo izbrali iz spletnega kataloga Halder.For this bachelor’s thesis, we used the Solidworks software to design 3D clamping jigs for clamping of connecting rods. From the forging of the connecting rod to the final product, we conceived two intermediate CNC machining procedures. For the first CNC machining, we created two different 3D models of clamping jigs and compared them with each other. For the second CNC machining, we made one 3D model of the clamping jig. The priorities of the clamping jig were to fix the workpiece perfectly during the machining, achieving cost-effectiveness and operational reliability, as well as easy replacement of deformed or damaged parts. The standard parts of the clamping jigs were selected from an online catalogue by Halder

Function-Based Mutation-Resistant Synthetic Signaling Device Activated by HIV‑1 Proteolysis

The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) virus is a major problem since it evades the function of antibodies and chemical inhibitors. Here, we demonstrate a viral detection strategy based on synthetic biology principles to detect a specific viral function rather than a particular viral protein. The resistance caused by mutations can be circumvented since the mutations that cause the loss of function also incapacitate the virus. Many pathogens encode proteases that are essential for their replication and that have a defined substrate specificity. A genetically encoded sensor composed of a fused membrane anchor, viral protease target site, and an orthogonal transcriptional activator was engineered into a human cell line. The HIV-1 protease released the transcriptional activator from the membrane, thereby inducing transcription of the selected genes. The device was still strongly activated by clinically relevant protease mutants that are resistant to protease inhibitors. In the future, a similar principle could be applied to detect also other pathogens and functions

Coupling CRISPR interference with FACS enrichment: New approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production.

Difficulties in obtaining and maintaining the desired level of the critical quality attributes (CQAs) of therapeutic proteins as well as the pace of the development are major challenges of current biopharmaceutical development. Therapeutic proteins, both innovative and biosimilars, are mostly glycosylated. Glycans directly influence the stability, potency, plasma half-life, immunogenicity, and effector functions of the therapeutic. Hence, glycosylation is widely recognized as a process-dependent CQA of therapeutic glycoproteins. Due to the typically high heterogeneity of glycoforms attached to the proteins, control of glycosylation represents one of the most challenging aspects of biopharmaceutical development. Here, we explored a new glycoengineering approach in therapeutic glycoproteins development, which enabled us to achieve the targeted glycoprofile of the Fc-fusion protein in a fast manner. Coupling CRISPRi technology with lectin-FACS sorting enabled downregulation of the endogenous gene involved in fucosylation and further enrichment of CHO cells producing Fc-fusion proteins with reduced fucosylation levels. Enrichment of cells with targeted glycoprofile can lead to time-optimized clone screening and speed up cell line development. Moreover, the presented approach allows isolation of clones with varying levels of fucosylation, which makes it applicable to a broad range of glycoproteins differing in target fucosylation level

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity