A modular degron library for synthetic circuits in mammalian cells

Tight control over protein degradation is a fundamental requirement for cells to respond rapidly to various stimuli and adapt to a fluctuating environment. Here we develop a versatile, easy-to-handle library of destabilizing tags (degrons) for the precise regulation of protein expression profiles in mammalian cells by modulating target protein half-lives in a predictable manner. Using the well-established tetracycline gene-regulation system as a model, we show that the dynamics of protein expression can be tuned by fusing appropriate degron tags to gene regulators. Next, we apply this degron library to tune a synthetic pulse-generating circuit in mammalian cells. With this toolbox we establish a set of pulse generators with tailored pulse lengths and magnitudes of protein expression. This methodology will prove useful in the functional roles of essential proteins, fine-tuning of gene-expression systems, and enabling a higher complexity in the design of synthetic biological systems in mammalian cells

High-titer production of lathyrane diterpenoids from sugar by engineered Saccharomyces cerevisiae

Euphorbiaceae are an important source of medically important diterpenoids, such as the anticancer drug ingenol-3-angelate and the antiretroviral drug prostratin. However, extraction from the genetically intractable natural producers is often limited by the small quantities produced, while the organic synthesis of terpene-derived drugs is challenging and similarly low-yielding. While transplanting the biosynthetic pathway into a heterologous host has proven successful for some drugs, it has been largely unsuccessful for diterpenoids due to their elaborate biosynthetic pathways and lack of genetic resources and tools for gene discovery. We engineered casbene precursor production in S. cerevisiae, verified the ability of six Euphorbia lathyris and Jatropha curcas cytochrome P450s to oxidize casbene, and optimized the expression of these P450s and an alcohol dehydrogenase to generate jolkinol C, achieving ~800 mg/L of jolkinol C and over 1 g/L total oxidized casbanes in millititer plates, the highest titer of oxidized diterpenes in yeast reported to date. This strain enables the semisynthesis of biologically active jolkinol C derivatives and will be an important tool in the elucidation of the biosynthetic pathways for ingenanes, tiglianes, and lathyranes. These findings demonstrate the ability of S. cerevisiae to produce oxidized drug precursors in quantities that are sufficient for drug development and pathway discovery

Genetically encoded betaxanthin-based small-molecular fluorescent reporter for mammalian cells

We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification

Programmable DARPin-based receptors for the detection of thrombotic markers

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein

Targeting protein–ligand neosurfaces with a generalizable deep learning tool

Molecular recognition events between proteins drive biological processes in living systems1. However, higher levels of mechanistic regulation have emerged, in which protein–protein interactions are conditioned to small molecules2, 3, 4–5. Despite recent advances, computational tools for the design of new chemically induced protein interactions have remained a challenging task for the field6, 7. Here we present a computational strategy for the design of proteins that target neosurfaces, that is, surfaces arising from protein–ligand complexes. To develop this strategy, we leveraged a geometric deep learning approach based on learned molecular surface representations8, 9 and experimentally validated binders against three drug-bound protein complexes: Bcl2–venetoclax, DB3–progesterone and PDF1–actinonin. All binders demonstrated high affinities and accurate specificities, as assessed by mutational and structural characterization. Remarkably, surface fingerprints previously trained only on proteins could be applied to neosurfaces induced by interactions with small molecules, providing a powerful demonstration of generalizability that is uncommon in other deep learning approaches. We anticipate that such designed chemically induced protein interactions will have the potential to expand the sensing repertoire and the assembly of new synthetic pathways in engineered cells for innovative drug-controlled cell-based therapies10

High-titer production of lathyrane diterpenoids from sugar by engineered Saccharomyces cerevisiae

Euphorbiaceae are an important source of medically important diterpenoids, such as the anticancer drug ingenol-3-angelate and the antiretroviral drug prostratin. However, extraction from the genetically intractable natural producers is often limited by the small quantities produced, while the organic synthesis of terpene-derived drugs is challenging and similarly low-yielding. While transplanting the biosynthetic pathway into a heterologous host has proven successful for some drugs, it has been largely unsuccessful for diterpenoids due to their elaborate biosynthetic pathways and lack of genetic resources and tools for gene discovery. We engineered casbene precursor production in S. cerevisiae, verified the ability of six Euphorbia lathyris and Jatropha curcas cytochrome P450s to oxidize casbene, and optimized the expression of these P450s and an alcohol dehydrogenase to generate jolkinol C, achieving ~800mg/L of jolkinol C and over 1g/L total oxidized casbanes in millititer plates, the highest titer of oxidized diterpenes in yeast reported to date. This strain enables the semisynthesis of biologically active jolkinol C derivatives and will be an important tool in the elucidation of the biosynthetic pathways for ingenanes, tiglianes, and lathyranes. These findings demonstrate the ability of S. cerevisiae to produce oxidized drug precursors in quantities that are sufficient for drug development and pathway discovery

Artificial receptors for mammalian synthetic biology

Synthetic biology has made great progress in engineering various components of cell signaling. These approaches are particularly relevant for generating therapeutic cells. The focus of this thesis concerns the development of tools for sensing biotechnologically or therapeutically important stimuli, such as disease markers, and for reprogramming cell behavior in response. The generalized extracellular molecule sensor (GEMS) design is based on the use of antibody fragments as ligand binding domains. The simultaneous binding of two such domains to a ligand causes the extracellular receptor domains to dimerize. This dimerization leads to a reorientation of the intracellular receptor domains, activating cellular signaling pathways. GEMS enabled the generation of cells that sense the cancer biomarker prostate-specific antigen (PSA) and activate transgene expression in response. The GEMS design was further used to construct receptors that sense heart damage. Cardiac troponin I is released by damaged cardiomyocytes, for example after acute myocardial infarction. Receptors that activate signaling pathways associated with tissue regeneration upon binding cardiac troponin I, may be a promising strategy for the development of cell therapy applications for heart disease. For some applications of artificial receptors, activating endogenous signaling pathways might cause unintended side effects. Therefore, we developed a synthetic signaling pathway based on phosphorylated histidine and aspartate residues in mammalian cells. This system is unlikely to interfere with endogenous signal processing, as the regulation of protein function by phosphorylation in mammalian cells is almost exclusively regulated by phosphorylated tyrosine, threonine and serine. All projects in this thesis provide insights into receptor development for different applications, and also enrich the toolbox of synthetic biology for cell signaling

Periscope. Galvano-cautery for Sterilizing Root-canals.

Editors: Aug. 1859-July 1865, J. D. White, J. H. McQuillen, G. J. Ziegler.--Aug. 1865-Dec. 1871, J. H. McQuillen, G. J. Ziegler.--Jan. 1872-May 1891, J. W. White.--July 1891-Apr. 1930, E. C. Kirk (with L. P. Anthony, Dec. 1917-Apr. 1930).--May 1930-Dec. 1936, L. P. Anthony.Vols. 1-13 are called "new series."Merged in Jan. 1937 with: Journal of the American Dental Association, ISSN 1048-6364, to form: Journal of the American Dental Association and dental cosmos, ISSN 0375-8451

The Closing of the Perforated Apex of the Root, with Subsequent Filling of the Root-Canal by a New Method.

Editors: Aug. 1859-July 1865, J. D. White, J. H. McQuillen, G. J. Ziegler.--Aug. 1865-Dec. 1871, J. H. McQuillen, G. J. Ziegler.--Jan. 1872-May 1891, J. W. White.--July 1891-Apr. 1930, E. C. Kirk (with L. P. Anthony, Dec. 1917-Apr. 1930).--May 1930-Dec. 1936, L. P. Anthony.Vols. 1-13 are called "new series."Merged in Jan. 1937 with: Journal of the American Dental Association, ISSN 1048-6364, to form: Journal of the American Dental Association and dental cosmos, ISSN 0375-8451