scholarly journals Modular cell-free expression plasmids to accelerate biological design in cells

2020 ◽  
Author(s):  
Ashty S. Karim ◽  
Fungmin Eric Liew ◽  
Shivani Garg ◽  
Bastian Vögeli ◽  
Blake J. Rasor ◽  
...  
Keyword(s):  
Vector System ◽  
Free Expression ◽  
Expression Vectors ◽  
Model Organisms ◽  
Industrial Biotechnology ◽  
Biological Design ◽  
Cellular Expression ◽  
Community Science

AbstractIndustrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy to use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have the desired metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has emerged as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for C. autoethanogenum expression with efficiencies ranging from 68-90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

scholarly journals Modular cell-free expression plasmids to accelerate biological design in cells

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ashty S Karim ◽  
Fungmin (Eric) Liew ◽  
Shivani Garg ◽  
Bastian Vögeli ◽  
Blake J Rasor ◽  
...  
Keyword(s):  
Vector System ◽  
Free Expression ◽  
Expression Vectors ◽  
Model Organisms ◽  
Industrial Biotechnology ◽  
Biological Design ◽  
In Vivo Experiments ◽  
Cellular Expression

Abstract Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms—organisms that are easy to use like Escherichia coli—often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have desirable metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes, and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has matured as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institute’s DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of functional enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for Clostridium autoethanogenum expression with efficiencies ranging from 68% to 90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

scholarly journals In vitroprototyping and rapid optimization of biosynthetic enzymes for cellular design

2019 ◽  
Author(s):  
Ashty S. Karim ◽  
Quentin M. Dudley ◽  
Alex Juminaga ◽  
Yongbo Yuan ◽  
Samantha A. Crowe ◽  
...  
Keyword(s):  
Model Organisms ◽  
Industrial Biotechnology ◽  
Biosynthetic Enzymes ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Design Build ◽  
Mix And Match ◽  
Cell Free Protein Synthesis

AbstractMicrobial cell factories offer an attractive approach for production of biobased products. Unfortunately, designing, building, and optimizing biosynthetic pathways remains a complex challenge, especially for industrially-relevant, non-model organisms. To address this challenge, we describe a platform forin vitroPrototyping andRapidOptimization ofBiosyntheticEnzymes (iPROBE). In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE with two examples. First, we tested and ranked 54 different pathways for 3-hydroxybutyrate production, improvingin vivoproduction inClostridiumby 20-fold to 14.63 ± 0.48 g/L and identifying a new biosynthetic route to(S)-(+)-1,3-butanediol. Second, we used iPROBE and data-driven design to optimize a 6-stepn-butanol pathway, increasing titers 4-fold across 205 pathways, and showed strong correlation between cell-free and cellular performance. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

Biological and biomedical applications of collagenous biomaterials

1990 ◽  
Vol 48 (3) ◽  
pp. 856-857
Author(s):  
Yasushi P. Kato ◽  
Michael G. Dunn ◽  
Frederick H. Silver ◽  
Arthur J. Wasserman
Keyword(s):  
Biomedical Applications ◽  
Soft Tissues ◽  
Collagen Fibers ◽  
Bone Collagen ◽  
Cover Slip ◽  
Fibroblast Migration ◽  
Cellular Expression ◽  
Defect Repair

Collagenous biomaterials have been used for growing cells in vitro as well as for augmentation and replacement of hard and soft tissues. The substratum used for culturing cells is implicated in the modulation of phenotypic cellular expression, cellular orientation and adhesion. Collagen may have a strong influence on these cellular parameters when used as a substrate in vitro. Clinically, collagen has many applications to wound healing including, skin and bone substitution, tendon, ligament, and nerve replacement. In this report we demonstrate two uses of collagen. First as a fiber to support fibroblast growth in vitro, and second as a demineralized bone/collagen sponge for radial bone defect repair in vivo.For the in vitro study, collagen fibers were prepared as described previously. Primary rat tendon fibroblasts (1° RTF) were isolated and cultured for 5 days on 1 X 15 mm sterile cover slips. Six to seven collagen fibers, were glued parallel to each other onto a circular cover slip (D=18mm) and the 1 X 15mm cover slip populated with 1° RTF was placed at the center perpendicular to the collagen fibers. Fibroblast migration from the 1 x 15mm cover slip onto and along the collagen fibers was measured daily using a phase contrast microscope (Olympus CK-2) with a calibrated eyepiece. Migratory rates for fibroblasts were determined from 36 fibers over 4 days.

scholarly journals A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

2020 ◽  
Author(s):  
Yueyang Wang ◽  
Alan Y. Hsu ◽  
Eric M. Walton ◽  
Ramizah Syahirah ◽  
Tianqi Wang ◽  
...  
Keyword(s):  
Cell Motility ◽  
Neutrophil Migration ◽  
Transgenic Fish ◽  
Subcellular Location ◽  
Vector System ◽  
Specific Gene ◽  
Tissue Specific ◽  
Biological Studies

AbstractTissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

scholarly journals MicroRNAs in Valvular Heart Diseases: Biological Regulators, Prognostic Markers and Therapeutical Targets

2021 ◽  
Vol 22 (22) ◽  
pp. 12132
Author(s):  
Francesco Nappi ◽  
Adelaide Iervolino ◽  
Sanjeet Singh Avtaar Singh ◽  
Massimo Chello
Keyword(s):  
Mitral Valve ◽  
Mitral Valve Prolapse ◽  
Heart Diseases ◽  
Osteoblastic Differentiation ◽  
New Drugs ◽  
Expression Vectors ◽  
Chordae Tendineae ◽  
Valvular Heart Diseases

miRNAs have recently attracted investigators’ interest as regulators of valvular diseases pathogenesis, diagnostic biomarkers, and therapeutical targets. Evidence from in-vivo and in-vitro studies demonstrated stimulatory or inhibitory roles in mitral valve prolapse development, aortic leaflet fusion, and calcification pathways, specifically osteoblastic differentiation and transcription factors modulation. Tissue expression assessment and comparison between physiological and pathological phenotypes of different disease entities, including mitral valve prolapse and mitral chordae tendineae rupture, emerged as the best strategies to address miRNAs over or under-representation and thus, their impact on pathogeneses. In this review, we discuss the fundamental intra- and intercellular signals regulated by miRNAs leading to defects in mitral and aortic valves, congenital heart diseases, and the possible therapeutic strategies targeting them. These miRNAs inhibitors are comprised of antisense oligonucleotides and sponge vectors. The miRNA mimics, miRNA expression vectors, and small molecules are instead possible practical strategies to increase specific miRNA activity. Advantages and technical limitations of these new drugs, including instability and complex pharmacokinetics, are also presented. Novel delivery strategies, such as nanoparticles and liposomes, are described to improve knowledge on future personalized treatment directions.

scholarly journals Astrogliosis in an Experimental Model of Hypovitaminosis B12: A Cellular Basis of Neurological Disorders due to Cobalamin Deficiency

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2261
Author(s):  
Zuzanna Rzepka ◽  
Jakub Rok ◽  
Justyna Kowalska ◽  
Klaudia Banach ◽  
Justyna Magdalena Hermanowicz ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Cobalamin Deficiency ◽  
Confocal Laser ◽  
In Vivo Model ◽  
Cellular Expression ◽  
Wide Range ◽  
Vitro Model

Cobalamin deficiency affects human physiology with sequelae ranging from mild fatigue to severe neuropsychiatric abnormalities. The cellular and molecular aspects of the nervous system disorders associated with hypovitaminosis B12 remain largely unknown. Growing evidence indicates that astrogliosis is an underlying component of a wide range of neuropathologies. Previously, we developed an in vitro model of cobalamin deficiency in normal human astrocytes (NHA) by culturing the cells with c-lactam of hydroxycobalamin (c-lactam OH-Cbl). We revealed a non-apoptotic activation of caspases (3/7, 8, 9) in cobalamin-deficient NHA, which may suggest astrogliosis. The aim of the current study was to experimentally verify this hypothesis. We indicated an increase in the cellular expression of two astrogliosis markers: glial fibrillary acidic protein and vimentin in cobalamin-deficient NHA using Western blot analysis and immunocytochemistry with confocal laser scanning microscopy. In the next step of the study, we revealed c-lactam OH-Cbl as a potential non-toxic vitamin B12 antagonist in an in vivo model using zebrafish embryos. We believe that the presented results will contribute to a better understanding of the cellular mechanism underlying neurologic pathology due to cobalamin deficiency and will serve as a foundation for further studies.

scholarly journals Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1523
Author(s):  
Isabelle Anna Zink ◽  
Erika Wimmer ◽  
Christa Schleper
Keyword(s):  
Comparative Analysis ◽  
Molecular Mechanisms ◽  
Model Organisms ◽  
Special Focus ◽  
Natural Environments ◽  
Type I ◽  
Accessory Proteins ◽  
Type Iii

Prokaryotes are constantly coping with attacks by viruses in their natural environments and therefore have evolved an impressive array of defense systems. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system found in the majority of archaea and about half of bacteria which stores pieces of infecting viral DNA as spacers in genomic CRISPR arrays to reuse them for specific virus destruction upon a second wave of infection. In detail, small CRISPR RNAs (crRNAs) are transcribed from CRISPR arrays and incorporated into type-specific CRISPR effector complexes which further degrade foreign nucleic acids complementary to the crRNA. This review gives an overview of CRISPR immunity to newcomers in the field and an update on CRISPR literature in archaea by comparing the functional mechanisms and abundances of the diverse CRISPR types. A bigger fraction is dedicated to the versatile and prevalent CRISPR type III systems, as tremendous progress has been made recently using archaeal models in discerning the controlled molecular mechanisms of their unique tripartite mode of action including RNA interference, DNA interference and the unique cyclic-oligoadenylate signaling that induces promiscuous RNA shredding by CARF-domain ribonucleases. The second half of the review spotlights CRISPR in archaea outlining seminal in vivo and in vitro studies in model organisms of the euryarchaeal and crenarchaeal phyla, including the application of CRISPR-Cas for genome editing and gene silencing. In the last section, a special focus is laid on members of the crenarchaeal hyperthermophilic order Sulfolobales by presenting a thorough comparative analysis about the distribution and abundance of CRISPR-Cas systems, including arrays and spacers as well as CRISPR-accessory proteins in all 53 genomes available to date. Interestingly, we find that CRISPR type III and the DNA-degrading CRISPR type I complexes co-exist in more than two thirds of these genomes. Furthermore, we identified ring nuclease candidates in all but two genomes and found that they generally co-exist with the above-mentioned CARF domain ribonucleases Csx1/Csm6. These observations, together with published literature allowed us to draft a working model of how CRISPR-Cas systems and accessory proteins cross talk to establish native CRISPR anti-virus immunity in a Sulfolobales cell.

scholarly journals Regulated hAAT Expression from a Novel rAAV Vector and Its Application in the Prevention of Type 1 Diabetes

2019 ◽  
Vol 8 (9) ◽  
pp. 1321 ◽  
Author(s):  
Hongxia Ma ◽  
Yuanqing Lu ◽  
Keith Lowe ◽  
Lonneke van der Meijden-Erkelens ◽  
Clive Wasserfall ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Fine Tuning ◽  
Vector System ◽  
Regulation System ◽  
Raav Vector

We, and others, have previously achieved high and sustained levels of transgene expression from viral vectors, such as recombinant adeno-associated virus (rAAV). However, regulatable transgene expression may be preferred in gene therapy for diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), in which the timing and dosing of the therapeutic gene product play critical roles. In the present study, we generated a positive feedback regulation system for human alpha 1 antitrypsin (hAAT) expression in the rAAV vector. We performed quantitative kinetics studies in vitro and in vivo demonstrating that this vector system can mediate high levels of inducible transgene expression. Transgene induction could be tailored to occur rapidly or gradually, depending on the dose of the inducing drug, doxycycline (Dox). Conversely, after withdrawal of Dox, the silencing of transgene expression occurred slowly over the course of several weeks. Importantly, rAAV delivery of inducible hAAT significantly prevented T1D development in non-obese diabetic (NOD) mice. These results indicate that this Dox-inducible vector system may facilitate the fine-tuning of transgene expression, particularly for hAAT treatment of human autoimmune diseases, including T1D.

scholarly journals c-erbA encodes multiple proteins in chicken erythroid cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
J Bigler ◽  
R N Eisenman
Keyword(s):  
Expression Vector ◽  
In Vitro Translation ◽  
Vector System ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Avian Erythroblastosis Virus ◽  
Related Proteins ◽  
Translational Mechanisms

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

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