Bu çalışma, sırasıyla NSF RUI Hibeler MCB-0950372 ve DMR-1105310 başlıklı &quot;Black Widow Spider ipek ve Spider Tutkal Silks Mekanik Davranışı Moleküler Karakterizasyonu&quot; tarafından desteklenmiştir.

<ol>
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A., Lewis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+mechanical+characterization+of+aciniform+silk:+uniformity+of+iterated+sequence+modules+in+a+novel+member+of+the+spider+silk+fibroin+gene+family.">Molecular and mechanical characterization of aciniform silk: uniformity of iterated sequence modules in a novel member of the spider silk fibroin gene family.</a> Mol. Biol. Evol. 21, 1950-1959 (2004).</li><li>Lazaris, A., Arcidiacono, S., Huang, Y., Zhou, J. F., Duguay, F., Chretien, N., Welsh, E. A., Soares, J. W., Karatzas, C. 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N., Lazaris, A., Richman, A., Brandle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+dragline+silk+proteins+in+transgenic+tobacco+leaves:+accumulation+and+field+production.">Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production.</a> Plant Biotechnology Journal. 2, 431-438 (2004).</li><li>Scheller, J., Guhrs, K. H., Grosse, F., Conrad, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+spider+silk+proteins+in+tobacco+and+potato.">Production of spider silk proteins in tobacco and potato.</a> Nat. Biotechnol. 19, 573-577 (2001).</li><li>An, B., Hinman, M. B., Holland, G. P., Yarger, J. L., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+beta-sheets+formation+in+synthetic+spider+silk+fibers+by+aqueous+post-spin+stretching.">Inducing beta-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching.</a> Biomacromolecules. 12, 2375-2381 (2011).</li><li>Elices, M., Guinea, G. V., Plaza, G. R., Karatzas, C., Riekel, C., Agullo-Rueda, F., Daza, R., Perez-Rigueiro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioinspired Fibers Follow the Track of Natural Spider Silk. Macromolecules. 44, 1166-1176 (2011).</li><li>Scheller, J., Guhrs, K. H., Grosse, F., Conrad, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+spider+silk+proteins+in+tobacco+and+potato.">Production of spider silk proteins in tobacco and potato.</a> Nature Biotechnology. 19, (2001).</li><li>Kojic, N., Kojic, M., Gudlavalleti, S., McKinley, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+removal+during+synthetic+and+Nephila+fiber+spinning.">Solvent removal during synthetic and Nephila fiber spinning.</a> Biomacromolecules. 5, 1698-1707 (2004).</li><li>Jeffery, F., La Mattina, C., Tuton-Blasingame, T., Hsia, Y., Gnesa, E., Zhao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+of+Black+Widow+Spider+Silk-producing+Glands.">Microdissection of Black Widow Spider Silk-producing Glands.</a> J. Vis. Exp. (47), e2382 (2011).</li><li>Blasingame, E., Tuton-Blasingame, T., Larkin, L., Falick, A. M., Zhao, L., Fong, J., Vaidyanathan, V., Visperas, A., Geurts, P., Hu, X., La Mattina, C., Vierra, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyriform+spidroin+1,+a+novel+member+of+the+silk+gene+family+that+anchors+dragline+silk+fibers+in+attachment+discs+of+the+black+widow+spider,+Latrodectus+hesperus.">Pyriform spidroin 1, a novel member of the silk gene family that anchors dragline silk fibers in attachment discs of the black widow spider, Latrodectus hesperus.</a> J. Biol. Chem. 284, 29097-29108 (2009).</li><li>La Mattina, C., Reza, R., Hu, X., Falick, A. M., Vasanthavada, K., McNary, S., Yee, R., Vierra, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+minor+ampullate+silk+proteins+are+constituents+of+prey+wrapping+silk+in+the+cob+weaver+Latrodectus+hesperus.">Spider minor ampullate silk proteins are constituents of prey wrapping silk in the cob weaver Latrodectus hesperus.</a> Biochemistry. 47, 4692-4700 (2008).</li><li>Hsia, Y., Gnesa, E., Jeffery, F., Tang, S., Vierra, C., Cuppoletti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+Silk+Composites+and+Applications.">Spider Silk Composites and Applications.</a> Metal, Ceramic and Polymeric Composites for Various Uses. 2, 303-324 (2011).</li></ol>

Çıkar çatışması ilan etti.

Bu metodoloji bükülmüş Sentetik elyaflar doğal liflerle karşılaştırıldığında aynı büyüklük sırasına mekanik vardır. Biriktirme ve sonrası dönüş beraberlik süreçleri makineleşme ile insan hatası miktarını azaltarak, numuneler arasındaki deneysel varyasyon daha kontrollü ve büyük ölçüde azaltılmış bulunmaktadır. Bizim metodoloji örümcek gen ailesinin diğer üyelerinin cDNAlarının gelen kodlanmış rekombinant proteinler bükülmüş olan diğer lif...

Örümcek ipek yüksek gerilimli çelik, Kevlar ve naylon gibi çeşitli sentetik malzemelerin dışarı gerçekleştirir olağanüstü mekanik özelliklere sahiptir. 1 Örümcekler çeşitli mekanik özellikleri gösterilecek en az 6-7 farklı fiber türleri, çekme mukavemeti ve genişletilebilirlik değişen miktarlarda tasarlanmış her dönmeye özgül biyolojik görevleri gerçekleştirmek için. 2 Araştırma bilim adamları hızla Çünkü yamyamlık 3,4. nedeniyle üstün mekanik özellikleri, biyouyumlu ve bunların toksik olmayan ve yeşil-maddi doğanın nesil Biyomalzeme olarak örümcek ipekleri kullanımı ve takip edilir arachnids ve zehirli doğa, tarım yoluyla örümcek ipekleri hasat endüstriyel ölçekte üretim için gerekli talepleri karşılamak için pratik bir strateji değildir. Bu nedenle, bilim gelen sentetik liflerin iplik in vitro ile birleştiğinde transjenik organizmalarda rekombinant ipek protein üretimi yönelmiştirse saflaştırılmış protein. tam uzunlukta rekombinant örümcek ipek proteinleri 5-8 İfade onların oldukça tekrarlayıcı doğası ve fiziksel uzunluğu (&gt; 15 kb) dahil kendi gen dizilerinin iç özellikleri, GC-zengin içerik verilen teknik olarak zor ve yanlı oldu alanin ve glisin kodon kullanımı. Bugüne kadar 9-11, en ​​laboratuarları kısmi cDNA dizileri veya sentetik genleri kullanarak büyük ampullate ipek proteinleri MaSp1 veya MaSp2 ile kesilmiş formlar ifade üzerine odaklanmıştır. 12-15 İplik sentetik örümcek ipekleri gerektiren zorlu bir süreçtir çeşitli bilimsel disiplinler ve eğirme sürecinin inceliklerini ustalığı ve bilgisi tamamen Video gösterimi tarafından kamuoyuna ortaya konamamıştır. Aslında, dünya genelinde laboratuarları sadece bir avuç örümcek ipeği cDNAlarının ifade ipek proteinleri arındırmak, sentetik lifler dönmeye ve post-spin beraberlik gerçekleştirmek için uzmanlığa sahip ve daha sonra nihayet kendi biyomateryal özelliklerini incelemektedir. 8,Iplik, sentetik lifler için 16,17 Farklı yaklaşımlar, ıslak ve kuru eğirme gibi elektro yöntemleri kapsayan var 16,18,19 Tüm işlemler tek bir ortak amacı var -. Mekanik özelliklere sahip sentetik örümcek ipliği üreten bir protokolünün geliştirilmesi rakip doğal konuları Büyük ölçekli ticari üretim işlemleri için. Burada bir ıslak eğirme yöntemi kullanarak bir laboratuar ölçekte yapay örümcek ipek üretmek için bir prosedür tanımlamaktır. Diğer eğirme yöntemleri ile karşılaştırıldığında, ıslak eğirme lif analizi için en tutarlı sonuçlar ortaya koymuştur. Onların saflaştırma takiben bakterilerde rekombinant ipek proteinleri, bir ifade ile, bu işlemi başlangıcından ana hatlarını ve sonra ile konuları verimleri &quot;olarak-bükülmüş&quot; lifler uygulanan bir post-sıkma çekme yöntemi de dahil olmak üzere, iplik için protein hazırlanması adımları tarif Doğal örümcek ipekleri kalitesi yaklaşmak malzeme özellikleri. Bizim methodology yakından ipek lifleri doğal eğirme süreci taklit etmek için tasarlanmıştır ve bu mimari ve Dahası küre ve koçanı-dokuma örümcekler. 20-22 gelen ipek üreten bezlerin fonksiyonu uzmanlığımız üzerine ağır çekiyor, biz gerekli ile sonuçlandırmak Araştırmacılar nihai gücü, nihai gerilme ve liflerin tokluk hesaplamak için izin gerilme-uzama eğrileri, çizmek için bir tensometre kullanarak sentetik malzeme özellikleri belirlemek için adımlar. Son olarak, ancak önemli değeri, iplik, biriktirme ve çizim aparatları oldukça karmaşık ve pahalı özel ekipman satın alma daha ticari parçalar kullanılarak ev inşa olabilir.

<table border="1"><tbody><tr><td> Reaktif / Ekipman </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> pBAD / TOPO ThioFusion İfade Kiti </td><td> Invitrogen </td><td> K370-01 </td><td></td></tr><tr><td> 10x Fastbreak Hücre Lizis Reaktif, </td><td> Promega </td><td> V857C </td><td></td></tr><tr><td> Ni-NTA Agaroz </td><td> Qiagen </td><td> 30210 </td><td> Tamponlar için talimatları içerir </td></tr><tr><td> ProteoSilver Gümüş Stain Kit </td><td> Sigma-Aldrich </td><td> PROTSIL1-1KT </td><td></td></tr><tr><td> FreeZone Lyophilizer </td><td> Labconco </td><td> 7960041 </td><td> FreeZone 12Plus </td></tr><tr><td> Heksafloroizopropanol (HFIP) </td><td> Sigma-Aldrich </td><td> 52512 </td><td></td></tr><tr><td> Şırınga </td&gt; <td> Hamilton </td><td> 7657-01 </td><td> 250 uL </td></tr><tr><td> Iğne </td><td> Hamilton </td><td> 7780-01 </td><td> 26s Ölçer, Blunt sonunda çıkarılabilir iğne </td></tr><tr><td> Şırınga Pompası </td><td> Harvard Apparatus </td><td> 702208 </td><td> 11Plus </td></tr><tr><td> Dijital Kaliper </td><td> Carrera </td><td> CP5906 </td><td> 0-150 mm aralığında </td></tr><tr><td> Paslanmaz çelik forseps </td><td> Dünya Hassas Aletler </td><td> 501764 </td><td> Mini Dumont # M5S </td></tr><tr><td> Motor </td><td> Doğa Mill </td><td> 7090529 </td><td> 12VDC, 2 rpm hız </td></tr><tr><td> Lineer Aktüatör </td><td> Warner Elektrik </td><td> 01-D024-0050-A06-LP-IP65 </td><td> 24VDC, 6 inç aralığında </td></tr><tr><td> Mikroskop Kesme </td><td> Leica Microsystems </td><td> Leica MZ16 </td><td></td></tr><tr><td> Dijital microscope kamera </td><td> Leica Microsystems </td><td> DFC320 </td><td> Yazılım: Leica Application Suite v2.8.1 </td></tr><tr><td> Vannas makas </td><td> Dünya Hassas Aletler </td><td> 500260 </td><td></td></tr><tr><td> Microtensometer </td><td> Aurora Bilimsel </td><td> 310C </td><td> 5N Çift Mod Sistemi </td></tr></tbody></table>

synthetic spider silk production on a laboratory scale

Toplumun ilerlemesi ve kaynakları kıt hale geldikçe, yeni teknolojiler geliştirmek için giderek önem kazanmaktadır yüksek performans özellikleri ile mühendis nesil biyomalzemeler söyledi. Bu yeni yapı malzemeleri geliştirme maliyet-etkin, hızlı olması ve işleme yöntemleri ve çevre dostu ve sürdürülebilir ürünler içermesi gerekmektedir. Örümcekler biyomimikri rakip iyi suni ve doğal malzemeler için yeni nesil mühendislik malzemelerinin zengin bir kaynak sunan, çeşitli mekanik özellikleri ile farklı elyaf türleri çok sayıda dönerler. Doğal örümcek ipeği büyük miktarlarda toplanması pratik olduğundan, sentetik ipek üretimi konuları sınırsız kaynağı erişimi olan bilim adamları sağlamak yeteneğine sahiptir. Eğirme süreci akıcı ve mükemmel olabilir, bu nedenle, yapay örümcek lifler vücut zırhı uzanan geniş bir uygulama yelpazesi, cerrahi dikiş için kullanılabilme potansiyeline sahiptirs, halat ve kablolar, lastikler, müzik aletleri için dizeleri ve havacılık ve uzay teknolojisi için kompozitler. Sentetik ipek üretim süreci ilerletmek için ve döndürmek için Spin kendi malzeme özelliklerini, düşük varyans gösterilecek fiberler elde etmek amacıyla, bakteriler, saflaştırma ve protein konsantrasyonu rekombinant örümcek ipek proteinlerinin ifadesini entegre bir ıslak eğirme-protokolü geliştirilen , fiber ekstrüzyon ve mekanik bir post-sıkma işlemi takip. Bu bir laboratuar ölçekte suni ipek lifleri spin ve analiz etmek için bir adım adım süreci ortaya çıkarmaktadır ilk görsel sunumudur. Ayrıca aynı iplik uyuşturucu bükülmüş lifler arasında değişkenlik giriş aza indirmek için ayrıntılı bilgi sağlar. Topluca, bu yöntemler, doğal örümcek ipek aşan yüksek kalitede elyaf yol açan, suni ipek, üretim işlemi itmek olacaktır.

Watch this Scientific Journal Video about Synthetic Spider Silk Production on a Laboratory Scale at JoVE.com

Synthetic Spider Silk Production on a Laboratory Scale

Örümcek ipek ve üstün mekanik özellikleri ve biyokimyasal rağmen, bu malzeme geleneksel yollarla, büyük miktarlarda hasat edilemez. Burada nesil Biyomalzeme olarak örümcek ipeği üretimi ve kullanımı eğitimi araştırmacılar için önemli bir süreç olan yapay örümcek ipeği lifleri, spin etkili bir strateji tanımlamak.

Bir Laboratuvar Ölçekli Sentetik Örümcek İpek Üretimi

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

synthetic-spider-silk-production-on-a-laboratory-scale

Research

JoVE Journal

Bioengineering

26.6K Görüntüleme.  University of the Pacific. Örümcek ipek ve üstün mekanik özellikleri ve biyokimyasal rağmen, bu malzeme geleneksel yollarla, büyük miktarlarda hasat edilemez. Burada nesil Biyomalzeme olarak örümcek ipeği üretimi ve kullanımı eğitimi araştırmacılar için önemli bir süreç olan yapay örümcek ipeği lifleri, spin etkili bir strateji tanımlamak.

Video: Synthetic Spider Silk Production on a Laboratory Scale

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Tek Sentetik Curli Operon Oluşturma tarafından Mühendislik Bağlanan Bakteriler

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Biyomühendislik

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly &alpha;-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable &beta;-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.

Electrospun Rekombinant Örümcek İpek Proteinlerin Nonwoven Ağlar dahil Hava Filtresi Cihazları

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Biyomedikal Uygulamalar için İpek-ipek Protein Alaşım Materyalleri Tasarlama

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

Üretim ve Monovalent Kuantum Noktalarının Hedefleme

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

İnsan Adenovirüs Tip 5 dayanarak Yüksek Kapasiteli Adenoviral Vektörler klonlanması ve Büyük Ölçekli Üretim

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Polarize Sinir Doku Engineered 3D İpek-kolajen bazlı Modeli

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

Laboratuvar ölçekte karıştırılmış Biyoreaktörler güç giriş ölçümlerde

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Isı ve basınç kullanarak sulu solvasyon yoluyla rekombinant örümcek Silks malzeme oluşumu

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate&#160;as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 &#956;L) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 &#956;M) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput&#160;gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

Sentetik Biyoloji ve Doğal Ürün Uygulamaları için Yüksek Verimli Streptomices Transkripsiyon-Çeviri Araç Seti

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.