Name: synthetic spider silk production on a laboratory scale
Uploaded: 2012-07-18T12:00:00
Description: Watch this Scientific Journal Video about Synthetic Spider Silk Production on a Laboratory Scale at JoVE.com

This work was supported by NSF RUI Grants MCB-0950372 and DMR-1105310 entitled "Molecular Characterization of Black Widow Spider Silks and Mechanical Behavior of Spider Glue Silks," respectively.

<img alt="Figure 11" src="/files/ftp_upload/4191/4191fig1overview.jpg" /> 
Graphical Overview: Biomimicry of the Spinning Process
Biomimicry of the natural spider silk production pathway: a route to manufacture synthetic silk. This image shows the major ampullate gland from the golden orb weaver, Nephila clavipes, and the components utilized for natural silk production (white text). The tail region synthesizes large quantities of silk proteins that are t...

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	<li>Gosline, J. M., Guerette, P. A., Ortlepp, C. S., Savage, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+design+of+spider+silks:+from+fibroin+sequence+to+mechanical+function.">The mechanical design of spider silks: from fibroin sequence to mechanical function.</a> J. Exp. Biol. 202, 3295-3303 (1999).</li><li>Foelix, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of spiders. , (1996).</li><li>Vollrath, F., Knight, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+crystalline+spinning+of+spider+silk.">Liquid crystalline spinning of spider silk.</a> Nature. 410, 541-548 (2001).</li><li>Spiess, K., Lammel, A., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+spider+silk+proteins+for+applications+in+biomaterials.">Recombinant spider silk proteins for applications in biomaterials.</a> Macromol. Biosci. 10, 998-1007 (2010).</li><li>Stark, M., Grip, S., Rising, A., Hedhammar, M., Engstrom, W., Hjalm, G., Johansson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroscopic+fibers+self-assembled+from+recombinant+miniature+spider+silk+proteins.">Macroscopic fibers self-assembled from recombinant miniature spider silk proteins.</a> Biomacromolecules. 8, 1695-1701 (2007).</li><li>Lazaris, A., Huang, Y., Zhou, J. F., Duguay, F., Chretien, N., Welsh, E. A., Soares, J. W., Karatzas, C. N. <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+Fibers+Spun+from+Soluble+Recombinant+Silk+Produced+in+Mammalian+Cells.">Spider Silk Fibers Spun from Soluble Recombinant Silk Produced in Mammalian Cells.</a> Science. 295, 472-476 (2002).</li><li>Teule, F., Cooper, A. R., Furin, W. A., Bittencourt, D., Rech, E. L., Brooks, A., 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=A+protocol+for+the+production+of+recombinant+spider+silk-like+proteins+for+artificial+fiber+spinning.">A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.</a> Nat. Protoc. 4, 341-355 (2009).</li><li>Gnesa, E., Hsia, Y., Yarger, J. L., Weber, W., Lin-Cereghino, J., Lin-Cereghino, G., Tang, S., Agari, K., 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=Conserved+C-Terminal+Domain+of+Spider+Tubuliform+Spidroin+1+Contributes+to+Extensibility+in+Synthetic+Fibers.">Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers.</a> Biomacromolecules. , (2011).</li><li>Hayashi, C. Y., Shipley, N. H., 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=Hypotheses+that+correlate+the+sequence,+structure,+and+mechanical+properties+of+spider+silk+proteins.">Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins.</a> Int. J. Biol. Macromol. 24, 271-275 (1999).</li><li>Xu, M., 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=Structure+of+a+protein+superfiber:+Spider+Dragline+Silk.">Structure of a protein superfiber: Spider Dragline Silk.</a> Proc. Natl. Acad. Sci. 87, 7120-7124 (1990).</li><li>Hayashi, C. Y., Blackledge, T. 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. N. <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+fibers+spun+from+soluble+recombinant+silk+produced+in+mammalian+cells.">Spider silk fibers spun from soluble recombinant silk produced in mammalian cells.</a> Science. 295, 472-476 (2002).</li><li>Arcidiacono, S., Mello, C., Kaplan, D., Cheley, S., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+recombinant+spider+silk+expressed+in+Escherichia+coli.">Purification and characterization of recombinant spider silk expressed in Escherichia coli.</a> Appl. Microbiol. Biotechnol. 49, 31-38 (1998).</li><li>Menassa, R., Zhu, H., Karatzas, C. 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. 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Synthetic fibers spun from this methodology are mechanically on the same order of magnitude compared to the natural fibers. By decreasing the amount of human error by mechanizing the spooling and post spin draw processes, the experimental variation between samples are more controlled and greatly reduced. 
Our methodology offers the potential to investigate the mechanical properties of other fibers that are spun from recombinant proteins encoded from the cDNAs of other members of spider gene fa...

Spider silk has extraordinary mechanical properties that out performs several manmade materials, including high-tensile steel, Kevlar and Nylon.1 Spiders spin at least 6-7 different fiber types that display diverse mechanical properties, each designed with varying amounts of tensile strength and extensibility to perform specific biological tasks.2 Research scientists are rapidly pursuing the use of spider silks as next generation biomaterials because of their outstanding mechanical properties, their biocompatibility, and their non-toxic and green-material nature.3,4 Because of the cannibalistic and venomous nature of arachnids, harvesting spider silks through farming is not a practical strategy to meet the demands necessary for industrial scale manufacturing. Therefore, scientists have turned to the production of recombinant silk proteins in transgenic organisms coupled with in vitro spinning of synthetic fibers from these purified proteins.5-8 Expression of full-length recombinant spider silk proteins has been technically difficult given the intrinsic properties of their gene sequences, which include their highly repetitive nature and physical lengths (&gt;15 kb), GC-rich content and biased alanine and glycine codon usage.9-11 To date, most labs have focused on expressing truncated forms of the major ampullate silk proteins MaSp1 or MaSp2 using partial cDNA sequences or synthetic genes.12-15 Spinning synthetic spider silks is a challenging process that requires mastery and knowledge in several scientific disciplines, and the intricacies of the spinning process have not been fully revealed to the general public by video representation. In fact, only a handful of labs across the globe have the expertise to express the spider silk cDNAs, purify the silk proteins, spin synthetic fibers and perform post-spin draw, and then finally test their biomaterial properties.8, 16,17 Different approaches for spinning synthetic fibers have encompassed wet and dry spinning as well as electrospinning methods.16,18,19 All procedures have one goal in common - development of a protocol that produces synthetic spider silk with mechanical properties that rival natural threads for large-scale commercial manufacturing processes.
Here we describe the procedure to generate artificial spider silks on a laboratory scale using a wet-spinning methodology. Relative to other spinning methods, wet spinning has produced the most consistent results for fiber analysis. We outline this procedure beginning with the expression of the recombinant silk proteins in bacteria, followed by their purification, and then describe the protein preparation steps for spinning, including a post-spin draw methodology applied to "as-spun" fibers that yields threads with material properties that approach the quality of natural spider silks. Our methodology is designed to closely mimic the natural spinning process of silk fibers and it draws heavily upon our expertise of the architecture and function of the silk-producing glands from orb- and cob-weaving spiders.20-22 Furthermore, we conclude with the necessary steps to determine the material properties of the synthetic fibers using a tensometer to plot stress-strain curves, which allow investigators to calculate the ultimate strength, ultimate strain, and toughness of fibers. Lastly, but of significant value, the spinning, spooling, and drawing apparatuses can be home-built using commercially available parts, rather than purchasing elaborate and costly customized equipment.

<table border="1">
 <tbody>
 <tr>
 <td>Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>pBAD/TOPO ThioFusion Expression Kit</td>
 <td>Invitrogen</td>
 <td>K370-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FastBreak Cell Lysis Reagent, 10x</td>
 <td>Promega</td>
 <td>V857C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ni-NTA Agarose</td>
 <td>Qiagen</td>
 <td>30210</td>
 <td>Includes instructions for buffers</td>
 </tr>
 <tr>
 <td>ProteoSilver Silver Stain Kit </td>
 <td>Sigma-Aldrich</td>
 <td>PROTSIL1-1KT</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FreeZone Lyophilizer</td>
 <td>Labconco</td>
 <td>7960041</td>
 <td>FreeZone 12Plus</td>
 </tr>
 <tr>
 <td>Hexafluoroisopropanol (HFIP)</td>
 <td>Sigma-Aldrich</td>
 <td>52512</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Hamilton</td>
 <td>7657-01</td>
 <td>250 &mu;L</td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>Hamilton</td>
 <td>7780-01</td>
 <td>26s Gauge, Blunt end removable needle</td>
 </tr>
 <tr>
 <td>Syringe Pump</td>
 <td>Harvard Apparatus</td>
 <td>702208</td>
 <td>11Plus</td>
 </tr>
 <tr>
 <td>Digital Caliper</td>
 <td>Carrera</td>
 <td>CP5906</td>
 <td>0-150 mm range</td>
 </tr>
 <tr>
 <td>Stainless steel forceps</td>
 <td>World Precision Instruments</td>
 <td>501764</td>
 <td>Mini Dumont #M5S</td>
 </tr>
 <tr>
 <td>Motor</td>
 <td>Nature Mill</td>
 <td>7090529</td>
 <td>12VDC, 2 rpm speed</td>
 </tr>
 <tr>
 <td>Linear Actuator</td>
 <td>Warner Electric</td>
 <td>01-D024-0050-A06-LP-IP65</td>
 <td>24VDC, 6 inch range</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Leica Microsystems</td>
 <td>Leica MZ16</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital microscope camera</td>
 <td>Leica Microsystems</td>
 <td>DFC320</td>
 <td>Software: Leica Application Suite v2.8.1</td>
 </tr>
 <tr>
 <td>Vannas scissors</td>
 <td>World Precision Instruments</td>
 <td>500260</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microtensometer</td>
 <td>Aurora Scientific</td>
 <td>310C</td>
 <td>5N Dual-Mode System</td>
 </tr>
 </tbody>
</table>

synthetic spider silk production on a laboratory scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

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

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

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

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Bioengineering

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

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.

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.

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.

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.

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.

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.

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.

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 ...

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.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.