The authors would like to gratefully acknowledge funding from the Utah Science and Technology Research (USTAR) initiative.

1. Recombinant spider silk mixture preparation from lyophilized protein stocks
<ol>
	<li>Determine the necessary formulation and volume required for the intended material formations. Typical formulations range from 3% (w/v) up to 15% (w/v). Using this selection, calculate the appropriate rSSp, concentrations, and ratios.
	<ol>
		<li>Use the following respective formulations to prepare each material described in this protocol: hydrogels/sponges/lyogels, 6% (w/v) 50:50 MaSp1:MaSp2; films/coatings, 5% (w/v) 80:20 MaSp1:MaSp2; adhesives, 12% (w/v) 50:50 MaSp1:MaSp2; fibers, 12.5% (w/v) 80:20 MaSp1:MaSp2. 
		NOTE: Even though most formulations are better fitted for specific forms and materials, there is a wide range of formulations that can often overlap. Additionally, the final rSSp materials can also be tailored during formation and processing to produce the desired properties. Generally, each protein will require investigation into appropriate or useful parameters.</li>
	</ol>
	</li>
	<li>Select a clean and new 8 mL autoclavable borosilicate glass culture vial with a rubber lined screw cap.</li>
	<li>Remove the cap and place the empty vial on an analytical balance. Tare the mass of the empty vial so that the balance reads zero mass.</li>
	<li>Add the desired lyophilized rSSp powder to the empty vial for each specific material.
	<ol>
		<li>Use these specific masses of each protein type for each material, when preparing a 2 mL of solution: hydrogels/sponges/lyogels, 60 mg of MaSp1 and 60 mg of MaSp2; films/coatings, 80 mg of MaSp1 and 20 mg of MaSp2; adhesives, 120 mg of MaSp1 and 120 mg of MaSp2; fibers, 200 mg of MaSp1 and 50 mg of MaSp2.</li>
	</ol>
	</li>
	<li>Add the desired amount of ultrapure water, at least 2 mL, to the vial that already contains the weighted rSSp powders. 
	NOTE: A minimal volume of 2 mL is recommended for all solvation procedures.</li>
	<li>Seal the vial cap and briskly vortex the contents to create a dispersed, and homogenous, rSSp mixture that is now ready for the solvation procedure. Additional homogenization approaches such as sonication or impeller mixing can be employed with, or in addition, the vortex mixing.</li>
</ol>
2. Recombinant spider silk solvation
CAUTION: High heats and pressures are generated during the solvation procedure. Proper personal protective equipment, especially goggles, long sleeves, and heat resistant gloves are required for this process.
<ol>
	<li>Perform a final check of the vial, or vessel, cap to ensure that it has been firmly and securely tightened. Then transfer the suspended rSSp mixture to a conventional microwave oven. 
	NOTE: Microwave units within the power range of 700 to 1,500 Watts, possessing smaller internal chamber capacities, and rotating platforms are recommended to provide better solvation conditions.</li>
	<li>Begin operation of the microwave with 5 s bursts at full power. After each burst briefly open the door and carefully mix/swirl the vial to prevent settling and maintain the suspended mixture.</li>
	<li>Repeat this microwave process until the mixture and/or solution has obtained a temperature of at least 130 &#176;C, when measured with an infrared thermometer directly against the solution containing portions of the vial. Repeat this process until all of the solid particulates have been completely dissolved and are no longer visible. 
	NOTE: It is suggested to allow the vial and solution to cool occasionally, especially if the formulation has a high concentration of rSSp present. Temperatures exceeding 200 &#176;C increase the risk of vial seal failure. Special attention must also be given to prevent the superheated mixture/solution from touching the seal, which will also result in vial containment failure.</li>
	<li>After successfully solvating the rSSp mixture into a solution allow the temperature of the solution and the vial cap to cool below 100 &#176;C (boiling point) before opening.</li>
</ol>
3. Hydrogels
<ol>
	<li>Prepare a hydrogel from the solution after removing it from the microwave and allowing it to cool and set. Cast the hydrogel into specific geometries prior to allowing it to fully cool. 
	NOTE: Different rSSps will require varied amounts of times to transition to a hydrogel. For example, MaSp2-like sequences tend to form hydrogels more rapidly in comparison to MaSp1-like sequences. Protein concentrations, salinity, and pH also directly affect the rate of transition to a hydrogel.</li>
</ol>
4. Sponges
<ol>
	<li>Prepare a rSSp sponges by first allowing the primary solvated solution to form a hydrogel.</li>
	<li>Place the hydrogel in a water bath, place this bath in the freezer at -20 &#176;C, and wait until the bath is frozen completely.</li>
	<li>Complete the sponge formation process by removing the frozen hydrogel and water bath from the freezer and thawing at 25 &#176;C. The resultant sponge can now be removed from the thawed water.</li>
</ol>
5. Lyogel
<ol>
	<li>Prepare a rSSp lyogel by directly freezing a formed hydrogel, either with or without a water bath, and transferring the frozen hydrogel sample to a lyophilizer (freeze dryer).</li>
	<li>Remove the final lyophilized gel material from the vessel that the moisture sublimation occurred in.</li>
</ol>
6. Films and coatings
<ol>
	<li>Use one of the three following methods: solution casting, solution spraying, or dip coating to produce films or coatings of rSSp.
	<ol>
		<li>Cast the solubilized silk solution into/onto PDMS forms of the desired shape.</li>
		<li>Pour and spread 200 &#181;L of the film solution and allow this to dry before peeling them off of the PDMS substrate for testing or treatment.</li>
		<li>After allowing these to dry, remove the formed films for mechanical testing or post-treat the films to improve the mechanical properties.</li>
	</ol>
	</li>
	<li>To prepare a coating, or a film that cannot be removed from the substrate, use either spray or dip coating to produce a thin film layer. 
	NOTE: To spray coat, this protocol has found success with a master airbrush model paint sprayer.
	<ol>
		<li>Form a dip coating by simply submerging the substrate of choice into the solubilized rSSp and repeat after drying to achieve the desired thickness.</li>
		<li>Perform an initial spray coat before applying a dip coat to increase the consistency and effectiveness of the final coating.</li>
	</ol>
	</li>
</ol>
7. Adhesives
NOTE: The formation of adhesives is achieved through one of the following methods.
<ol>
	<li>Directly add the solubilized rSSp onto a substrate and then apply a second substrate over the top of the solution. Firmly clamp the pieces together and then dry the samples in an oven with a minimal temperature of 25 &#176;C for at least 16 h.</li>
	<li>Alternatively, spray the two substrate surfaces with a spray coating and then clamp the substrates together.</li>
	<li>Applying the rSSp through the dip method of coating the substrates and sticking the substrates can also be used to prepare and adhesive.</li>
</ol>
8. Wet-Spun fibers
<ol>
	<li>Load the solubilized dope solution into a concentric syringe with Luer-Lok tip through a 19 G glide needle. Eject air bubbles and let the dope sit at the Luer-Lok end of the syringe.</li>
	<li>Insert at least 25 mm of PEEK tubing, internal diameter 0.01 inch, into the PEEK tubing&#8217;s one-piece finger tight Fittings for 1/16 inch OD and 10/32 cone. Attach this fitting to a PEEK tubing to Luer-Lok female adapter.
	<ol>
		<li>Replace the 19 gauge needle with this set up on the loaded syringe.</li>
	</ol>
	</li>
	<li>Fill a tall, clear glass bath with 99% pure isopropanol to use for coagulation bath.
	<ol>
		<li>Fill the stretch baths, located below the stretch godets. These will have 80:20 isopropanol: distilled water in the first stretch bath, and 20:80 isopropanol: distilled water in the second stretch bath.</li>
	</ol>
	</li>
	<li>Set the godet stretch system such that the first godet after the coagulation bath and the first godet in the first stretch bath are rotating at the same speed.
	<ol>
		<li>Initiate the first stretch by adjusting the speeds of the final godet in stretch bath 1, the middle upper godet, and the first godet in stretch bath 2 to the same speed. This speed will be 2x as fast as the initial fiber removal speed.</li>
		<li>Initiate the second stretch by adjusting the speeds of the final godet in stretch bath 2, the last upper godet, and the winder to the same speed. This speed will be 2x as fast as the speed used for the first stretch or 4x the initial fibers removal speed.</li>
		<li>Place nitrile gloves on the outside of the intermediate godets to keep the fiber from slipping.</li>
	</ol>
	</li>
	<li>Start to slowly extrude the solution into the coagulation bath. In an automated system set the extrusion rate to match a removal speed of 10 mm/s.
	<ol>
		<li>Allow fiber extrusion to become uniform before pulling the fibers out of the bath using with a thin metal hook or forceps. Verify removing the fiber from the bath created a loop between the PEEK tubing tip and path the fiber leaving the bath the bath.</li>
	</ol>
	</li>
	<li>Guide the retrieved fiber through the series of godets such that the fiber is submerged in the stretch baths but drying in the air between the stretch baths and before going onto a spool. This drying is achieved by the higher placed intermediate godets. 
	NOTE: The fiber removal rate and/or extrusion rate will need to be adjusted based upon the protein concentration, additives, and protein type to allow ample coagulation time without pooling fibers on the bottom of the coagulation bath.</li>
	<li>Attach the fully stretched fiber to the spool on the winder mechanism using tape.</li>
</ol>

<ol>
	<li>Huemmerich, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Structure+Elements+of+Spider+Dragline+Silks+and+Their+Contribution+to+Protein+Solubility.">Primary Structure Elements of Spider Dragline Silks and Their Contribution to Protein Solubility.</a> Biochemistry. 43 (42), 13604-13612 (2004).</li><li>Schacht, K., 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=Controlled+Hydrogel+Formation+of+a+Recombinant+Spider+Silk+Protein.">Controlled Hydrogel Formation of a Recombinant Spider Silk Protein.</a> Biomacromolecules. 12 (7), 2488-2495 (2011).</li><li>Jones, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+Than+Just+Fibers:+An+Aqueous+Method+for+the+Production+of+Innovative+Recombinant+Spider+Silk+Protein+Materials.">More Than Just Fibers: An Aqueous Method for the Production of Innovative Recombinant Spider Silk Protein Materials.</a> Biomacromolecules. 16 (4), 1418-1425 (2015).</li><li>Tucker, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+Physical+Properties+of+Recombinant+Spider+Silk+Films+Using+Organic+and+Aqueous+Solvents.">Mechanical and Physical Properties of Recombinant Spider Silk Films Using Organic and Aqueous Solvents.</a> Biomacromolecules. 15 (8), 3158-3170 (2014).</li><li>Harris, T. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Sticky+Situation:+An+Investigation+of+Robust+Aqueous-Based+Recombinant+Spider+Silk+Protein+Coatings+and+Adhesives.">A Sticky Situation: An Investigation of Robust Aqueous-Based Recombinant Spider Silk Protein Coatings and Adhesives.</a> Biomacromolecules. 17 (11), 3761-3772 (2016).</li><li>Jones, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+Heat+and+Pressure+for+Solubilization+of+Recombinant+Spider+Silk+Proteins+in+Aqueous+Solution.">Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.</a> International Journal of Molecular Sciences. 17 (11), 1955 (2016).</li><li>Copeland, C. G., Bell, B. E., Christensen, C. D., 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=Development+of+a+Process+for+the+Spinning+of+Synthetic+Spider+Silk.">Development of a Process for the Spinning of Synthetic Spider Silk.</a> ACS Biomaterials Science and Engineering. 1 (7), 557-584 (2015).</li><li>Arcidiacono, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+Processing+and+Fiber+Spinning+of+Recombinant+Spider+Silks.">Aqueous Processing and Fiber Spinning of Recombinant Spider Silks.</a> Macromolecules. 35 (4), 1262-1266 (2002).</li><li>Work, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Major+Ampullate+Silk+Fiber+Formation+by+Orb-Web-Spinning+Spiders.">Mechanisms of Major Ampullate Silk Fiber Formation by Orb-Web-Spinning Spiders.</a> Transactions of the American Microscopical Society. 96 (2), 170-189 (1977).</li><li>Decker, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+Destruction+of+Endotoxin+in+Synthetic+Spider+Silk+Proteins.">Method for the Destruction of Endotoxin in Synthetic Spider Silk Proteins.</a> Scientific Reports. 8 (12166), 1-6 (2018).</li></ol>

The authors declare no conflict of interest.

After recombinant spider silk proteins are purified they have to then be prepared in a solution that can be used for material formation. By mixing lyophilized spider silk protein with water and exposing this mixture to microwave irradiation, to generate heat and pressure, it possible to prepare a rSSp solution. A wide variety of material forms can be produced from this simple and efficient method of rSSp solubilization. Each material has to be uniquely prepared and processed to achieve the desired outcome and properties....

Spider silks have garnered the interest of material scientists for their impressive combination of strength, elasticity, and biocompatibility. Recreating fibers has traditionally been the thrust of the research. This effort was hampered by the recombinant spider silk protein (rSSp) insolubility in water as well as the inability of traditional solvation techniques (chaotropic agents and detergents) to achieve aqueous solvation. Further, techniques that have been developed for solvating versions of rSSp do not work on all rSSp variants and also require substantial manipulation and time that often results in protein loss1,2. This has largely resulted in the field utilizing 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) as a solvent from which to form fibers, and other limited material forms. The advantage being that all known rSSp are soluble in HFIP, providing data uniformity between each research group. The disadvantage is that HFIP is a toxic solvent that is expensive and impractical to scale due to health concerns and environmental considerations.
A novel approach to rSSp solvation was developed that bridged the technological gap between the harsh organic solvent HFIP and other techniques that selectively worked for rSSp solvation. The combination of specific heats and pressures was applied to suspensions of rSSp and water. The results were near 100% solvation and recovery of the rSSp, as well as high protein concentrations; a variety of materials forms were determined to be possible from these formulations that were not all achievable using HFIP or other organic solvents3,4,5,6. The objective of this approach is to efficiently and easily solubilize purified and dried recombinant spider proteins in an aqueous solution that can then be utilized for the production of a variety of material forms.
Fibers, films, coatings, adhesives, hydrogels, lyogels, microspheres, and sponge materials are all readily accomplishable from the same aqueous rSSp solution using this method. The continued evolution of this method, not only with additional rSSp but with other proteins, could lead to new material forms and alternative protein purification and solubilization avenues.

<table border="0" cellpadding="0" cellspacing="0" style="width:697px;" width="698">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">3 mL Syringe with Luer-Lok Tip</td>
			<td style="width:148px;">BD</td>
			<td style="width:119px;">309657</td>
			<td style="width:215px;">Other size syringes can be used but to keep the tips on, it is advised to use luer-lok tips</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4 mL culture vial, clear with rubber lined cap</td>
			<td style="width:148px;">Wheaton</td>
			<td style="width:119px;">225142</td>
			<td style="width:215px;">Minimum dope volume is 1mL, max is 2mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">8 mL culture vial, clear with rubber lined cap</td>
			<td style="width:148px;">Wheaton</td>
			<td style="width:119px;">225144</td>
			<td style="width:215px;">Minimum dope volume is 2mL, max is 4mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">99% Isopropyl Alcohol, Reagent ACS/USP Grade</td>
			<td style="width:148px;">Pharmco-Aaper</td>
			<td style="width:119px;">231000099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Freezone 4.5 Plus</td>
			<td style="width:148px;">Labconco</td>
			<td style="width:119px;">7386030</td>
			<td>Freeze Dryer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luer Adapter Female Luer x 10-32 Female, Tefzel (ETFE)</td>
			<td style="width:148px;">IDEX</td>
			<td style="width:119px;">P-629</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Microwave</td>
			<td style="width:148px;">Magic Chef</td>
			<td style="width:119px;">HMD1110B</td>
			<td style="width:215px;">120V, 60Hz AC; 1000 watts; 1.1 cu. ft. capacity; with glass turn table</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">One-Piece Fingertight 10-32 Coned, for 1/16&#34; OD</td>
			<td style="width:148px;">IDEX</td>
			<td style="width:119px;">F-120X</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PEEK Tubing 1/16&#34; OD x 0.010&#34; ID</td>
			<td style="width:148px;">IDEX</td>
			<td style="width:119px;">1531B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sprayer: Master Airbrush</td>
			<td style="width:148px;">Master Airbrush</td>
			<td style="width:119px;">TC-60</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

From the described method of solubilization of rSSp, a variety of material forms can be achieved as seen in Figure 1. The method of solubilization is to apply heat and pressure, generated by a conventional microwave, to a suspension of rSSp and water. When critical temperatures and pressures are achieved, the protein will solubilize. From this solubilized rSSp solution, the required conditions are presented for seven material forms: hydrogels, lyogels, sponge...

Watch this Scientific Journal Video about Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure at JoVE.com

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

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 ...

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5.1K Views.  Utah State University. 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.

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

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

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.

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 ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

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 ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen&#8482; QuantiGene&#8482; Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP&#174; magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&#38;E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads for drug delivery applications. Despite their promise, polymersomes are limited in application due to their spherical shape, which is not readily taken up by cells, as demonstrated by solid nanoparticle scientists. This article describes a salt-based method for increasing the aspect ratios of spherical poly(ethylene glycol) (PEG)- based polymersomes. This method can elongate polymersomes and ultimately control their final shape by adding sodium chloride in post-formation dialysis. Salt concentration can be varied, as described in this method, based on the hydrophobicity of the block copolymer being used as the base for the polymersome and the target shape. Elongated nanoparticles have the potential to better target the endothelium in larger diameter blood vessels, like veins, where margination is observed. This protocol can expand therapeutic nanoparticle applications by utilizing elongation techniques in tandem with the dual-loading, long-circulating benefits of polymersomes.

Polymersomes are self-assembled polymeric vesicles that are formed in spherical shapes to minimize Gibb's Free Energy. In the case of drug delivery, more elongated structures are beneficial. This protocol establishes methods to create more rod-like polymersomes, with elongated aspect ratios, using salt to induce osmotic pressure and reduce internal vesicle volumes.

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads ...