Latest revision as of 04:00, 19 September 2015

iGEM UCLA

BACKGROUND

METHODOLOGY

RESULTS

BIOBRICKS

Materials Processing

Background

Abstract

Following genetic design of our constructs, we must express and process them into functional materials. Here, we highlight the methods that we used to take our silks from DNA to proteins and ultimately to fibers and films.

Introduction

To create functionalized fibers, we co-spun the NCSilkGFP with native Bombyx mori silk. The NCSilkGFP was designed such that the N and C termini have affinity to b.mori silk, allowing it to bind to the native silk upon spinning.

Methods for co-spinning silk with the NCSilkGFP

In brief, we followed the standard process as highlighted in the literature ^[1] to produce a concentrated aqueous solution, or dope, of commercially purchased b.mori silk. We then added aqueous NCSilkGFP to the concentrated b.mori dope so that the final solution had 750 grams of b.mori silk to 1 gram of NCSilkGFP. We then extruded this dope into a coagulation bath of 90% v/v isopropanol and water and collected the resulting fiber on a godet. Detailed steps are as follows:

Degumming

Silk is comprised of two main proteins, fibroin and sericin. Fibroin is the structural protein of the silk and our protein of interest. Sericin serves as the ‘glue’ of the silk. Degumming separates and removes sericin and is an essential preparation step before dissolving the silk. We found that commercially degummed silk was not properly degummed for our purposes and this step had to be carried out in-lab.

To degum, we boiled 2.5 grams of B.mori silk in 0.02M sodium carbonate solution for 30 minutes.

Solubilization

The resilience of silk to many different solvents is well known. In order to dissolve silk, we used lithium bromide, a strong chaotrope^[1] that disrupts the hydrogen bonds and thus secondary structure of the silk.

We dissolved our silk in 9.3M LiBr at 60 C for 4 hours.

Silk solubilized in 9.3M lithium bromide.

We dialyzed our silk in SnakeSkin Dialysis Tubing of 3.5 kDa molecular weight cutoff against double deionized water. We dialyzed for 48 hours with a total of 6 dialysis bath changes. Following dialysis, we centrifuged the solution to remove any flocculents and other insoluble proteins.

Concentration Dialysis

The previous dialysis results in an aqueous silk solution with concentration ranging from 4% w/v to 8% w/v (40 - 80 mg/mL). This is too dilute to spin into a fiber, so the solution must be concentrated by dialyzing against a highly concentrated polymer solution. The polymer solution draws water out from the silk solution by osmotic pressure.

To concentrate, we dialyzed 10 mL of aqueous silk solution in a Slide-A-Lyzer dialysis cassette of 3.5 kDa Molecular weight cutoff against a solution of 10% w/v 10,000 molecular weight PEG for 18 hours. This will yield an aqueous silk solution of 15-18% w/v (150-200 mg/mL) concentration. Dialyzing the solution for too long will overconcentrate the silk dope, which will lead to the silk forming a gel in the cassette. Once a silk has gelled, it is no longer usable in any further processing steps.

19% w/v silk dope after concentration dialysis.

Spiking with the NCSilkGFP

NCSilkGFP was added to the silk dope such that the resulting solution had a 1:750 mass to mass ratio of NCSilkGFP to native B.mori silk.

Spinning the composite silk

In order to form fibers from silk, soluble silk protein solutions must be much like how they are in natural spider spinnerets. The majority of spinning methods entail pushing, or extruding, silk solution through very thin channels. During this extrusion, shear forces on the silk solution cause the amino acids of the proteins to align in a way that allows the strong beta sheets of the silk structure to form. Multiple proteins are similarly aligned, causing separate proteins to interact and form larger structures.^[2]

We used a 21 gauge needle to load the silk dope into a 1 mL BD syringe with luer lok. We then replaced the needle with PEEK tubing of 0.127 mm inner diameter. We used a syringe pump to extrude the silk at a rate of 10 uL/min into a coagulation bath of 90% v/v isopropanol and water. After the fiber formed, we drew it out of the bath and wound it around a pair of tweezers that we spun by hand to collect the fiber as it formed. In later iterations of our spinning apparatus, we wound the fiber around a motorized godet to collect the fiber as it formed in order to automate the process. We designed the godet in OpenSCAD and 3D printed it in a Stratasys UPrint SE.

An early iteration of our silk spinning setup where we wound our fibers by hand around a pair of tweezers.

A later version of our setup. The glass dish contains the isopropanol bath. The godet is driven by a stepper motor.

A closeup of our spinner setup. The godet draws the fiber from the bath after it has formed.

Closeup of the co-spun silk wound around the godet.

Co-spun silk after removing from the godet. A small volume (~500 uL) of dope yields a very long contiguous length of fiber, but in order to remove from the godet without inadvertently stretching the fiber, we had to cut it.

Measuring fiber diameter and fluorescence

We used an EVOS light microscope to image the fibers under white light and blue excitation light. We used ImageJ to determine fluorescence of the fiber.

Testing the composite silk

We tested our co-spun fibers in an Instron model 5564 table mounted system with a 10kN load cell. To prepare our fibers for testing, we mounted them on cardstock frames with 10 mm gauge length. We then loaded the carded fibers into the Instron with pneumatic grips and cut the sides of the cardstock frame. We then had the machine extend the fiber at a rate of 0.1 mm/min and measure the tensile force of the fiber.

An example of the frames that we used to prepare the fibers for Instron testing.

Image of the fiber loaded into the Instron

Results

Fiber radius

We took images along three points on the fiber and used ImageJ to measure the diameter of the fiber at each point. We then averaged the measurements and found the fiber's radius to be 13.9 +/- 0.3 um.

Fiber fluorescence

We used an EVOS FL digital microscope to take microscopic images of our fibers. Natural silk showed some background fluorescence, but the co-spun fiber with NCSilkGFP had considerably stronger fluorescence, as determined by visual assessment. Unfortunately, we were unable to quantify the difference in fluorescence.

Fluorescence microscopy of the same fiber. 470 nm wavelength excitation light was used at 100% intensity in the EVOS FL digital microscope.

For comparison, this is fluorescence microscopy of native Bombyx mori silk. 470 nm wavelength excitation light was used at 100% intensity in the EVOS FL digital microscope.

Fiber Strength

Stress Strain curve for the co-spun silk with NCSilkGFP.

Our fiber had a Young's modulus of 5.39 GPa. The Young's modulus is a measure of the fiber's elasticity. The fiber's ultimate strength, or stress at which failure occurs, was 103.4 +/- 6.6 MPa. The toughness of the fiber, which is the total energy that the fiber absorbed before breaking, was 1.47 megaJoules. The maximum strain, or deformation, that the fiber tolerated before breaking was 2.2% of the original unstressed length (10mm).

Overall, this fiber that we co-spun was extremely stiff and brittle and did not match the properties of natural silk. However, this could be largely attributed to the fact that we did not stretch the fiber after spinning, which is known to be a crucial process that confers silk with much of its natural strength and elasticity.^[3] It has been determined in the literature that silk reconstituted from silk dopes and stretched to several times its length can have properties matching or even exceeding those of natural silk fibers.^[6] Stretching is known to cause further formation of beta sheet crystals in the fiber, as well as align them in the same axis which allows them to form more hydrogen bonds with each other, thereby increasing the fiber's overall strength.^[6] We still need to stretch our co-spun silk after spinning in order to determine if the NCSilkGFP actually interferes with the structure and strength of the reconstituted silk.

Stress Strain curves for the natural B.mori silk and the co-spun silk with NCSilkGFP. It is very clear that our co-spun fiber is vastly inferior to the natural silk in every measure

Methods for Producing Recombinant Silk Films

Dialyzing silk to remove ions and prepare for lyophilization

Following recombinant protein expression and purification, the solution contains many ions and other molecules, which are easily removed by dialysis. We dialyzed the solution in a SnakeSkin tube with 3.5 kDa MWCO against 5mM ammonium bicarbonate for 24 hours with 2 total bath changes. Dialyzing against ammonium bicarbonate also prepares the solution for lyophilization, as ammonium bicarbonate facilitates the sublimation of water in the lyophilizer.^[5]

Lyophilization

We froze the dialyzed silk solution using liquid nitrogen and then lyophilized the frozen sample in a LabConco FreeZone 4.5-105. Lyophilization allows us to recover all of the protein that was purified, which is around 5 to 8 mg from 1 L of recombinant cell culture.

The lyophilized silk can be seen on the walls of the tube. In this form, the silk is pure and without any solvent.

Dissolving in HFIP

Very few solvents are capable of dissolving pure silk. HFIP, a highly polar organic solvent, is one of them.^[1] We dissolved our recombinant silk in HFIP to obtain a dilute silk dope of 5% w/v (50 mg/mL).

Casting films and water annealing

We pipetted 100 uL of silk dope into a small weigh boat and let it dry in a fume hood for 24 hours. We then removed the film from the weigh boat and placed it into a vacuum desiccator along with water in a separate container. Vacuum was then applied to the desiccator, and the film was allowed to anneal for 24 hours. The water annealing process encourages further formation of the silk's secondary structure, giving the film stability in aqueous environments as well as added strength.^[4]

Measuring thickness of the film

A Vernier micrometer was used to measure the thickness of the film.

Mechanical testing

Just as with the fibers, strips of the fiber were mounted onto cardstock frames with 10mm gauge lengths. The mounted strips were then loaded into the Instron for testing.

Results for Recombinant Silk Films

Unfortunately, as of 18 September 2015, we haven't gotten around to assaying the mechanical properties of our silk films yet. Any updates and data obtained after the wiki freeze (18 September 2015) will be uploaded to our team's OpenWetWare page. Please note that any and all data hosted on that page is ineligible for consideration by the iGEM wiki judges.

References

^[1]Rockwood, D., Preda, R., Yücel, T., Wang, X., Lovett, M., & Kaplan, D. (2011). Materials fabrication from Bombyx mori silk fibroin. Nat Protoc Nature Protocols, 1612-1631. ^[2]

Teulé, F., Cooper, A., Furin, W., Bittencourt, D., Rech, E., Brooks, A., & Lewis, R. (n.d.). A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nat Protoc Nature Protocols, 341-355.

^[3]

Albertson, A., Teulé, F., Weber, W., Yarger, J., & Lewis, R. (n.d.). Effects of different post-spin stretching conditions on the mechanical properties of synthetic spider silk fibers. Journal of the Mechanical Behavior of Biomedical Materials, 225-234.

^[4]

Hu, X., Shmelev, K., Sun, L., Gil, E., Park, S., Cebe, P., & Kaplan, D. (2011). Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing. BioMacroMolecules, 1686-1696.

^[5]

Ausubel, F. (1990). Current protocols in molecular biology. New York: Greene Pub. Associates and Wiley-Interscience :.

^[6]