The overall goal of this procedure is to produce synthetic spider silk by mimicking the natural spider silk spinning process to replicate the process of spinning silk in the lab. Large amounts of recombinant silk protein are produced in bacteria following purification by chromatography and concentration by lyophilization. The protein is solubilized and the spinning dope is pushed through a syringe with a small diameter needle into a dehydrating alcohol bath.
Finally, the synthetic as spun fibers are subject to a post spin draw to enhance the molecular orientation of the protein chains. The main advantage of this technique over existing methods like electro spinning or dry spinning, is the lower cost ease of manipulation and the potential to expand the procedure into a large scale production format. The implications of this technique extend toward developing a new class of biomaterials.
These materials can be used for a broad range of different applications, such as sutures, body armor, as well as strings for musical instruments. Though this method can provide insight into the material properties of spider silks, it can also be applied to studying other types of biopolymers spun from recombinant proteins. Generally, individuals new to this technique will struggle because it requires expertise in many disciplines, including bioengineering, biophysics, cellular, and molecular biology and biochemistry.
We first had the idea for this method when we needed to mimic the natural spinning process performed by the spider in an attempt to produce synthetic fibers To generate the plasmid that will be used for spider silk production. The desired spider silk CDNA is amplified using specific primer sets and ligated into a prokaryotic expression vector, such as PBA topo thio fusion. This vector has an end terminal thio redoxin tag to facilitate silk protein solubilization and a C terminal six x his tag for protein purification.
Once ligated, the ligation products are transformed into competent e coli cells and a plasmid containing colony is cultured. Spider silk protein expression is then induced by the addition of aose and cells are pelleted and stored at minus 80 degrees Celsius until use to ly the bacterial cells at 20 milliliters of one x lysis buffer to the cell pellet resus suspend the cells completely next to promote further cell lysis. Add lysozyme to a concentration of one milligram per milliliter to digest chromosomal DNA, which reduces the viscosity of the solution at 100 micrograms of DNA.
Then place the sample on an orbital shaker and gently rock for 20 minutes at room temperature. Sonicate the solution for one minute at setting 19 to clarify the solution centrifuge at 16, 000 Gs for 10 minutes at four degrees Celsius. Following the spin check to see that the supernatant is non viscous and clear, if the solution is viscous and cloudy, add more DNAs than sonicate and reell at the supernatant.
Once the supernatant is clear, transfer it into a clean 25 milliliter BioRad poly prep column for nickel NTA affinity column chromatography. To purify the fusion protein, add one milliliter of nickel NTA slurry containing 0.5 milliliter beads to the column tightly secure the cap and stop cock, and then place the column on a rocker for one hour to equilibrate and allow binding of the six X his tag to the nickel NTA beads. Set the column upright on a stand and allow the nickel NTA beads to settle for approximately two minutes.
When the beads are completely settled, a light blue layer can be seen at the bottom. Remove the cap and allow the solution to flow through the stop cock. Collect the solution in a 15 milliliter conical tube.
For further analysis, add 20 milliliters of one x wash buffer and allow the beets to settle. Open the stopcock and collect the flow through in four five milliliter aliquots. For further analysis, store the aliquots at four degrees Celsius or minus 80 degrees Celsius for short or long-term storage respectively.
Note that additional wash volumes can be used to reduce contaminating protein, however, this may reduce overall protein yield. Next, add 20 milliliters of one x elution buffer and allow the resin to settle open the stop cock and allow the solution to flow through. Collect this solution which contains 20 milliliters in four five milliliter aliquots for further analysis.
Store at four degrees Celsius or minus 80 degrees Celsius for short or long-term storage respectively. Note that additional elution volumes can be collected from the nickel NTA resin if needed. Next, separate the proteins based upon their molecular masses.
Using SDS page analysis, visualize the proteins with silver or kumasi brilliant blue R two 50 only pure fractions should be used for the following steps. Confirm the identity of the purified protein by Western blot analysis to remove salts and concentrate the protein, perform dialysis and lyophilize the sample according to the instructions in the accompanying document, and store the freeze dried samples at minus 80 degrees Celsius. The purpose of the next steps is to prepare the dope and syringe for the spinning process working under a safety hood.
Carefully add the appropriate amount of HEXA fluoro, ISOPROPANOL or HFIP to each tube of dried protein powder to obtain 200 milligrams per milliliter to 500 milligrams per milliliter, or 20%to 50%week per volume. Cap the tube as soon as possible after adding HFIP para, film the tubes and place them on a rocker to solubilize the protein vortex to facilitate solubilization. This may take up to two days.
Once solubilized centrifuge the sample at 16, 000 Gs for two to three minutes. Once the protein is in solution, it is referred to as spinning dope. The solution will be incredibly viscous.
Make sure there are no aggregates present as it may clog the syringe. In the next step, carefully load at least 25 microliters of the spinning dope into a Hamilton Gast tight syringe, equipped with a 127 micron in her diameter needle, holding the syringe with the needle up to press the plunger. To remove any air bubbles, it is very important to remove the air bubbles since they will create inconsistencies in the fiber.
Next, lock the syringe to a Harvard apparatus, 11 plus syringe pump. Place a 400 milliliter beaker filled with 95%isopropanol on a movable stand such that the tip of the syringe just breaks the surface of the alcohol. Set the syringe pump to 15 microliters per minute and press start.
The extrusion of the fiber. Should take about one to two minutes depending on the starting volume of the spinning dope. Once all of the solution has been spun into fiber, allow the fiber to sit in the isopropanol for 20 minutes to fully equilibrate.
Next, the sample is collected using a spooling device. The spooling device used here was created by gluing metal combs to a digital caliper. As shown in this image, apply double-sided tape to both sides of the comb on the spooling device.
Then attach the spooling device to a motor. Set the motor to spool the threads at two RPM. Next, using forceps, gently grab one end of the fiber and slowly pull it out of the isopropanol.
Attach it to the edge of one of the comb arms on the spool. Using the double-sided tape, The next few steps should be done without stopping to prevent the fibers from drying out. Turn on the motor and use a glass rod to gently guide the fiber onto the spooling device.
Do not allow the fiber to double up and stack. The double-sided tape. Should prevent the entire fiber from slipping and allows for selective stretching of the interior segments of the fibers within the caliper arms.
Once the entire fiber is spooled, detach the spool from the motor and apply glue to the edge of each silk fiber on the double-sided tape. This fastens the spider silk onto the apparatus. Attach the spool to the linear actuator to perform the post spin draw.
Determine the initial length of the fibers using the C.This is the internal length of the fiber. It does not include the length glued and wrapped around the spool. Lower the spool into a 75%isopropanol bath.
Allow the fibers to equilibrate for 10 minutes. Set the linear actuator speed to 1.5 millimeters per second. Based on the initial length, calculate the final length for the desired post bin draw ratio.
The amount stretched can be controlled based on the speed or the length depending on the setup. For example, with an initial length of 15 millimeters and a desired post bin draw ratio of three x, the final length should be 45 millimeters. This can also be calculated as powering the linear actuator for 20 seconds at a rate of 1.5 millimeters per second.
Second, once the post bin draw is complete, slowly raise the spool out of the isopropanol. Allow the droplets of isopropanol to evaporate for one minute to collect samples. First, cut one end of the fiber while holding the other end with forceps.
Cut that end and mount the fiber on cardstock or foil frames for testing purposes. If further post bin draw ratios are desired, lower the spool back into the 75%isopropanol bath. Allow the fibers to equilibrate for 10 minutes and then proceed to the next post bin.
Draw ratio. Record the humidity and temperature, which may affect the fibers. Mechanical properties.
Allow the collected samples to equilibrate to the standard lab environment. 40%humidity and 25 degrees Celsius are ideal for at least one hour before mechanical testing. With the fiber mounted on the cardstock frame, apply super glue to the edges of cardstock or foil to secure the fiber on the frame.
Take note of the size of the opening of the frame. This is the initial length of the tested fiber. A one inch opening frame is shown here using a light microscope with a 100 x or greater magnification take diameter measurements along the longitudinal fiber axis.
At least three measurements should be recorded. The higher the magnification used and the more measurements taken, the better the accuracy. Secure the cardstock frame to a tenter mechanical loading frame.
Then cut the frame on both sides so the tension is only running through the fiber. Tear the tenter and collect the data. A standard strain rate of 2%per second is recommended.
Mechanical data collection procedure may vary depending upon the brand of tenter used for the studies. Using the data collected in the average diameter, a stress strain curve can be plotted in Microsoft Excel. The broken fiber can now be mounted on a scanning electron microscope, Stu for morphological analysis and breakpoint diameter measurements.
The fiber segment away from the breakpoint can be used to estimate and check the initial diameter measured by the light microscope. The spider silk protein MASP one was expressed and purified from bacteria to determine the purity of the protein prior to spinning the fiber, the proteins were subject to size fractionation. Using SDS page analysis followed by visualization with silver staining as shown here, the six solutions reveal the majority of the sample releases within the first four fractions.
The spider silk protein TUSP one was also produced according to the protocol, presented herein and spun into fibers to determine the mechanical properties of synthetic tubular formm Silks fibers were spun and increasing postin draw ratios were performed. The colors in this image show fibers that were subject to different postin draw ratios ranging from 2.5 x to six x. Fibers show low variation within their ratio group.
As postman draw ratios increase the strength of the fiber is increased while extensibility is decreased. However, the fibers displayed equivalent levels of toughness to assess the ultra structure of the fibers scanning electron microscopy, images of fibers spun from recombinant tsp one proteins were acquired as can be seen here. The smooth external surface can be seen at 500 x magnification at 5, 000 decks.
The dense interior core can be observed from a natural break of fiber. This suggests that the recombinant proteins are assembling and packing well during the extrusion process. Once mastered, this technique can be done in 10 days if performed properly.
While attempting this procedure, it's important to remove all the salt from the purified recombinant protein before the freeze drying step and to also make sure the correct spinning dope concentration is used to prevent the needle from clogging during the extrusion process Following this procedure. Other fibroin, such as mes, P one TUSB one, and P ysb one can be spun into individual fibers or as composites in order to produce a wide range of biomaterials After its development. This technique paved the way for researchers in the field of bioengineering and material science to explore the use of synthetic spider silks for applications that have implications in the military medical field, arts, athletics, and automotive industry.
After watching this video, you should have a better understanding how to spin synthetic silk fibers or composite materials using proteins purified from bacteria. Don't forget that working with AFib can be extremely hazardous and precautions such as working under a fume hood should always be used while performing this procedure.
