Team:UCLA/Project/Functionalizing Fibers

From 2014.igem.org

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          <h1>Functionalizing Fibers</h1>
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          <h2>Co-Spinning</h2>
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          <p>Another method of producing functionalized fibers is to simultaneously spin, or "co-spin" ordinary silk proteins with other proteins. These proteins will be expressed separately, but mixed together and spun into a fiber from a single dope.
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For our particular project, the proteins that we plan to co-spin are natural Bombyx mori silkworm silk and a recombinant protein fusion consisting of a silk-GFP hybrid. There are two recombinant fusions that we are testing: the first is a single MaSp2 subunit of Nephila clavipes silk genetically fused to superfolder GFP, and the second is superfolder GFP flanked by the N and C termini of Bombyx mori silk.
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[IMAGE of MaSp2-sfGFP silk amino acid sequence here]
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[IMAGE of N terminus - sfGFP - C terminus amino acid sequence here]
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sfGFP in these experiments can be swapped out for any other funcitonal protein that is small enough in size so as to not interfere with the silk fiber formation when co-spun with natural silk.
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The silk sequences in the protein fusions function somewhat as affinity domains, encouraging the fusions to bing to the native silk proteins at homologous amino acid sequences during the co-spinning process.
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This has been previously accomplished in vivo in recombinant silkworms, and we aim to translate the process into an in vitro protocol. </p>
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          <h2>Future Directions</h2>
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          <p>Attaching functional peptides to silk can have promising applications. A team in Japan, led by Dr. Tamura, has been able to successfully express recombinant silk-GFP fusion fibers from genetically engineered silkworms, suggesting that it is indeed possible to produce functional materials out of recombinant silk (Iizuka et al.) Although several groups have been able to express and isolate silk from microbial organisms (Telue et al.), the functionalization aspect has yet to be thoroughly investigated in model microbial organisms such as E. coli. Our goal this year has been to create a protein fusion in E. coli that allows GFP to intercalate into silk fibers. If this works, it would serve as a proof of principle for the next step of our project- replacing GFP with SpyTag, to have an “all-purpose” protein fusion intercalated into our fiber. SpyTag is a peptide that irreversibly forms an amide bond with its partner protein, SpyCatcher. Theoretically, this would allow us the flexibility to produce a number of various fibers via post-translational treatment. For example, a SpyTag-silk fiber could be treated with a SpyCatcher-RFP fusion to produce a red fluorescent fiber without ever having to directly fuse RFP to the silk at the genetic level.</p>
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          <h2>Functionalization Constructs</h2>
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          <p>Functional recombinant silk fibers can be produced through several means. One of the approaches our team took to make such fiber is by fusing GFP to one subunit of MASP2. In theory, the MASP2 subunit would facilitate intermolecular interactions between the GFP-MASP2 protein fusion, allowing the GFP to intercalate into the final fiber when extruded via co-spinning. The part was produced using standard iGEM Assembly RFC10, after replacing the stop codon with two nucleotides at the end of GFP to keep everything in frame. The final construct consists of a 6x His Tag fused to the N-terminus of GFP in the GFP-MASP2 fusion, flanked by the BioBrick prefix and suffix. </p>
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<h1> <font size="5"> PROGRAMMING SILK: Functionalizing silk fibers </font> </h1>
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<p>To give functionality to our fibers, we will be genetically fusing various proteins onto our spider silk. In effect, these fusion proteins will have dual function: that of the fused protein as well as that of natural spider silk. Areas like medicine, art, and the fashion industry could immensely benefit from incorporating these dynamic fusion proteins into their current practices.
 
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<p>To begin with, we will fuse Green Fluorescent Protein (GFP) onto the silk. This will not only be a proof of concept for future experiments, but it will produce a tangible product. Converting this product into, say, a glowing silk t-shirt, is only a couple steps away.</p>
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<p> Next we will attach streptavidin to our silk. Streptavidin is a well-characterized protein useful in molecular biology for its high affinity towards its binding partner, biotin. Both proteins and small molecules can be "biotinylated," allowing them to bind to streptavidin, or in our case, the streptavidin-silk fusion. A simple test we can do to verify this fusion protein is indeed being formed involves biotinylated GFP to act as a visual indicator. After this, we can even try biotinylating enzymes that can function in the body. Silk is ideal in drug delivery as it is sturdy yet biodegradable, and it can act as a scaffold for these enzymes to work in humans. </p>
 
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<p> With these experiments, we aim to demonstrate that this approach of functionalizing silk fibers can be extended to many or all other proteins. The other two projects involve the critical steps of optimizing our spider silk and the spinning process, but it is with this functionalization of the silk fibers where we can truly see the potential of silk. </p>
 
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Revision as of 01:41, 17 October 2014

iGEM UCLA




























Functionalizing Fibers

Co-Spinning

Another method of producing functionalized fibers is to simultaneously spin, or "co-spin" ordinary silk proteins with other proteins. These proteins will be expressed separately, but mixed together and spun into a fiber from a single dope. For our particular project, the proteins that we plan to co-spin are natural Bombyx mori silkworm silk and a recombinant protein fusion consisting of a silk-GFP hybrid. There are two recombinant fusions that we are testing: the first is a single MaSp2 subunit of Nephila clavipes silk genetically fused to superfolder GFP, and the second is superfolder GFP flanked by the N and C termini of Bombyx mori silk. [IMAGE of MaSp2-sfGFP silk amino acid sequence here] [IMAGE of N terminus - sfGFP - C terminus amino acid sequence here] sfGFP in these experiments can be swapped out for any other funcitonal protein that is small enough in size so as to not interfere with the silk fiber formation when co-spun with natural silk. The silk sequences in the protein fusions function somewhat as affinity domains, encouraging the fusions to bing to the native silk proteins at homologous amino acid sequences during the co-spinning process. This has been previously accomplished in vivo in recombinant silkworms, and we aim to translate the process into an in vitro protocol.

Future Directions

Attaching functional peptides to silk can have promising applications. A team in Japan, led by Dr. Tamura, has been able to successfully express recombinant silk-GFP fusion fibers from genetically engineered silkworms, suggesting that it is indeed possible to produce functional materials out of recombinant silk (Iizuka et al.) Although several groups have been able to express and isolate silk from microbial organisms (Telue et al.), the functionalization aspect has yet to be thoroughly investigated in model microbial organisms such as E. coli. Our goal this year has been to create a protein fusion in E. coli that allows GFP to intercalate into silk fibers. If this works, it would serve as a proof of principle for the next step of our project- replacing GFP with SpyTag, to have an “all-purpose” protein fusion intercalated into our fiber. SpyTag is a peptide that irreversibly forms an amide bond with its partner protein, SpyCatcher. Theoretically, this would allow us the flexibility to produce a number of various fibers via post-translational treatment. For example, a SpyTag-silk fiber could be treated with a SpyCatcher-RFP fusion to produce a red fluorescent fiber without ever having to directly fuse RFP to the silk at the genetic level.

Functionalization Constructs

Functional recombinant silk fibers can be produced through several means. One of the approaches our team took to make such fiber is by fusing GFP to one subunit of MASP2. In theory, the MASP2 subunit would facilitate intermolecular interactions between the GFP-MASP2 protein fusion, allowing the GFP to intercalate into the final fiber when extruded via co-spinning. The part was produced using standard iGEM Assembly RFC10, after replacing the stop codon with two nucleotides at the end of GFP to keep everything in frame. The final construct consists of a 6x His Tag fused to the N-terminus of GFP in the GFP-MASP2 fusion, flanked by the BioBrick prefix and suffix.

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