Team:UCLA/Project/Functionalizing Fibers

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           <h1 align="middle" style="position:relative;top:0%;text-decoration:none;font-family:helvetica;font-size:150%;background-color:#0A64A4;">FUNCTIONALIZATION</h1>
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           <h1>Functionalizing Fibers</h1>
           <h1>Functionalizing Fibers</h1>
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           <h2>Co-Spinning</h2>
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           <h2>Project Goals</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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           <p>Due to its remarkable physical properties, silk has a wide array of potential applicationsWe hope to further the potential of silk by conjugating it to other functional peptides. This approach allows us to greatly expand the potential of silk. For example, 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<sup><a href="#references">[1]</a></sup>. Although several groups have been able to express and isolate silk from microbial organisms<sup><a href="#references">[2]</a></sup>, the functionalization aspect has yet to be thoroughly investigated in model microbial organisms such as <i>E. coli</i>.</p>
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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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<p>One of our goal this year has been to functionalize silk by genetically conjugating it with GFP and getting it to glow green. 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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[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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           <h2>Co-Spinning</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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           <p>A 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. This is an alternative to simply fusing the silk to a protein, and spinning only the protein fusion. This is useful because the fused protein can often interfere with the silk’s physical properties. Therefore, spinning the functionalized proteins with normal silk proteins helps maintain the physical integrity of the resulting fiber. In order to get the functional peptides to associate with the silk, we attach the peptide to either a short silk repeat or some other sequence with affinity for silk.</p>
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<figure style= "margin: 10px; float: right;"><img src= "http://2014.igem.org/wiki/images/c/cc/TamuraFigure.jpg" /><figcaption style="margin: auto;"></figcaption></figure>
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<p>For our particular project, the proteins that we plan to co-spin are natural <i>Bombyx mori</i> 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 <i>Nephila clavipes</i> silk genetically fused to superfolder GFP, and the second is superfolder GFP flanked by the N and C termini of <i>Bombyx mori</i> silk.</p>
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<figure align="middle" style= "position:relative; margin: 10px; float: right;">
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<a href = "http://2014.igem.org/wiki/images/2/2b/B_mori_N-GFP-C_terminus.PNG">
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<img src= "http://2014.igem.org/wiki/images/2/2b/B_mori_N-GFP-C_terminus.PNG" width = 50%/></a>
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<figcaption style="margin: auto;">The sequence for one of our silk-GFP hybrids. This is based off of a sequence used <i>in vivo</i> in experiments by Iizuka, et al. <sup><a href="#references">[1]</a></sup></figcaption></figure>
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<p></p><p></p>
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<p>sfGFP in these experiments can be swapped out for any other functional protein that is small enough in size so as to not interfere with the silk fiber formation when co-spun with natural silk.</p>
 +
<p>The silk sequences in the protein fusions function somewhat as affinity domains, encouraging the fusions to bind to the native silk proteins at homologous amino acid sequences during the co-spinning process.</p>
 +
<p>This has been previously accomplished <i>in vivo</i> in recombinant silkworms, but we aim to translate the process into an <i>in vitro</i> protocol<sup><a href="#references">[1]</a></sup>.</p>
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           <h2>Functionalization Constructs</h2>
           <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>  
+
           <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 and the native silk proteins, 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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          <h2>What We've Done</h2>
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          <p>This summer we have established protocols for processing <i>Bombyx mori</i> silk for co spinning. (see protocol page and processing silk page).  We have also designed, ordered, and assembled functional silk sequences.  These sequences include the N terminus-sfGFP-C terminus fusion, as well as a sfGFP-MaSp 2 fusion (see above).
 +
We synthesized the N terminus-sfGFP-C terminus fusion as a gBlock from IDT.  We designed the sequence so that the GFP in between the N and C termini could be swapped out for other functional proteins using Assembly Standard 23.  This is possible because the GFP is flanked by Bsa1 restriction sites that generate overhangs that are complementary with EcoR1 and Spe1.
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We are assembling the sfGFP-MaSp2 fusion by introducing an extra two nucleotides the the end of the registry sfGFP coding sequence to keep the sequence in frame, and then using standard assembly to fuse the two sequences. 
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We are ready to clone both of these sequences, and will ligate them into the shipping backbone very soon.  The next step will be to express these proteins, purify them, and and then co spin them with the processed <i>Bombyx mori</i> silk.</p>
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<h2>References</h2>
<p>[1] Iizuka, T., Sezutsu, H., Tatematsu, K.-i., Kobayashi, I., Yonemura, N., Uchino, K., Nakajima, K., Kojima, K., Takabayashi, C., Machii, H., Yamada, K., Kurihara, H., Asakura, T., Nakazawa, Y., Miyawaki, A., Karasawa, S., Kobayashi, H., Yamaguchi, J., Kuwabara, N., Nakamura, T., Yoshii, K. and Tamura, T. (2013), Colored Fluorescent Silk Made by Transgenic Silkworms. Adv. Funct. Mater., 23: 5232–5239. doi: 10.1002/adfm.201300365</p>
<p>[1] Iizuka, T., Sezutsu, H., Tatematsu, K.-i., Kobayashi, I., Yonemura, N., Uchino, K., Nakajima, K., Kojima, K., Takabayashi, C., Machii, H., Yamada, K., Kurihara, H., Asakura, T., Nakazawa, Y., Miyawaki, A., Karasawa, S., Kobayashi, H., Yamaguchi, J., Kuwabara, N., Nakamura, T., Yoshii, K. and Tamura, T. (2013), Colored Fluorescent Silk Made by Transgenic Silkworms. Adv. Funct. Mater., 23: 5232–5239. doi: 10.1002/adfm.201300365</p>
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Latest revision as of 03:31, 18 October 2014

iGEM UCLA



























Functionalizing Fibers

Project Goals

Due to its remarkable physical properties, silk has a wide array of potential applications. We hope to further the potential of silk by conjugating it to other functional peptides. This approach allows us to greatly expand the potential of silk. For example, 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[1]. Although several groups have been able to express and isolate silk from microbial organisms[2], the functionalization aspect has yet to be thoroughly investigated in model microbial organisms such as E. coli.

One of our goal this year has been to functionalize silk by genetically conjugating it with GFP and getting it to glow green. 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.

Co-Spinning

A 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. This is an alternative to simply fusing the silk to a protein, and spinning only the protein fusion. This is useful because the fused protein can often interfere with the silk’s physical properties. Therefore, spinning the functionalized proteins with normal silk proteins helps maintain the physical integrity of the resulting fiber. In order to get the functional peptides to associate with the silk, we attach the peptide to either a short silk repeat or some other sequence with affinity for silk.

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.

The sequence for one of our silk-GFP hybrids. This is based off of a sequence used in vivo in experiments by Iizuka, et al. [1]

sfGFP in these experiments can be swapped out for any other functional 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 bind 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, but we aim to translate the process into an in vitro protocol[1].

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 and the native silk proteins, 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.

What We've Done

This summer we have established protocols for processing Bombyx mori silk for co spinning. (see protocol page and processing silk page). We have also designed, ordered, and assembled functional silk sequences. These sequences include the N terminus-sfGFP-C terminus fusion, as well as a sfGFP-MaSp 2 fusion (see above). We synthesized the N terminus-sfGFP-C terminus fusion as a gBlock from IDT. We designed the sequence so that the GFP in between the N and C termini could be swapped out for other functional proteins using Assembly Standard 23. This is possible because the GFP is flanked by Bsa1 restriction sites that generate overhangs that are complementary with EcoR1 and Spe1. We are assembling the sfGFP-MaSp2 fusion by introducing an extra two nucleotides the the end of the registry sfGFP coding sequence to keep the sequence in frame, and then using standard assembly to fuse the two sequences. We are ready to clone both of these sequences, and will ligate them into the shipping backbone very soon. The next step will be to express these proteins, purify them, and and then co spin them with the processed Bombyx mori silk.

References

[1] Iizuka, T., Sezutsu, H., Tatematsu, K.-i., Kobayashi, I., Yonemura, N., Uchino, K., Nakajima, K., Kojima, K., Takabayashi, C., Machii, H., Yamada, K., Kurihara, H., Asakura, T., Nakazawa, Y., Miyawaki, A., Karasawa, S., Kobayashi, H., Yamaguchi, J., Kuwabara, N., Nakamura, T., Yoshii, K. and Tamura, T. (2013), Colored Fluorescent Silk Made by Transgenic Silkworms. Adv. Funct. Mater., 23: 5232–5239. doi: 10.1002/adfm.201300365


[2] Teulé, Florence, et al. "A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning." Nature protocols 4.3 (2009): 341-355.


[3] Rockwood, Danielle N., et al. "Materials fabrication from Bombyx mori silk fibroin." Nature protocols 6.10 (2011): 1612-1631.