Difference between revisions of "Team:UCLA/Project/Functionalizing Silk"

 
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     <a href="#background"><div class= "page_header_box" id= "box1">
 
     <a href="#background"><div class= "page_header_box" id= "box1">
 
           <h1 align="middle" style="position:relative;top:0%;text-decoration:none;font-family:helvetica;font-size:150%;background-color:#0A64A4;">BACKGROUND</h1>
 
           <h1 align="middle" style="position:relative;top:0%;text-decoration:none;font-family:helvetica;font-size:150%;background-color:#0A64A4;">BACKGROUND</h1>
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<!--BACKGROUND-->
 
<!--BACKGROUND-->
 
<div class= "content_subsection" id="background">
 
<div class= "content_subsection" id="background">
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<h1>Creating Functional Fibers</h1>
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<h1>Silk Functionalization: Developing the Next Generation of High Performance Fibers</h1>
 
<h2>Background</h2>
 
<h2>Background</h2>
 
<h4>Abstract</h4>
 
<h4>Abstract</h4>
 
<p>
 
<p>
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'''Silk fibers''' possess the potential to be transformed into '''functional biomaterials''' that can be exploited in an array of biomedical applications, from aiding '''nanoscale drug delivery''' to simulating '''medical sutures'''. However, traditional methods of incorporating functional domains into fibers involve '''difficult, costly, and time-consuming''' processes. We propose an '''in vitro, co-spinning''' method to quickly and '''efficiently''' functionalize silk fibers. In essence, we spin a mixture of '''wild-type silk dope spiked with a small volume of functional domain'''. This functional domain which will '''bind''' to the native silk proteins when co-spun, thereby incorporating itself into the final '''synthetic fiber'''. To ensure proper binding of our functional domain, we created a '''co-spinning module'''. This module is a genetic construct consisting of our gene of interest flanked on either side by the '''N and C terminal domains''' of '''Bombyx mori''' (silkworm silk). When co-spun, the termini on our synthetic protein will bind to the respective termini in the native silk proteins, thereby functionalizing the fiber. Our goal is to '''develop, optimize and experimentally validate''' our co-spinning module, and assess its potential as a '''scalable''' and '''powerful tool''' to manufacture silk fibers with an array of functional capacities.  
+
<b>Silk fibers</b> possess the potential to be transformed into functional biomaterials that can be exploited in an array of biomedical applications, from aiding nanoscale drug delivery to simulating medical sutures. However, traditional methods of incorporating functional domains into fibers involve difficult, costly, and time-consuming processes. We propose an in vitro, co-spinning method to quickly and efficiently functionalize silk fibers. In essence, we spin a mixture of wild-type silk dope spiked with a small volume of functional domain. This functional domain which will bind to the native silk proteins when co-spun, thereby incorporating itself into the final synthetic fiber. To ensure proper binding of our functional domain, we created a co-spinning module. This module is a genetic construct consisting of our gene of interest flanked on either side by the N and C terminal domains of <i>Bombyx mori</i> (silkworm silk). When co-spun, the termini on our synthetic protein will bind to the respective termini in the native silk proteins, thereby functionalizing the fiber. Our goal is to develop, optimize and experimentally validate our co-spinning module, and assess its potential as a scalable and powerful tool to manufacture silk fibers with an array of functional capacities.  
 
</p>  
 
</p>  
 +
 +
<figure style="float: left; margin: auto"><img style="width: 400px; height: 200px" src= "https://static.igem.org/mediawiki/2015/8/88/Bombyx_mori.jpeg" />
 +
<figcaption  style="margin: auto; width: 400px;" align="left">
 +
<b></b><i>Bombyx mori</i> larva
 +
<br/>Wolinsky, National Geographic Creative
 +
</figcaption>
 +
</figure>
 +
 +
<figure style="float: right; margin: auto"><img style="width: 400px; height: 200px" src= "https://static.igem.org/mediawiki/2015/a/af/Bombyx_mori_fibroin.jpeg" />
 +
<figcaption  style="margin: auto; width: 400px;" align="left">
 +
<b></b> <i>Bombyx mori</i> silk proteins consist of sericin, a group of glycoproteins encapsulating fibroin. Fibroin serves as a vehicle for incorporating desired functionality.
 +
<br/>Nato, Hiroshima University
 +
</figcaption>
 +
</figure>
 +
 +
<p style="clear: both;">
 +
 +
 
<h4>Introduction</h4>
 
<h4>Introduction</h4>
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<p>
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</p>
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Native silk fibers exhibit great tensile strength, elasticity, and flexibility. These mechanical properties, coupled with the non-immunogenic behavior of silk proteins, render silk fibers a worthy candidate in the realm of biomedical applications. Due to the potential of these fibers as a vehicle for nanoscale drug delivery or a scaffold for tissue engineering, there is much interest in attaching domains onto these fibers to achieve desired functionality. Bombyx mori, commonly known as silkworm, contain silk proteins comprised of fibroin, the core fiber that provides silkworm silk it’s structure. Fibroin is the main protein of interest that we aim to attach functional domains onto. Fibroin serves as a vehicle for functionality.  
+
 
 +
Native silk fibers exhibit great tensile strength, elasticity, and flexibility. These mechanical properties, coupled with the non-immunogenic behavior of silk proteins, render silk fibers a worthy candidate in the realm of biomedical applications. Due to the potential of these fibers as a vehicle for nanoscale drug delivery or a scaffold for tissue engineering, there is much interest in attaching domains onto these fibers to achieve desired functionality. <i>Bombyx mori</i>, commonly known as silkworm, contain silk proteins comprised of fibroin, the core fiber that provides silkworm silk it’s structure. Fibroin is the main protein of interest that we aim to attach functional domains onto. Fibroin serves as a vehicle for functionality.  
 
</p>
 
</p>
 
<p>
 
<p>
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Previously, to functionalize fibroin proteins, scientists have relied on chemical conjugation of silk peptides or breeding transfected silkworms that express transgenes encoding functional domains. However, these methods have inherent limitations. Attaching big, bulky functional domains directly onto the ends of fibroin proteins can disrupt Beta-sheet formation between the fibers. Preserving secondary structure is critical to preserve functionality. Moreover, native Bombyx mori silk genes are incredibly repetitive, which makes cloning of transgenes complex and time consuming. The breeding of transfected silkworms can be summed up as costly and laborious. These disadvantages prevent chemical conjugation and in vivo expression from becoming  sustainable, cost efficient ways of manufacturing silk fibers with functional capacities. As a result, there is much need to develop a new method of manufacturing functionalized fibers.  
+
Previously, to functionalize fibroin proteins, scientists have relied on chemical conjugation of silk peptides or breeding transfected silkworms that express transgenes encoding functional domains. However, these methods have inherent limitations. Attaching big, bulky functional domains directly onto the ends of fibroin proteins can disrupt Beta-sheet formation between the fibers. Preserving secondary structure is critical to preserve functionality. Moreover, native <i>Bombyx mori</i> silk genes are incredibly repetitive, which makes cloning of transgenes complex and time consuming. The breeding of transfected silkworms can be summed up as costly and laborious. These disadvantages prevent chemical conjugation and in vivo expression from becoming  sustainable, cost-efficient ways of manufacturing silk fibers with functional capacities. As a result, there is much need to develop a new method to manufacture functional fibers.  
 
</p>
 
</p>
 
</div>
 
</div>
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<div class= "content_subsection" id="methodology">
 
<div class= "content_subsection" id="methodology">
 
<h2>Methodology</h2>
 
<h2>Methodology</h2>
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<ol type="A">
 
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<li>
 
−
Iterative capped assembly is similar to Golden Gate assembly, which uses unique sticky ends to assemble gene fragments in a specific order. Whereas Golden Gate is a one-pot reaction with all the pieces ligated simultaneously, ICA is a more “controlled” variation where pieces are assembled one at a time. ICA relies on using 3 different versions of the monomer to be assembled, each of which has different sticky ends such that the monomers must be assembled in an A-B-C fashion. This prevents monomers from self-ligating. Type IIS restriction enzymes (such as BsaI), which cleave outside of the recognition site are used to generate these.
 
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</li>
 
  
 +
<figure style="float: left"><img style="width: 500px; height: 130px" src= "https://static.igem.org/mediawiki/2015/6/66/SfGFP_construct.jpeg" />
 +
<figcaption  style="margin: auto; width: 500px;" align="left">
 +
<b>Fig 1:</b> Our novel, co-spinning module, with our gene of interest, sfGFP, inserted between the non-repetitive N and C terminal domains of <i>Bombyx mori</i>, along with appropriate Bsa1 restriction sites. Also not shown is a 6X histidine tag, used for later protein purification processes. 
 +
</figure>
  
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<figure><img style="margin-left: 25%; width: 500px;" src= "https://static.igem.org/mediawiki/2015/b/b9/Fig_1_ICA_Methodology.png" />
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<p>
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<figcaption  style="margin: auto; width: 100%;">
+
The theory behind the co-spinning methodology is to attach a functional domain onto the native silk fiber without disrupting the natural mechanical properties or non-immunogenic behavior of wild-type silk. To achieve this, we've created a genetic construct, entitled our "co-spinning module", consisting of super folder Green Fluorescent Protein (sfGFP) flanked on either sides by the N and C terminal domains of <i>Bombyx mori</i>. These termini are critical in maintaining the structural integrity of the fibers. Specifically, the N terminal domains of individual fibroin proteins bind to their identical counterparts on adjacent fibroin proteins, thereby establishing disulfide linkages between the fibers. The C terminal domains on the fibroin proteins aid the fibers in responding to decreases in pH levels and mechanical stresses, conditions that induce the stacking of Beta sheets into an actual fiber. The repetitive motifs in between the terminal domains are hydrophobic regions that cluster together, separate from the hydrophilic N terminal domains, thus simulating a micelle. From our co-spinning module, we create a synthetic protein that emulates the composition of native fibroin, with the repetitive motifs between the domains replaced with our functional domain, sfGFP. When co-spun, the N terminal domains on our synthetic protein will recognize and bind to their counterparts on the native fibroin proteins, maintaining the micellar structure. The repetitive regions on the native fibroin and the sfGFP on our synthetic protein are coiled structures that, when exposed to the mechanical stresses induced by in vitro syringe extrusion, will flatten and stack up into Beta sheets, and Beta sheet formation is the key indication of proper fiber formation.
−
<b>Fig 1:</b>In ICA, the initiator of the growing DNA chains are immobilized on magnetic streptavidin beads to facilitate removal. The first monomer is ligated to the initiator. (Step A) Subsequent ligation steps incorporate capping oligos, which prevent the extension of incorrect chains: those that failed to ligate in the previous round. (Steps B, C) Ligation continues until the construct reaches the final desired length. The final step incorporates a terminator oligo. (Final Step) Only DNA constructs of the correct size can ligate to the terminator, as all incorrect constructs will have been capped.
+
</p>
 +
 
 +
<br/>
 +
<figure style="float: left"><img style="width: 600px; height: 325px" src= "https://static.igem.org/mediawiki/2015/e/e6/Xpressmethod2.jpeg" />
 +
<figcaption  style="margin: auto; width: 600px;" align="left">
 +
<b>Fig 2:</b> An outline of our expression techniques, using E-coli chassis. The 6X histidine tag attached to our co-spinning module enables us to use IMAC, immobilized metal affinity chromatography with nickel resin beads.
 +
</figure>
 +
 
 +
<br/>
 +
 
 +
<figure style="float: left"><img style="width: 500px; height: 300px" src= "https://static.igem.org/mediawiki/2015/e/e5/Finalcospin.jpeg" />
 +
<figcaption  style="margin: auto; width: 500px;" align="left">
 +
<b>Fig 3:</b> In our co-spinning methodology, <i>Bombyx mori</i> silk dope is spiked with a small volume (3 ul) of functional domain. The N termini on our expressed co-spinning module protein bind to the N-termini in the wildtype silk fibroin, and these hydrophilic heads arrange in a micellar structure, isolating the dangling coils of repetitive regions and sfGFP. After in-vitro syringe extrusion, the sheer forces applied induces Beta sheet formation.  
 
</figcaption>
 
</figcaption>
 
</figure>
 
</figure>
  
−
<li>
+
<figure style="float: right"><img style="width: 500px; height: 300px" src= "https://static.igem.org/mediawiki/2015/c/c5/Glowing_silk.png" />
−
A basic biobrick for ICA consists of a gene monomer flanked by BsaI recognition and cleavage sites. While each of the core monomer is identical, the restriction sites are oriented such that digestion with BsaI yields distinct sticky ends for each of the three types of units. These pieces must be digested to reveal the sticky ends before assembly.
+
<figcaption  style="margin: auto; width: 500px;" align="left">
−
</li>
+
<b>Fig 4:</b> Co-spinning module validated with our first, proof of concept co-spin of sfGFP with wild type <i>Bombyx mori</i> silk dope.  
−
<li>
+
</figcaption>
−
Accessory pieces required in ICA include the initiator, streptavidin coated beads, the terminator, and the capping oligos.
+
</figure>
−
    <ol type="i">
+
−
    <li>
+
−
    The initiator is a dsDNA fragment made by annealing two ssDNA oligos together. The initiator is designed such that one end is biotinylated, for conjugation to streptavidin coated beads. The other end has a sticky overhang and is designed to anneal to the forward sticky end of the ‘A-type’ monomer unit. This end is 5’-phosphorylated to enable ligation. The initiator also contains a primer binding site that can be used for PCR amplification, as well as other accessory sequences such as affinity tags and the biobrick prefix.
+
−
    </li>
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−
    <li>
+
−
    Streptavidin coated beads serve as a solid support for the elongating DNA chain during ICA. The biotinylated end of the initiator binds to streptavidin to anchor the nascent construct. The ability to physically separate the DNA from solution is needed due to repeated wash and ligation steps used during ICA.
+
−
    </li>
+
−
    <li>
+
−
    The terminator is constructed similarly to the initiator, but lacks biotinylation. One end of the terminator is compatible to the reverse sticky end of the ‘C-type’ monomer unit. This end is 5’-phosphorylated to enable ligation. The terminator also contains a primer binding site that can be used for PCR amplification, as well as the biobrick suffix.
+
−
    </li>
+
−
    <li>
+
−
      The capping oligos are comprised of a single 5’-phosphorylated ssDNA oligo that can form a stable stem loop structure with a unique sticky end. There are three distinct caps, each of which can bind to the A, B, or C sticky ends
+
−
    </li>
+
−
    </ol>
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−
</li>
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−
<li>
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−
In each extension step, the next sequential monomer (A, B, or C) is added onto the growing chain. Chains that failed to extend during the previous extension step are capped using a hair-pin oligo that prevents subsequent extension. These capped chains are still present in the mixture for the duration of ICA, but do not participate in any ligation event, and are not amplified in the final PCR. Each final construct is flanked by a biotinylated initiator oligo which allows immobilization onto streptavidin beads, and a terminator oligo. These two oligos provide primer annealing sites which can be used to amplify the sequence using conventional PCR.
+
−
</li>
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−
<li>
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−
A generalized workflow is demonstrated below:
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−
    <ol type="i">
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−
    <li>
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−
    The initiator, terminator, and capping oligos are prepared ahead of time by mixing the relevant oligos and ramping down from 95 C to form the working oligos.
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−
    </li>
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−
    <li>
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−
    Monomers of each type (A, B, C) are digested from plasmid with BsaI and purified prior to ICA. These are termed working monomers.
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−
    </li>
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    <li>
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    The initiator is attached to the streptavidin coated beads.
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−
    </li>
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    <li>
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    An ‘A-type’ monomer is ligated to the end of the initiator. Afterwards, any unreacted fragments, as well as the ligase are washed off.
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−
    </li>
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−
    <li>
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−
    Next, the ‘B-type’ monomer is ligated to the end of the growing chain. In this reaction mixture, an ‘A-type’ cap is also included, to terminate any chains that failed to extend in the previous ‘A-type’ ligation. Again, unreacted fragments are removed by washing.
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−
    </li>
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    <li>
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−
      Next, the ‘C-type’ monomer is ligated. This reaction mixture contains the ‘B-type’ cap.
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    </li>
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    <li>
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      Next the ‘A-type’ monomer is ligated. This time, the ‘C-type’ cap is also included in the mixture.
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    </li>
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    <li>
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    This proceeds in a repetitive fashion until the desired construct length is reached.
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    </li>
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    <li>
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    The final constructed is eluted off the beads. The eluate is used as a template for PCR to amplify the construct. Only complete constructs that contain the initiator and terminator are amplified. Capped constructs do not amplify.
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    </li>
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    <li>
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−
    The amplified construct can now be used for downstream cloning.
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    </li>
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    </ol>
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</li>
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</ol>
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</div>
 
</div>
  
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<p style="clear: both;">
  
 
<!--RESULTS-->
 
<!--RESULTS-->
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<h2>Results</h2>
 
<h2>Results</h2>
 
<p>
 
<p>
−
Using ICA, we have generated 10 silk constructs. These include constructs of pure MaSp2 ranging from 3-15 mers, pure MaSp1 of 9 and 12-mers and 12-mers of MaSp1/2 hybrids in 3 different ratios.
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Successful in vitro co-spin of wild type <i>Bombyx mori</i> silk dope with our expressed co-spinning module protein to produce a fiber that visibly fluoresces!
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</p>
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−
<h4>Future Directions</h4>
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−
<p>
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−
While we were able to construct many sequences of varying length and composition using ICA, we were unable to explore its maximum potential for cloning repetitive genes. We have not established an upper limit on the number of monomers able to be assemble using ICA. In addition, we have not explored the extended use of ICA to create extremely large (greater than 50 monomer units)
+
 
</p>
 
</p>
 +
 +
<figure align="middle""><img style="width: 600px; height: 250px" src= "https://static.igem.org/mediawiki/2015/0/0b/Uclaigem2015_tamura005_fluorescence0x.jpg" />
 +
<figcaption  style="margin: auto; width: 500px;" align="left">
 +
<b>Fig 5:</b> Our co-spun synthetic fiber under Evos FL digital fluorescence microscopy.
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</figcaption>
 +
</figure>
 +
 
<h4>Achievements</h4>
 
<h4>Achievements</h4>
 
<ol>
 
<ol>
 
<li>
 
<li>
−
Successfully improved last year’s biobrick BBa_K1384000
+
Successful development and experimental validation of our co-spinning module as a tool to incorporate functional domains into wild-type <i>Bombyx mori</i> silk dope, with is then processed into a functional, synthetic fiber.  
−
    <ol type="a">
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−
    <li>
+
−
    Redesigned MaSp1 and MaSp2 monomers with modified sticky ends, and cloned these into biobricks.
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−
    </li>
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−
    </ol>
+
 
</li>
 
</li>
 
<li>
 
<li>
−
Created a collection of parts to be used for Iterative Capped Assembly
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Designed and sequence-verified our novel co-spinning module with super folder GFP, (sfGFP) sandwiched between the N and C terminal domains of <i>Bombyx mori</i>.
 
</li>
 
</li>
 
<li>
 
<li>
−
Demonstrated that ICA is adaptable to silk using our designed sticky ends.
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Successful cloning of co-spinning module with E-coli chassis, and successful amplification of part with Polymerase Chain Reaction (PCR).  
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    <ol type="a">
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−
    <li>
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−
    Optimized ICA for use with spider silk genes to enable fast, efficient assembly of repetitive constructs.
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−
    </li>
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−
    </ol>
+
 
</li>
 
</li>
 
<li>
 
<li>
−
Used ICA to assemble a variety of silk genetic constructs of different length and composition to examine their properties.
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Successful expression and purification of sfGFP protein with N and C termini attached on either side.
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    <ol type="a">
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−
    <li>
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−
    Used ICA to create 10 different silk constructs ranging from 3-mers to 15-mers, and constructs with MaSp1 and MaSp2 in ratios of [1:2], [1:1] and [2:1].
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−
    </li>
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−
    </ol>
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</li>
 
</li>
 
</ol>
 
</ol>
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</div>
 
  
  
 +
<figure style="float: left"><img style="width: 500px; height: 300px" src= "https://static.igem.org/mediawiki/2015/6/6c/Tamura_DNA_gel.png" />
 +
<figcaption  style="margin: auto; width: 500px;" align="left">
 +
<b>Fig 6:</b> DNA gel verifies successful cloning and PCR amplification of NC-sfGFP genetic construct. NC is an abbreviation for N and C terminal domains of <i>Bombyx mori</i>. A bright band at approximately 1650 kb, the expected insert size. Cloning and amplification done in both vectors, psB1A2 (first lane) and psB1C3 (third lane) yields comparable results.
 +
</figcaption>
 +
</figure>
 +
 +
<figure style="float: right"><img style="width: 500px; height: 300px" src= "https://static.igem.org/mediawiki/2015/d/d7/Tamura_sds_page.png" />
 +
<figcaption  style="margin: auto; width: 500px;" align="left">
 +
<b>Fig 7:</b> SDS PAGE verifies successful expression and purification of our NC-sfGFP co-spinning module, with a singular, dark band at 66kD.
 +
</figcaption>
 +
</figure>
 +
 +
 +
<p style="clear: both;">
 +
 +
<h4>Future Directions</h4>
 +
<p>
 +
Now that we have established a proof of concept with sfGFP, you can imagine how we can swap sfGFP with other functional domains to spin out synthetic fibers that exhibit an array of functionality! In order to verify this BioBrick as a screening platform to assay for functional peptide affinity, a wide variety of co-spinning modules must be constructed.  Namely, co-spinning modules digested and ligated with albumin-binding domain (ABD), immunoglobin G binding domain (ImGBD) and avidin binding domains (AvBD) may be of critical use in both experimental design and developing a functional application of co-spinning in a wide variety of biomedical applications.
 +
</p>
 +
<figure align="middle""><img style="width: 600px; height: 250px" src= "https://static.igem.org/mediawiki/2015/c/c5/Otherdomains2.jpeg" />
 +
<figcaption  style="margin: auto; width: 600px;" align="left">
 +
<b>Fig 8:</b> Schematic outlining future functional domains to test, including albumin-binding domain (ABD), avidin binding domain (AvBD) and immunoglobin G binding (ImGBD).
 +
</figcaption>
 +
</figure>
 +
 +
</div>
  
 
<!--BIOBRICKS-->
 
<!--BIOBRICKS-->
 
<div class= "content_subsection" id="biobricks">
 
<div class= "content_subsection" id="biobricks">
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<h2>List of Biobricks</h2>
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<h2>List of Biobricks</h2>  
 
<ul>
 
<ul>
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<li>
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MaSp2 AB:              BBa_K1763002
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<li>http://parts.igem.org/Part:BBa_K1763444 </li>
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</li>
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−
<li>
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−
MaSp2 BC: BBa_K1763003
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−
</li>
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−
<li>
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−
MaSp2 CA: BBa_K1763004
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−
</li>
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−
<li>
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−
MaSp2 SeqAB: BBa_K1763009
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−
</li>
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−
<li>
+
−
MaSp1 AB: BBa_K1763010
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−
</li>
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−
<li>
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−
MaSp1 BC: BBa_K1763011
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−
</li>
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−
<li>
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−
MaSp1 CA: BBa_K1763012
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−
</li>
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−
<li>
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−
MaSp1 SeqAB2:         BBa_K1763423
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−
</li>
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−
<li>
+
−
M2-3(1C3): BBa_K1763424
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−
</li>
+
−
<li>
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−
M2-3(T7): BBa_K1763425
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−
</li>
+
−
<li>
+
−
M2-6(1C3): BBa_K1763426
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−
</li>
+
−
<li>
+
−
M2-6(T7): BBa_K1763427
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−
</li>
+
−
<li>
+
−
M2-9(1C3): BBa_K1763428
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−
</li>
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−
<li>
+
−
M2-9(T7): BBa_K1763429
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−
</li>
+
−
<li>
+
−
M2-12(1C3): BBa_K1763430
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−
</li>
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−
<li>
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−
M2-12(T7): BBa_K1763431
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−
</li>
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−
<li>
+
−
M2-15(1C3): BBa_K1763432
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−
</li>
+
−
<li>
+
−
M2-15(T7): BBa_K1763433
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−
</li>
+
−
<li>
+
−
M1-9(1C3): BBa_K1763434
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−
</li>
+
−
<li>
+
−
M1-9(T7): BBa_K1763435
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−
</li>
+
−
<li>
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M1-12(1C3): BBa_K1763436
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M1-12(T7): BBa_K1763437
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M1/2[2:1]-12(1C3): BBa_K1763438
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M1/2[2:1]-12(T7): BBa_K1763439
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M1/2[1:1]-12(1C3): BBa_K1763440
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M1/2[1:1]-12(T7): BBa_K1763441
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M1/2[1:2]-12(1C3): BBa_K1763442
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M1/2[1:2]-12(T7): BBa_K1763443
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Briggs A., Rios X., Chari R., Luhan Y., Zhang F., Mali P., and Church G. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. <i>Nucleic Acids Research.</i> 2012;<b>40</b>(15): e117
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Teulé, F. et al. Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties. Proc. Natl. Acad. Sci. U.S.A. 109, 923–8 (2012).
 
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Hinman, M.B., Lewis, R. Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. J. Biol. Chem. 1992;267: 19320–19324.
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Teulé, F. et al. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nat Protoc 4, 341–55 (2009).
 
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Tokareva O., Michalczechen-Lacerda V., Rech E., and Kaplan D. Recombinant DNA production of spider silk proteins. <i>Microbial Biotechnology.</i> 2013;<b>6</b>(6): 651-663
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Jansson, R. Strategies for Functionalization of Recombinant Spider Silk. 11, 76 (Acta Universitatis agriculturae Sueciae, 2015).
 
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Xu, M., Lewis, R.V. Structure of a protein superfiber: spider dragline silk. PNAS;1990;87, 7120.
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Kojima, K. et al. A new method for the modification of fibroin heavy chain protein in the transgenic silkworm. Biosci. Biotechnol. Biochem. 71, 2943–51 (2007).
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<b>Fig 1:</b>In ICA, the initiator of the growing DNA chains are immobilized on magnetic streptavidin beads to facilitate removal. The first monomer is ligated to the initiator. (Step A) Subsequent ligation steps incorporate capping oligos, which prevent the extension of incorrect chains: those that failed to ligate in the previous round. (Steps B, C) Ligation continues until the construct reaches the final desired length. The final step incorporates a terminator oligo. (Final Step) Only DNA constructs of the correct size can ligate to the terminator, as all incorrect constructs will have been capped.
 
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<b>Fig 2:</b>Downstream Cloning after Iterative Capped Assembly. After elution from the beads, the ICA constructs are amplified using PCR primers that anneal to the initiator and the terminator. These primer binding sites are unique in the construct, and can be found nowhere else in the sequence. The only constructs that are amplified are those that have the initiator and terminator. All other constructs, while present, are excluded from amplification. After amplification, the construct can be cloned into a vector using traditional techniques.
 
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<b>Fig 4:</b>Schematic of examples of  initiator (a), terminator (b), and capping oligos (c), used in our ICA project. The cap shown has the B-type sticky end.
 
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<b>Fig 6:</b>Gel image of constructs we created this summer using ICA. pSB1C3 Plasmids containing the sequence verified construct were digested using XbaI and PstI. Results were run on 1% TAE gel. The expected band size for the pSB1C3 is ~2070. Expected sizes for inserts fragments are indicated on the right hand side.
 
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<h2>Functionalizing Silk Fibers</h2>
 
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'''Silk fibers''' possess the potential to be transformed into '''functional biomaterials''' that can be exploited in an array of biomedical applications, from aiding '''nanoscale drug delivery''' to simulating '''medical sutures'''. However, traditional methods of incorporating functional domains into fibers involve '''difficult, costly, and time-consuming''' processes. We propose an '''in vitro, co-spinning''' method to quickly and '''efficiently''' functionalize silk fibers. In essence, we spin a mixture of '''wild-type silk dope spiked with a small volume of functional domain'''. This functional domain which will '''bind''' to the native silk proteins when co-spun, thereby incorporating itself into the final '''synthetic fiber'''. To ensure proper binding of our functional domain, we created a '''co-spinning module'''. This module is a genetic construct consisting of our gene of interest flanked on either side by the '''N and C terminal domains''' of '''Bombyx mori''' (silkworm silk). When co-spun, the termini on our synthetic protein will bind to the respective termini in the native silk proteins, thereby functionalizing the fiber. Our goal is to '''develop, optimize and experimentally validate''' our co-spinning module, and assess its potential as a '''scalable''' and '''powerful tool''' to manufacture silk fibers with an array of functional capacities.
 
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<h2>Achievements</h2>
 
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Successful in vitro co-spin of wild type Bombyx mori silk dope with synthetic protein to produce a fiber that visibly fluoresces!
 
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Designed and sequence-verified our novel co-spinning module with super folded GFP, (sfGFP) sandwiched between the N and C terminal domains of Bombyx mori.
 
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Successful cloning of co-spinning module with E-coli chassis, and successful amplification of part with Polymerase Chain Reaction (PCR).
 
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Successful expression and purification of sfGFP protein with N and C termini attached on either side.
 

Latest revision as of 03:32, 19 September 2015

iGEM UCLA




BACKGROUND

METHODOLOGY

RESULTS

BIOBRICKS























Silk Functionalization: Developing the Next Generation of High Performance Fibers

Background

Abstract

Silk fibers possess the potential to be transformed into functional biomaterials that can be exploited in an array of biomedical applications, from aiding nanoscale drug delivery to simulating medical sutures. However, traditional methods of incorporating functional domains into fibers involve difficult, costly, and time-consuming processes. We propose an in vitro, co-spinning method to quickly and efficiently functionalize silk fibers. In essence, we spin a mixture of wild-type silk dope spiked with a small volume of functional domain. This functional domain which will bind to the native silk proteins when co-spun, thereby incorporating itself into the final synthetic fiber. To ensure proper binding of our functional domain, we created a co-spinning module. This module is a genetic construct consisting of our gene of interest flanked on either side by the N and C terminal domains of Bombyx mori (silkworm silk). When co-spun, the termini on our synthetic protein will bind to the respective termini in the native silk proteins, thereby functionalizing the fiber. Our goal is to develop, optimize and experimentally validate our co-spinning module, and assess its potential as a scalable and powerful tool to manufacture silk fibers with an array of functional capacities.

Bombyx mori larva
Wolinsky, National Geographic Creative
Bombyx mori silk proteins consist of sericin, a group of glycoproteins encapsulating fibroin. Fibroin serves as a vehicle for incorporating desired functionality.
Nato, Hiroshima University

Introduction

Native silk fibers exhibit great tensile strength, elasticity, and flexibility. These mechanical properties, coupled with the non-immunogenic behavior of silk proteins, render silk fibers a worthy candidate in the realm of biomedical applications. Due to the potential of these fibers as a vehicle for nanoscale drug delivery or a scaffold for tissue engineering, there is much interest in attaching domains onto these fibers to achieve desired functionality. Bombyx mori, commonly known as silkworm, contain silk proteins comprised of fibroin, the core fiber that provides silkworm silk it’s structure. Fibroin is the main protein of interest that we aim to attach functional domains onto. Fibroin serves as a vehicle for functionality.

Previously, to functionalize fibroin proteins, scientists have relied on chemical conjugation of silk peptides or breeding transfected silkworms that express transgenes encoding functional domains. However, these methods have inherent limitations. Attaching big, bulky functional domains directly onto the ends of fibroin proteins can disrupt Beta-sheet formation between the fibers. Preserving secondary structure is critical to preserve functionality. Moreover, native Bombyx mori silk genes are incredibly repetitive, which makes cloning of transgenes complex and time consuming. The breeding of transfected silkworms can be summed up as costly and laborious. These disadvantages prevent chemical conjugation and in vivo expression from becoming sustainable, cost-efficient ways of manufacturing silk fibers with functional capacities. As a result, there is much need to develop a new method to manufacture functional fibers.

Methodology

Fig 1: Our novel, co-spinning module, with our gene of interest, sfGFP, inserted between the non-repetitive N and C terminal domains of Bombyx mori, along with appropriate Bsa1 restriction sites. Also not shown is a 6X histidine tag, used for later protein purification processes.

The theory behind the co-spinning methodology is to attach a functional domain onto the native silk fiber without disrupting the natural mechanical properties or non-immunogenic behavior of wild-type silk. To achieve this, we've created a genetic construct, entitled our "co-spinning module", consisting of super folder Green Fluorescent Protein (sfGFP) flanked on either sides by the N and C terminal domains of Bombyx mori. These termini are critical in maintaining the structural integrity of the fibers. Specifically, the N terminal domains of individual fibroin proteins bind to their identical counterparts on adjacent fibroin proteins, thereby establishing disulfide linkages between the fibers. The C terminal domains on the fibroin proteins aid the fibers in responding to decreases in pH levels and mechanical stresses, conditions that induce the stacking of Beta sheets into an actual fiber. The repetitive motifs in between the terminal domains are hydrophobic regions that cluster together, separate from the hydrophilic N terminal domains, thus simulating a micelle. From our co-spinning module, we create a synthetic protein that emulates the composition of native fibroin, with the repetitive motifs between the domains replaced with our functional domain, sfGFP. When co-spun, the N terminal domains on our synthetic protein will recognize and bind to their counterparts on the native fibroin proteins, maintaining the micellar structure. The repetitive regions on the native fibroin and the sfGFP on our synthetic protein are coiled structures that, when exposed to the mechanical stresses induced by in vitro syringe extrusion, will flatten and stack up into Beta sheets, and Beta sheet formation is the key indication of proper fiber formation.


Fig 2: An outline of our expression techniques, using E-coli chassis. The 6X histidine tag attached to our co-spinning module enables us to use IMAC, immobilized metal affinity chromatography with nickel resin beads.

Fig 3: In our co-spinning methodology, Bombyx mori silk dope is spiked with a small volume (3 ul) of functional domain. The N termini on our expressed co-spinning module protein bind to the N-termini in the wildtype silk fibroin, and these hydrophilic heads arrange in a micellar structure, isolating the dangling coils of repetitive regions and sfGFP. After in-vitro syringe extrusion, the sheer forces applied induces Beta sheet formation.
Fig 4: Co-spinning module validated with our first, proof of concept co-spin of sfGFP with wild type Bombyx mori silk dope.

Results

Successful in vitro co-spin of wild type Bombyx mori silk dope with our expressed co-spinning module protein to produce a fiber that visibly fluoresces!

Fig 5: Our co-spun synthetic fiber under Evos FL digital fluorescence microscopy.

Achievements

  1. Successful development and experimental validation of our co-spinning module as a tool to incorporate functional domains into wild-type Bombyx mori silk dope, with is then processed into a functional, synthetic fiber.
  2. Designed and sequence-verified our novel co-spinning module with super folder GFP, (sfGFP) sandwiched between the N and C terminal domains of Bombyx mori.
  3. Successful cloning of co-spinning module with E-coli chassis, and successful amplification of part with Polymerase Chain Reaction (PCR).
  4. Successful expression and purification of sfGFP protein with N and C termini attached on either side.
Fig 6: DNA gel verifies successful cloning and PCR amplification of NC-sfGFP genetic construct. NC is an abbreviation for N and C terminal domains of Bombyx mori. A bright band at approximately 1650 kb, the expected insert size. Cloning and amplification done in both vectors, psB1A2 (first lane) and psB1C3 (third lane) yields comparable results.
Fig 7: SDS PAGE verifies successful expression and purification of our NC-sfGFP co-spinning module, with a singular, dark band at 66kD.

Future Directions

Now that we have established a proof of concept with sfGFP, you can imagine how we can swap sfGFP with other functional domains to spin out synthetic fibers that exhibit an array of functionality! In order to verify this BioBrick as a screening platform to assay for functional peptide affinity, a wide variety of co-spinning modules must be constructed. Namely, co-spinning modules digested and ligated with albumin-binding domain (ABD), immunoglobin G binding domain (ImGBD) and avidin binding domains (AvBD) may be of critical use in both experimental design and developing a functional application of co-spinning in a wide variety of biomedical applications.

Fig 8: Schematic outlining future functional domains to test, including albumin-binding domain (ABD), avidin binding domain (AvBD) and immunoglobin G binding (ImGBD).

List of Biobricks

  • http://parts.igem.org/Part:BBa_K1763444

References

Teulé, F. et al. Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties. Proc. Natl. Acad. Sci. U.S.A. 109, 923–8 (2012).

Teulé, F. et al. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nat Protoc 4, 341–55 (2009).

Jansson, R. Strategies for Functionalization of Recombinant Spider Silk. 11, 76 (Acta Universitatis agriculturae Sueciae, 2015).

Kojima, K. et al. A new method for the modification of fibroin heavy chain protein in the transgenic silkworm. Biosci. Biotechnol. Biochem. 71, 2943–51 (2007).