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

 
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<div class= "content_subsection" id="background">
 
<div class= "content_subsection" id="background">
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<h1>Programming Spider Silk</h1>
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<h1>Honeybee Silk</h1>
 
<h2>Background</h2>
 
<h2>Background</h2>
 
<h4>Abstract</h4>
 
<h4>Abstract</h4>
 
<p>
 
<p>
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A major obstacle in creating recombinant spider silk is the highly repetitive nature of the genes that encode it. Silk genes are comprised of a repetitive core region containing ~100 repeats of a spidroin gene which precludes the use of traditional cloning techniques due to non-specificity in primer binding. While other techniques such as head-to-tail assembly or concatemerization have been developed to facilitate spider silk cloning, none of these techniques can assemble silk genes in a quick and directed manner. We have adapted the use of Iterative Capped Assembly (ICA) as a technique to construct silk genes in a rapid and sequence specific manner. By creating a number of different constructs this summer, we show that ICA can greatly facilitate the engineering of recombinant spider-silk.
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In addition to the more well known silks from spiders and silkworms, we decided to also explore silk from the honeybee <i>Apis mellifera</i>. We cloned the honeybee silk gene as well as several variants of it and submitted them as the first honeybee silk biobricks. To investigate its potential as a biomaterial, we expressed the silk protein and confirmed its presence using SDS PAGE.
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</p>  
 
</p>  
 
<h4>Introduction</h4>
 
<h4>Introduction</h4>
 
<p>
 
<p>
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Recombinant spider-silks with customizable properties present an appealing biomaterial that can be used in textiles, tissue scaffolds, and other unique applications. Spider silk is a proteinaceous fiber whose proteins consist of non-repetitive N and C terminal domains, and a highly repetitive central core that consists of up to 100 repeats of Spidroin1 (MaSp1) or Spidroin 2 (MaSp2). These spidroin repeats are directly responsible for the final properties and behavior of the spider silk. MaSp1 contributes strength to the fiber, while MaSp2 contributes elasticity. Importantly, the relative content of MaSp1 and MaSp2 monomers in silk proteins can dictate the fiber properties when spun. Engineering recombinant spider silk genes of varying lengths and spidroin content is nearly impossible using traditional cloning methods, due to the repetitive core. These repeats essentially forbid the use of primers to amplify said genes due to the possibility of non-specific priming. These obstacles have made engineering recombinant spider silk a difficult process.
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Silk from Apis Mellifera presents an intriguing alternative to silks from spiders or silkworms. Although it is not quite as strong as these other types of silks, working with honeybee silk has certain advantages over spider and silkworm silk. The size of the honeybee silk protein gene is considerably smaller than the silk genes of spiders or silkworms. More importantly, the gene sequence is non-repetitive, which allows us to synthesize and make modifications to the gene, without the complications that are inherent to repetitive DNA sequences. Honeybee silk also has a very different secondary and tertiary structure than spider and silkworm silks. It forms primary alpha helices, and four silk proteins come together to form a coiled coil structure.
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</p>
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<p>
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<figure align="middle"><img width="500px" height="400" src= "http://mbe.oxfordjournals.org/content/24/11/2424/F8.large.jpg" />
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Existing techniques seek to remove reliance of cloning on primer annealing (Tokareva et al, 2013). Generally, these techniques break the silk genetic into monomeric sequences, then assemble the monomers into the final construct. Head-to-tail cloning assembles gene constructs by ligating two plasmid halves together. Each half carries one of the silk monomers, and the resulting complete plasmid has been doubled. This technique can be used recursively to assemble increasingly large silk genes in a specified manner. Directional recursive ligation uses a similar tactic, where individual monomers are ligated one at a time into a receiving plasmid. Concatemerization is another technique where a pool of monomers are ligated in a single reaction, then cloned into plasmids. This particular technique is useful for creating a library of sizes and compositions.
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<figcaption  style="width: 100%; margin: auto;" align="middle">
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</p>
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<b>Fig 1:</b>Honeybee Silk Coiled Coil Structure
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<p>
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</figcaption>
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These existing techniques are not ideal for engineering recombinant silks, because they require repeated and extensive cloning for large constructs, as in the case of head-to-tail assembly and directional recursive ligation, or do not offer any control over the length or genetic composition, as is the case for concatemerization. As it currently stands, there is no one technique that offers rapid and controllable assembly of recombinant spider silk genes.
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</figure>
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</p>
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<p>
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Iterative Capped Assembly (ICA) is a cloning method that is used to sequentially assemble long,
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In wild type honeybee silk these coiled coils are formed from four similar, yet unique proteins, Amelf 1-4. However, a study has shown that using one of these proteins, (Amelf3) is sufficient to reproduce the physical properties of the wild type fibers.
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repetitive DNA sequences. This technique was developed by Briggs et al. in 2012 as a method
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A major goal of our project is to give biological fibers entirely new functionalities. Therefore, in addition to expressing wild type honeybee silk, we have also created constructs in which honeybee silk protein is fused to other proteins. Our first fusion construct is honeybee silk fused to super folder green fluorescent protein (sfGFP), which will act as our proof of principle that silk can be functionalized while still maintaining exceptional physical properties. Our second fusion is silk fused to SpyCatcher protein, which would allow for the capture of any protein modified to contain the short SpyTag peptide.  
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to assemble Transcription Activator-Like Effector Nucleases (TALENs) which are sequence
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specific DNA binding proteins that consist of multiple repetitive monomers. Each repeat
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monomer is responsible for binding to a specific nucleotide in the target sequence. Due to the
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repetitive nature of TALE genes, conventional PCR is unable to reliably amplify these sequences
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due to non-specific primer binding.
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</p>
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<p>
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Although ICA was developed using TALE construction as a model problem, this technique can
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be used to construct long, repetitive DNA constructs in a directly controllable fashion. ICA assembles repetitive sequences one monomer unit at a time, while preventing the elongation of
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incomplete nucleotide chains. The full length sequence is flanked by unique primer annealing
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sites, which allows the PCR amplification of the final product. This entire process is performed
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using a solid substrate, which greatly facilitates the construction of long sequences.
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</p>
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<p>
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This summer, we adapt the use of Iterative Capped Assembly (ICA) as a technique for cloning recombinant spider silks in a time-efficient and specific manner that is unparalleled by existing methods. Our experience shows that ICA can be used as a method for the quick assembly of spider silk genes.
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</p>
 
</p>
 
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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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<ol type="Honeybee Silk Genetics">
 
<li>
 
<li>
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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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Although previous literature has shown that wild type honeybee silk is composed of four highly similar proteins, we decided to only produce one. This decision was based on the result of Tara Sutherland's research, which claimed to be able to produce honeybee fibers with comparable physical properties to wild type fibers using only one of the four proteins. The protein they used was AmelF3, which is the protein we focused on.
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We obtained the sequence of the AmelF3 gene from genbank.( http://www.ncbi.nlm.nih.gov/gene/100192201), synthesized it using IDT’s synthesis service and used this construct as our baseline sequence from which to construct our biobricks. Because we aimed to express honeybee silk protein, we added regulatory elements such as promoters and ribosome binding sites to several our constructs. Two of our biobricks represent fusion proteins between silk and another functional protein. The first fusion biobrick is silk fused to sfGFP(<a href="http://parts.igem.org/Part:BBa_K1763015">BBa_K1763015</a>), which will serve as our proof of principle that honeybee silk can be functionalized while still retaining its mechanical properties. The second fusion biobrick is honeybee silk fused to SpyCatcher protein, which allow for capture of and protein modified to contain a Spytag peptide.(<a href="http://parts.igem.org/Part:BBa_K1763008">BBa_K1763008</a>) Additionally, we fused sfGFP to AmelF3. Please see our list of biobricks (<a href="https://2015.igem.org/Team:UCLA/Parts"?>Parts</a>) and the respective biobrick pages for more detailed design information and characterization of each biobrick.
 
</li>
 
</li>
  
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<li>
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In addition to creating honeybee genetic constructs, we needed protocols to express and purify honeybee silk proteins. We drew heavily from previous literature by T. Sutherland that had established a method of expressing and purifying honeybee silk proteins.  According to previous literature, honeybee silk proteins aggregate and form insoluble inclusion bodies within the e. coli cell. Therefore, it is reported that these proteins can be purified by lysing the cells and collecting the insoluble fraction. In order to determine protein concentration. Furthermore, because we wanted to use a T7 promoter to drive expression of our honeybee constructs, therefore, we used a strain of bacteria that expressed T7 polymerase, BL21 (DE3).  For more detailed descriptions of our protein expression and purification, please see our lab notebook entries.
  
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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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<figcaption  style="margin: auto; width: 100%;">
 
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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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</figcaption>
 
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</figure>
 
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<li>
 
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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.
 
 
</li>
 
</li>
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<li>
 
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Accessory pieces required in ICA include the initiator, streptavidin coated beads, the terminator, and the capping oligos.
 
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    <ol type="i">
 
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    <li>
 
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    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.
 
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    </li>
 
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    <li>
 
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    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.
 
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    </li>
 
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    <li>
 
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    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.
 
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    </li>
 
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    <li>
 
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      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
 
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    </li>
 
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    </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.
 
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</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>
 
  
 
</div>
 
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<h2>Results</h2>
 
<h2>Results</h2>
 
<p>
 
<p>
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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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We used standard restriction digest cloning to clone our five biobrick constructs. We sequence verified all our parts and submitted them to the registry.
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</p>
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Here is a diagram of all of our sequence verified parts.
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<h4>Future Directions</h4>
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<li>
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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)
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<figure align="middle"><img width="800px" height="450px" src= "https://2015.igem.org/File:Screen_Shot_2015-09-18_at_7.15.27_PM.png" />
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<figcaption  style="width: 100%; margin: auto;" align="middle">
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<b>Fig 2:</b>Schematic of our honeybee silk constructs
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</figcaption>
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</figure>
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From top to bottom they are the honeybee silk coding region, the coding region under control of lac promoter, the silk protein fused to Spycatcher, the coding region under control of a T7 promoter, and silk protein fused to sfGFP.
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We also successfully made competent BL21 (DE3) cells using the Zymo mix and go chemical competency kit. We performed a competency test and we determined that the competency of our cells was 3e7 colony forming units / microgram of DNA. (<a href="https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/12_July_2015"?>Parts</a>) 
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Finally, we were able to express our honeybee silk proteins and confirm its presence through SDS PAGE.
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<figure align="middle"><img width="800px" height="450" src= "https://2015.igem.org/File:UCLA_honeybee_Growth_optimization_37C.jpg?" />
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<figcaption  style="width: 100%; margin: auto;" align="middle">
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<b>Fig 3:</b>SDS PAGE optimization image
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</figcaption>
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</figure>
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We also obtained some rough estimate of protein yield by doing a BCA protein assay. This estimate may not be entirely reliable because we observed some contaminating bands on our SDS PAGE gels, indicating that there are proteins other than honeybee silk in the solution. From our BCA assay, we determined that we got around 12 mg of protein from a 300ml overnight cell culture.
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<figure align="middle"><img width="200px" height="500" src= "https://2015.igem.org/File:Honeybee_silk_SDS_PAGE.jpg" />
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<figcaption  style="width: 100%; margin: auto;" align="middle">
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<b>Fig 4:</b>SDS PAGE image of honeybee silk
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</figcaption>
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</figure>
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Future Directions
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Optimize protein expression and purification protocols for higher yields and greater purity
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Process purified silk proteins into materials such as films or fibers
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Assess the mechanical properties of these fibers
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Express and process functionalized silk proteins
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Assay functionalized honeybee silk materials for functionality 
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</p>
 
</p>
 
<h4>Achievements</h4>
 
<h4>Achievements</h4>
 
<ol>
 
<ol>
 
<li>
 
<li>
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Successfully improved last year’s biobrick BBa_K1384000
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Submitted and sequenced 5 honeybee silk biobricks
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    <ol type="a">
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    <li>
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    Redesigned MaSp1 and MaSp2 monomers with modified sticky ends, and cloned these into biobricks.
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    </li>
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    </ol>
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</li>
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<li>
 
<li>
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Created a collection of parts to be used for Iterative Capped Assembly
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Expressed honeybee silk protein and verified presence of SDS PAGE gel
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</li>
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<li>
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Demonstrated that ICA is adaptable to silk using our designed sticky ends.
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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>
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</li>
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<li>
 
<li>
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Used ICA to assemble a variety of silk genetic constructs of different length and composition to examine their properties.
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Had a great summer! 
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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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</ol>
 
</ol>
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<ul>
 
<ul>
 
<li>
 
<li>
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MaSp2 AB:               BBa_K1763002
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Apis mellifera (honeybee) silk fibroin 3: <a href="http://parts.igem.org/Part:BBa_K1763000">BBa_K1763000</a>                                     
 
</li>
 
</li>
 
<li>
 
<li>
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MaSp2 BC: BBa_K1763003
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Apis mellifera (honeybee) silk fibroin 3 + regulatory elements: <a href="http://parts.igem.org/Part:BBa_K1763001">BBa_K1763001></a>        
 
</li>
 
</li>
 
<li>
 
<li>
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MaSp2 CA: BBa_K1763004
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Apis mellifera (honeybee) silk fibroin 3 + T7 promoter: <a href="http://parts.igem.org/Part:BBa_K1763007">BBa_K1763007</a>
 
</li>
 
</li>
 
<li>
 
<li>
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MaSp2 SeqAB: BBa_K1763009
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Lac Promoter + Apis mellifera (honeybee) silk fibroin 3 + SpyCatcher: <a href="http://parts.igem.org/Part:BBa_K1763008">BBa_K1763008</a>
 
</li>
 
</li>
 
<li>
 
<li>
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MaSp1 AB: BBa_K1763010
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T7 Promoter + Apis mellifera (honeybee) silk fibroin 3 + sfGFP: <a href="http://parts.igem.org/Part:BBa_K1763015">BBa_K1763015</a>
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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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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>
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M2-3(1C3): BBa_K1763424
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M2-3(T7): BBa_K1763425
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<li>
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M2-6(1C3): BBa_K1763426
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M2-6(T7): BBa_K1763427
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</li>
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<li>
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M2-9(1C3): BBa_K1763428
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</li>
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<li>
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M2-9(T7): BBa_K1763429
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</li>
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<li>
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M2-12(1C3): BBa_K1763430
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M2-12(T7): BBa_K1763431
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M2-15(1C3): BBa_K1763432
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M2-15(T7): BBa_K1763433
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<li>
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M1-9(1C3): BBa_K1763434
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<li>
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M1-9(T7): BBa_K1763435
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<li>
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M1-12(1C3): BBa_K1763436
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</li>
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<li>
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M1-12(T7): BBa_K1763437
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</li>
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<li>
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M1/2[2:1]-12(1C3): BBa_K1763438
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</li>
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<li>
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M1/2[2:1]-12(T7): BBa_K1763439
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</li>
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<li>
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M1/2[1:1]-12(1C3): BBa_K1763440
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</li>
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M1/2[1:1]-12(T7): BBa_K1763441
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</li>
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M1/2[1:2]-12(1C3): BBa_K1763442
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</li>
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M1/2[1:2]-12(T7): BBa_K1763443
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</li>
 
</li>
 
</ul>
 
</ul>
  
 
</div>
 
</div>
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<h2>References</h2>
 
<h2>References</h2>
 
<p>
 
<p>
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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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Weisman, S., Haritos, V., Church, J., Huson, M., Mudie, S., Rodgers, A., Dumsday, G., Sutherland, T. Honeybee silk: Recombinant protin production, assembly, and fiber spinning. <i>Elsevier Ltd.</i> 2009.
 
</p>
 
</p>
 
<p>
 
<p>
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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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Sutherland, T., Church, J., Hu, X., Huson, M., Kaplan, D., Weisman, S. Single Honeybee Silk Protein Mimics Properties fo Multi-Protein Silk. <i>PLoS ONE</i> 2011. e16489
 
</p>
 
</p>
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<p>
 
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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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Xu, M., Lewis, R.V. Structure of a protein superfiber: spider dragline silk. PNAS;1990;87, 7120.
 
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<p>Sutherland, T. Conservation of Essential Design Features in Coiled Coil Silks.<i> Mol Biol Evol</i> 2007;24:2424-2432
  
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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 5:</b>Schematic of the three types of sticky ends we designed for ICA. Sticky end A is 5’-AGTT-3’. Sticky end B is 5’-TGTC-3’. Sticky end C is 5’-CGTG-3’. An assembled 3-mer construct AB+BC+CA is shown as an example of how these biobricks would be used.
 
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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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Latest revision as of 03:59, 19 September 2015

iGEM UCLA




BACKGROUND

METHODOLOGY

RESULTS

BIOBRICKS























Honeybee Silk

Background

Abstract

In addition to the more well known silks from spiders and silkworms, we decided to also explore silk from the honeybee Apis mellifera. We cloned the honeybee silk gene as well as several variants of it and submitted them as the first honeybee silk biobricks. To investigate its potential as a biomaterial, we expressed the silk protein and confirmed its presence using SDS PAGE.

Introduction

Silk from Apis Mellifera presents an intriguing alternative to silks from spiders or silkworms. Although it is not quite as strong as these other types of silks, working with honeybee silk has certain advantages over spider and silkworm silk. The size of the honeybee silk protein gene is considerably smaller than the silk genes of spiders or silkworms. More importantly, the gene sequence is non-repetitive, which allows us to synthesize and make modifications to the gene, without the complications that are inherent to repetitive DNA sequences. Honeybee silk also has a very different secondary and tertiary structure than spider and silkworm silks. It forms primary alpha helices, and four silk proteins come together to form a coiled coil structure.

Fig 1:Honeybee Silk Coiled Coil Structure
In wild type honeybee silk these coiled coils are formed from four similar, yet unique proteins, Amelf 1-4. However, a study has shown that using one of these proteins, (Amelf3) is sufficient to reproduce the physical properties of the wild type fibers. A major goal of our project is to give biological fibers entirely new functionalities. Therefore, in addition to expressing wild type honeybee silk, we have also created constructs in which honeybee silk protein is fused to other proteins. Our first fusion construct is honeybee silk fused to super folder green fluorescent protein (sfGFP), which will act as our proof of principle that silk can be functionalized while still maintaining exceptional physical properties. Our second fusion is silk fused to SpyCatcher protein, which would allow for the capture of any protein modified to contain the short SpyTag peptide.

Methodology

  1. Although previous literature has shown that wild type honeybee silk is composed of four highly similar proteins, we decided to only produce one. This decision was based on the result of Tara Sutherland's research, which claimed to be able to produce honeybee fibers with comparable physical properties to wild type fibers using only one of the four proteins. The protein they used was AmelF3, which is the protein we focused on. We obtained the sequence of the AmelF3 gene from genbank.( http://www.ncbi.nlm.nih.gov/gene/100192201), synthesized it using IDT’s synthesis service and used this construct as our baseline sequence from which to construct our biobricks. Because we aimed to express honeybee silk protein, we added regulatory elements such as promoters and ribosome binding sites to several our constructs. Two of our biobricks represent fusion proteins between silk and another functional protein. The first fusion biobrick is silk fused to sfGFP(BBa_K1763015), which will serve as our proof of principle that honeybee silk can be functionalized while still retaining its mechanical properties. The second fusion biobrick is honeybee silk fused to SpyCatcher protein, which allow for capture of and protein modified to contain a Spytag peptide.(BBa_K1763008) Additionally, we fused sfGFP to AmelF3. Please see our list of biobricks (Parts) and the respective biobrick pages for more detailed design information and characterization of each biobrick.
  2. In addition to creating honeybee genetic constructs, we needed protocols to express and purify honeybee silk proteins. We drew heavily from previous literature by T. Sutherland that had established a method of expressing and purifying honeybee silk proteins. According to previous literature, honeybee silk proteins aggregate and form insoluble inclusion bodies within the e. coli cell. Therefore, it is reported that these proteins can be purified by lysing the cells and collecting the insoluble fraction. In order to determine protein concentration. Furthermore, because we wanted to use a T7 promoter to drive expression of our honeybee constructs, therefore, we used a strain of bacteria that expressed T7 polymerase, BL21 (DE3). For more detailed descriptions of our protein expression and purification, please see our lab notebook entries.

Results

We used standard restriction digest cloning to clone our five biobrick constructs. We sequence verified all our parts and submitted them to the registry. Here is a diagram of all of our sequence verified parts.

  • Fig 2:Schematic of our honeybee silk constructs
    From top to bottom they are the honeybee silk coding region, the coding region under control of lac promoter, the silk protein fused to Spycatcher, the coding region under control of a T7 promoter, and silk protein fused to sfGFP. We also successfully made competent BL21 (DE3) cells using the Zymo mix and go chemical competency kit. We performed a competency test and we determined that the competency of our cells was 3e7 colony forming units / microgram of DNA. (Parts) Finally, we were able to express our honeybee silk proteins and confirm its presence through SDS PAGE.
    Fig 3:SDS PAGE optimization image
    We also obtained some rough estimate of protein yield by doing a BCA protein assay. This estimate may not be entirely reliable because we observed some contaminating bands on our SDS PAGE gels, indicating that there are proteins other than honeybee silk in the solution. From our BCA assay, we determined that we got around 12 mg of protein from a 300ml overnight cell culture.
    Fig 4:SDS PAGE image of honeybee silk
    Future Directions Optimize protein expression and purification protocols for higher yields and greater purity Process purified silk proteins into materials such as films or fibers Assess the mechanical properties of these fibers Express and process functionalized silk proteins Assay functionalized honeybee silk materials for functionality

    Achievements

    1. Submitted and sequenced 5 honeybee silk biobricks
    2. Expressed honeybee silk protein and verified presence of SDS PAGE gel
    3. Had a great summer!

    • List of Biobricks

    • Apis mellifera (honeybee) silk fibroin 3: BBa_K1763000
    • Apis mellifera (honeybee) silk fibroin 3 + regulatory elements: BBa_K1763001>
    • Apis mellifera (honeybee) silk fibroin 3 + T7 promoter: BBa_K1763007
    • Lac Promoter + Apis mellifera (honeybee) silk fibroin 3 + SpyCatcher: BBa_K1763008
    • T7 Promoter + Apis mellifera (honeybee) silk fibroin 3 + sfGFP: BBa_K1763015

    References

    Weisman, S., Haritos, V., Church, J., Huson, M., Mudie, S., Rodgers, A., Dumsday, G., Sutherland, T. Honeybee silk: Recombinant protin production, assembly, and fiber spinning. Elsevier Ltd. 2009.

    Sutherland, T., Church, J., Hu, X., Huson, M., Kaplan, D., Weisman, S. Single Honeybee Silk Protein Mimics Properties fo Multi-Protein Silk. PLoS ONE 2011. e16489

    Sutherland, T. Conservation of Essential Design Features in Coiled Coil Silks. Mol Biol Evol 2007;24:2424-2432