http://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&feed=atom&action=historyTeam:UCLA/Project/Customizing Silk - Revision history2024-03-29T14:39:02ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=393070&oldid=prevAnuvedverma at 02:59, 18 October 20142014-10-18T02:59:37Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Customizing Silk</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Customizing Silk</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h2>Background</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h2>Background</h2></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p>Spider silk is an exceptional natural material. It exhibits remarkable strength and has the potential to be a very useful medium for a variety of applications. The properties of spider silk come primarily from the structure and amino acid sequence of the silk protein. One spider silk protein is comprised of repetitive units, referred to as monomers. Repeats of identical monomers make up one spider silk protein, and these proteins come together to form a silk fiber. The monomer is therefore the basic unit of the silk polypeptide, and changing the sequence alters the physical properties of the spider silk.<sup href="#references">[1]</sup> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p>Spider silk is an exceptional natural material. It exhibits remarkable strength and has the potential to be a very useful medium for a variety of applications. The properties of spider silk come primarily from the structure and amino acid sequence of the silk protein. One spider silk protein is comprised of repetitive units, referred to as monomers. Repeats of identical monomers make up one spider silk protein, and these proteins come together to form a silk fiber. The monomer is therefore the basic unit of the silk polypeptide, and changing the sequence alters the physical properties of the spider silk.<sup<ins class="diffchange diffchange-inline">><a </ins>href="#references">[1]<ins class="diffchange diffchange-inline"></a></ins></sup> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: right;"><img src= "https://static.igem.org/mediawiki/2014/2/28/CustomizingSilk1.png" /><figcaption style="margin: auto;width:300px;">Fig 1: This image illustrates the amino acid motifs that compose various spider silk monomers. Different types of silk protein contain different motifs. These motifs define the physical characteristics of each monomer and the overall silk protein.<sup href="#references">[6]</sup></figcaption></figure></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: right;"><img src= "https://static.igem.org/mediawiki/2014/2/28/CustomizingSilk1.png" /><figcaption style="margin: auto;width:300px;">Fig 1: This image illustrates the amino acid motifs that compose various spider silk monomers. Different types of silk protein contain different motifs. These motifs define the physical characteristics of each monomer and the overall silk protein.<sup<ins class="diffchange diffchange-inline">><a </ins>href="#references">[6]<ins class="diffchange diffchange-inline"></a></ins></sup></figcaption></figure></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p>In fact, the sequence of the spider silk monomer differs slightly from species to species. Therefore, silk produced by different species have different physical properties. Some monomer sequences result in silks with greater strength, while other sequences result in silks with greater flexibility. Furthermore, individual spiders can produce multiple types of silk such as dragline and flagelliform silk, each with very different properties.<sup href="#references">[2]</sup></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p>In fact, the sequence of the spider silk monomer differs slightly from species to species. Therefore, silk produced by different species have different physical properties. Some monomer sequences result in silks with greater strength, while other sequences result in silks with greater flexibility. Furthermore, individual spiders can produce multiple types of silk such as dragline and flagelliform silk, each with very different properties.<sup<ins class="diffchange diffchange-inline">><a </ins>href="#references">[2]<ins class="diffchange diffchange-inline"></a></ins></sup></p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Much research has been done on the production of spider silk from recombinant bacteria.<sup href="#references">[3]</sup> We want to build on this work and take it a step further. Using the wide range of spider silk monomers as our building blocks, we want to be able to create hybrid silk proteins with specified physical properties. By mixing and matching these different monomer sequences within one polypeptide, we hope to be able to fine tune the properties of our produced silk. If this system could be standardized, it could be exceedingly useful for quickly producing silk to fit different a variety of different needs and purposes. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Much research has been done on the production of spider silk from recombinant bacteria.<sup<ins class="diffchange diffchange-inline">><a </ins>href="#references">[3]<ins class="diffchange diffchange-inline"></a></ins></sup> We want to build on this work and take it a step further. Using the wide range of spider silk monomers as our building blocks, we want to be able to create hybrid silk proteins with specified physical properties. By mixing and matching these different monomer sequences within one polypeptide, we hope to be able to fine tune the properties of our produced silk. If this system could be standardized, it could be exceedingly useful for quickly producing silk to fit different a variety of different needs and purposes. </p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h4 id="project_goals">Project Goals</h4><p>For our project, we have chosen to start out with two different silk monomers, Major Ampullate Spidroin 1 (MaSp1) and 2 (MaSp2). The MaSp1 monomer contributes strength to spider silk while the MaSp2 contributes more flexibility.<sup href="#references">[4]</sup> As a proof of principle we hope to show that by assembling these two repeats into one protein at different ratios, we can create silk with a spectrum of strength and extensibility. </p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h4 id="project_goals">Project Goals</h4><p>For our project, we have chosen to start out with two different silk monomers, Major Ampullate Spidroin 1 (MaSp1) and 2 (MaSp2). The MaSp1 monomer contributes strength to spider silk while the MaSp2 contributes more flexibility.<sup<ins class="diffchange diffchange-inline">><a </ins>href="#references">[4]<ins class="diffchange diffchange-inline"></a></ins></sup> As a proof of principle we hope to show that by assembling these two repeats into one protein at different ratios, we can create silk with a spectrum of strength and extensibility. </p> </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: left;"><img src= "https://static.igem.org/mediawiki/2014/a/ae/CustomizingSilk2.png" /><figcaption style="margin: auto;">Fig 2: Variations of the MaSp1 amino acid sequences between different spider species <sup href="#references">[6]</sup></figcaption></figure></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: left;"><img src= "https://static.igem.org/mediawiki/2014/a/ae/CustomizingSilk2.png" /><figcaption style="margin: auto;">Fig 2: Variations of the MaSp1 amino acid sequences between different spider species <sup<ins class="diffchange diffchange-inline">><a </ins>href="#references">[6]<ins class="diffchange diffchange-inline"></a></ins></sup></figcaption></figure></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div class= "content_subsection" id="ica"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <div class= "content_subsection" id="ica"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h2> Iterative Capped Assembly</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h2> Iterative Capped Assembly</h2></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p>Assembling spider silk monomers such as MaSp1 and MaSp2 together can prove to be rather difficult. Due to the repetitiveness of our silk monomers, common techniques such as Gibson assembly and direct synthesis of a full-length gene product could be ineffective and less efficient in building silk sequences. Therefore, a technique called iterative capped assembly (ICA), which allows rapid assembly of repeating monomers, would be a better option in working with our highly repetitive sequences. This technique has previously been applied in order to assemble other repetitive modules. For example, ICA has been demonstrated to efficiently assemble very repetitive transcription activator-like effector nucleases (TALENs) up to 21 monomers long<sup><a href="references">[5]</a></sup>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p>Assembling spider silk monomers such as MaSp1 and MaSp2 together can prove to be rather difficult. Due to the repetitiveness of our silk monomers, common techniques such as Gibson assembly and direct synthesis of a full-length gene product could be ineffective and less efficient in building silk sequences. Therefore, a technique called iterative capped assembly (ICA), which allows rapid assembly of repeating monomers, would be a better option in working with our highly repetitive sequences. This technique has previously been applied in order to assemble other repetitive modules. For example, ICA has been demonstrated to efficiently assemble very repetitive transcription activator-like effector nucleases (TALENs) up to 21 monomers long<sup><a href="<ins class="diffchange diffchange-inline">#</ins>references">[5]</a></sup>.</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>ICA allows the assembly of silk monomers in different ratios and orders into a custom gene sequence of modifiable length. Gene monomers are assembled individually into a growing chain that is anchored to a solid foundation through streptavidin-biotin interaction.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>ICA allows the assembly of silk monomers in different ratios and orders into a custom gene sequence of modifiable length. Gene monomers are assembled individually into a growing chain that is anchored to a solid foundation through streptavidin-biotin interaction.</p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: right;"><img src= "https://static.igem.org/mediawiki/2014/2/29/ICAhowitworks.png" /><figcaption style="margin: auto; width: 300px;">Fig. 3: Schematic of ICA from Briggs, et al.<sup>[5]</sup></figcaption></figure></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: right;"><img src= "https://static.igem.org/mediawiki/2014/2/29/ICAhowitworks.png" /><figcaption style="margin: auto; width: 300px;">Fig. 3: Schematic of ICA from Briggs, et al.<sup<ins class="diffchange diffchange-inline">><a href="#references"</ins>>[5]<ins class="diffchange diffchange-inline"></a></ins></sup></figcaption></figure></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We have designed 3 types of overhangs that the Bsa1 enzyme can generate. These are the A overhang, (AGCA), the B overhang (TGCA) and the C overhang (TGCT).</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>We have designed 3 types of overhangs that the Bsa1 enzyme can generate. These are the A overhang, (AGCA), the B overhang (TGCA) and the C overhang (TGCT).</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>A key aspect of ICA is that the gene to be assembled is fixed to a solid support as it is being ligated together. Streptavidin beads act as the solid support in this case. Conjugating our gene to the beads therefore necessitates an “initiator oligo”, a biotynilated sequence that contains both the biobrick prefix as well as one of the A,B, or C overhangs at its 3’ end. An advantage of fixing our growing sequence to the beads is that it allows us to remove all traces of the previous ligation in a “wash” step before adding the next silk monomer to the growing gene.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>A key aspect of ICA is that the gene to be assembled is fixed to a solid support as it is being ligated together. Streptavidin beads act as the solid support in this case. Conjugating our gene to the beads therefore necessitates an “initiator oligo”, a biotynilated sequence that contains both the biobrick prefix as well as one of the A,B, or C overhangs at its 3’ end. An advantage of fixing our growing sequence to the beads is that it allows us to remove all traces of the previous ligation in a “wash” step before adding the next silk monomer to the growing gene.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: left;"><img src= "https://static.igem.org/mediawiki/2014/0/04/ICA2.PNG" /><figcaption style="margin: auto;width: 300px;">Fig. 4: Immobilization of the initiator to magnetic beads. Figure from Briggs, et al.<sup>[5]</sup></figcaption></figure></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: left;"><img src= "https://static.igem.org/mediawiki/2014/0/04/ICA2.PNG" /><figcaption style="margin: auto;width: 300px;">Fig. 4: Immobilization of the initiator to magnetic beads. Figure from Briggs, et al.<sup<ins class="diffchange diffchange-inline">><a href="#references"</ins>>[5]<ins class="diffchange diffchange-inline"></a></ins></sup></figcaption></figure></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>When dealing with repetitive sequences, regular assembly techniques would give products of various lengths due to the uncontrolled ligation among pieces. Another powerful aspect of ICA is that it increases the frequency of producing a full-length sequence that we intend to build. This is achieved by adding capping oligos that ligate to a chain where a previous monomer piece fail to attach. Any incomplete or incorrect sequence due to unsuccessful ligations would be blocked from further extending, and thus monomers that are added later would ligate to the right sequences at a higher frequency.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>When dealing with repetitive sequences, regular assembly techniques would give products of various lengths due to the uncontrolled ligation among pieces. Another powerful aspect of ICA is that it increases the frequency of producing a full-length sequence that we intend to build. This is achieved by adding capping oligos that ligate to a chain where a previous monomer piece fail to attach. Any incomplete or incorrect sequence due to unsuccessful ligations would be blocked from further extending, and thus monomers that are added later would ligate to the right sequences at a higher frequency.</p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Once the full gene sequence has been generated, the terminator oligo containing the biobrick suffix is added to complete the assembly. The gene can then be released from the streptavidin beads by heating and disrupting the Streptavidin biotin interaction<sup>[5]</sup>. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Once the full gene sequence has been generated, the terminator oligo containing the biobrick suffix is added to complete the assembly. The gene can then be released from the streptavidin beads by heating and disrupting the Streptavidin biotin interaction<sup<ins class="diffchange diffchange-inline">><a href="#references"</ins>>[5]<ins class="diffchange diffchange-inline"></a></ins></sup>. </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>ICA is a proficient technique to standardize assembly of any custom gene sequence. Not only does it provide flexibility in producing custom gene sequence, it is also compatible with the iGEM biobrick system. The initiator contains the prefix sequence of a biobrick and the terminator contains the suffix sequence. These contain primer sites allow PCR amplification of the full-length constructs after the release from the beads. In addition, restriction sites within the initiator and terminator enable the insertion of the complete, assembled product into a biobrick backbone easily using Golden-Gate cloning. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>ICA is a proficient technique to standardize assembly of any custom gene sequence. Not only does it provide flexibility in producing custom gene sequence, it is also compatible with the iGEM biobrick system. The initiator contains the prefix sequence of a biobrick and the terminator contains the suffix sequence. These contain primer sites allow PCR amplification of the full-length constructs after the release from the beads. In addition, restriction sites within the initiator and terminator enable the insertion of the complete, assembled product into a biobrick backbone easily using Golden-Gate cloning. </p></div></td></tr>
</table>Anuvedvermahttp://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=392576&oldid=prevAnuvedverma at 02:55, 18 October 20142014-10-18T02:55:52Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h2> Iterative Capped Assembly</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h2> Iterative Capped Assembly</h2></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p>Assembling spider silk monomers such as MaSp1 and MaSp2 together can prove to be rather difficult. Due to the repetitiveness of our silk monomers, common techniques such as Gibson assembly and direct synthesis of a full-length gene product could be ineffective and less efficient in building silk sequences. Therefore, a technique called iterative capped assembly (ICA), which allows rapid assembly of repeating monomers, would be a better option in working with our highly repetitive sequences. This technique has previously been applied in order to assemble other repetitive modules. For example, ICA has been demonstrated to efficiently assemble very repetitive transcription activator-like effector nucleases (TALENs) up to 21 monomers long<sup>[5]</sup>.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p>Assembling spider silk monomers such as MaSp1 and MaSp2 together can prove to be rather difficult. Due to the repetitiveness of our silk monomers, common techniques such as Gibson assembly and direct synthesis of a full-length gene product could be ineffective and less efficient in building silk sequences. Therefore, a technique called iterative capped assembly (ICA), which allows rapid assembly of repeating monomers, would be a better option in working with our highly repetitive sequences. This technique has previously been applied in order to assemble other repetitive modules. For example, ICA has been demonstrated to efficiently assemble very repetitive transcription activator-like effector nucleases (TALENs) up to 21 monomers long<sup<ins class="diffchange diffchange-inline">><a href="references"</ins>>[5]<ins class="diffchange diffchange-inline"></a></ins></sup>.</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>ICA allows the assembly of silk monomers in different ratios and orders into a custom gene sequence of modifiable length. Gene monomers are assembled individually into a growing chain that is anchored to a solid foundation through streptavidin-biotin interaction.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>ICA allows the assembly of silk monomers in different ratios and orders into a custom gene sequence of modifiable length. Gene monomers are assembled individually into a growing chain that is anchored to a solid foundation through streptavidin-biotin interaction.</p></div></td></tr>
</table>Anuvedvermahttp://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=392458&oldid=prevAnuvedverma at 02:55, 18 October 20142014-10-18T02:55:01Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Customizing Silk</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h1>Customizing Silk</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h2>Background</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <h2>Background</h2></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p>Spider silk is an exceptional natural material. It exhibits remarkable strength and has the potential to be a very useful medium for a variety of applications. The properties of spider silk come primarily from the structure and amino acid sequence of the silk protein. One spider silk protein is comprised of repetitive units, referred to as monomers. Repeats of identical monomers make up one spider silk protein, and these proteins come together to form a silk fiber. The monomer is therefore the basic unit of the silk polypeptide, and changing the sequence alters the physical properties of the spider silk.<sup>[1]</sup> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p>Spider silk is an exceptional natural material. It exhibits remarkable strength and has the potential to be a very useful medium for a variety of applications. The properties of spider silk come primarily from the structure and amino acid sequence of the silk protein. One spider silk protein is comprised of repetitive units, referred to as monomers. Repeats of identical monomers make up one spider silk protein, and these proteins come together to form a silk fiber. The monomer is therefore the basic unit of the silk polypeptide, and changing the sequence alters the physical properties of the spider silk.<sup <ins class="diffchange diffchange-inline">href="#references"</ins>>[1]</sup> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: right;"><img src= "https://static.igem.org/mediawiki/2014/2/28/CustomizingSilk1.png" /><figcaption style="margin: auto;width:300px;">Fig 1: This image illustrates the amino acid motifs that compose various spider silk monomers. Different types of silk protein contain different motifs. These motifs define the physical characteristics of each monomer and the overall silk protein.<sup>[6]</sup></figcaption></figure></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: right;"><img src= "https://static.igem.org/mediawiki/2014/2/28/CustomizingSilk1.png" /><figcaption style="margin: auto;width:300px;">Fig 1: This image illustrates the amino acid motifs that compose various spider silk monomers. Different types of silk protein contain different motifs. These motifs define the physical characteristics of each monomer and the overall silk protein.<sup <ins class="diffchange diffchange-inline">href="#references"</ins>>[6]</sup></figcaption></figure></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p>In fact, the sequence of the spider silk monomer differs slightly from species to species. Therefore, silk produced by different species have different physical properties. Some monomer sequences result in silks with greater strength, while other sequences result in silks with greater flexibility. Furthermore, individual spiders can produce multiple types of silk such as dragline and flagelliform silk, each with very different properties.<sup>[2]</sup></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p>In fact, the sequence of the spider silk monomer differs slightly from species to species. Therefore, silk produced by different species have different physical properties. Some monomer sequences result in silks with greater strength, while other sequences result in silks with greater flexibility. Furthermore, individual spiders can produce multiple types of silk such as dragline and flagelliform silk, each with very different properties.<sup <ins class="diffchange diffchange-inline">href="#references"</ins>>[2]</sup></p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Much research has been done on the production of spider silk from recombinant bacteria.<sup>[3]</sup> We want to build on this work and take it a step further. Using the wide range of spider silk monomers as our building blocks, we want to be able to create hybrid silk proteins with specified physical properties. By mixing and matching these different monomer sequences within one polypeptide, we hope to be able to fine tune the properties of our produced silk. If this system could be standardized, it could be exceedingly useful for quickly producing silk to fit different a variety of different needs and purposes. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Much research has been done on the production of spider silk from recombinant bacteria.<sup <ins class="diffchange diffchange-inline">href="#references"</ins>>[3]</sup> We want to build on this work and take it a step further. Using the wide range of spider silk monomers as our building blocks, we want to be able to create hybrid silk proteins with specified physical properties. By mixing and matching these different monomer sequences within one polypeptide, we hope to be able to fine tune the properties of our produced silk. If this system could be standardized, it could be exceedingly useful for quickly producing silk to fit different a variety of different needs and purposes. </p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><h4 id="project_goals">Project Goals</h4><p>For our project, we have chosen to start out with two different silk monomers, Major Ampullate Spidroin 1 (MaSp1) and 2 (MaSp2). The MaSp1 monomer contributes strength to spider silk while the MaSp2 contributes more flexibility.<sup>[4]</sup> As a proof of principle we hope to show that by assembling these two repeats into one protein at different ratios, we can create silk with a spectrum of strength and extensibility. </p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><h4 id="project_goals">Project Goals</h4><p>For our project, we have chosen to start out with two different silk monomers, Major Ampullate Spidroin 1 (MaSp1) and 2 (MaSp2). The MaSp1 monomer contributes strength to spider silk while the MaSp2 contributes more flexibility.<sup <ins class="diffchange diffchange-inline">href="#references"</ins>>[4]</sup> As a proof of principle we hope to show that by assembling these two repeats into one protein at different ratios, we can create silk with a spectrum of strength and extensibility. </p> </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: left;"><img src= "https://static.igem.org/mediawiki/2014/a/ae/CustomizingSilk2.png" /><figcaption style="margin: auto;">Fig 2: Variations of the MaSp1 amino acid sequences between different spider species <sup>[6]</sup></figcaption></figure></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><figure style= "margin: 10px; float: left;"><img src= "https://static.igem.org/mediawiki/2014/a/ae/CustomizingSilk2.png" /><figcaption style="margin: auto;">Fig 2: Variations of the MaSp1 amino acid sequences between different spider species <sup <ins class="diffchange diffchange-inline">href="#references"</ins>>[6]</sup></figcaption></figure></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>References</h2></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h2>References</h2></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p dir="ltr"><sup><span>[1] </span></sup><span>Lewis, Randolph V. "Spider silk: ancient ideas for new biomaterials."&nbsp;</span><em>Chemical reviews</em><span>&nbsp;106.9 (2006): 3762-3774.</span></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p dir="ltr"><sup><span>[1] </span></sup><span>Lewis, Randolph V. "Spider silk: ancient ideas for new biomaterials."&nbsp;</span><em>Chemical reviews</em><span>&nbsp;106.9 (2006): 3762-3774.</span></p></div></td></tr>
</table>Anuvedvermahttp://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=369169&oldid=prevMichaelc1618 at 23:36, 17 October 20142014-10-17T23:36:50Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>ICA allows the assembly of silk monomers in different ratios and orders into a custom gene sequence of modifiable length. Gene monomers are assembled individually into a growing chain that is anchored to a solid foundation through streptavidin-biotin interaction.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>ICA allows the assembly of silk monomers in different ratios and orders into a custom gene sequence of modifiable length. Gene monomers are assembled individually into a growing chain that is anchored to a solid foundation through streptavidin-biotin interaction.</p></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>Silk monomers for ICA were built using PCR to attach <del class="diffchange diffchange-inline">bsaI </del>recognition site onto the <del class="diffchange diffchange-inline">MaSP </del>sequences, as well as different end extensions in the form of 4bp overhang that is essential for ligation. BsaI is a type IIs endonuclease that cleaves outside the recognition site and therefore generates overhangs that are still part of the native silk sequence. This leaves our digested products, or building blocks, free of any remaining recognition site, which is usually formed with other types of restriction enzymes. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>Silk monomers for ICA were built using PCR to attach <ins class="diffchange diffchange-inline">BsaI </ins>recognition site onto the <ins class="diffchange diffchange-inline">MaSp </ins>sequences, as well as different end extensions in the form of 4bp overhang that is essential for ligation. BsaI is a type IIs endonuclease that cleaves outside the recognition site and therefore generates overhangs that are still part of the native silk sequence. This leaves our digested products, or building blocks, free of any remaining recognition site, which is usually formed with other types of restriction enzymes. </p></div></td></tr>
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</table>Michaelc1618http://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=365907&oldid=prevAnuvedverma at 23:08, 17 October 20142014-10-17T23:08:28Z<p></p>
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</table>Anuvedvermahttp://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=365883&oldid=prevAnuvedverma at 23:08, 17 October 20142014-10-17T23:08:13Z<p></p>
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</table>Anuvedvermahttp://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=365396&oldid=prevAnuvedverma at 23:03, 17 October 20142014-10-17T23:03:20Z<p></p>
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</table>Anuvedvermahttp://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=362652&oldid=prevAnuvedverma at 22:35, 17 October 20142014-10-17T22:35:48Z<p></p>
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</table>Anuvedvermahttp://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=357634&oldid=prevAnuvedverma at 21:43, 17 October 20142014-10-17T21:43:28Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><<del class="diffchange diffchange-inline">p </del>id="project_goals"<del class="diffchange diffchange-inline">><h3</del>>Project Goals</<del class="diffchange diffchange-inline">h3</del>>For our project, we have chosen to start out with two different silk monomers, Major Ampullate Spidroin 1 (MaSp1) and 2 (MaSp2). The MaSp1 monomer contributes strength to spider silk while the MaSp2 contributes more flexibility.<sup>[4]</sup> As a proof of principle we hope to show that by assembling these two repeats into one protein at different ratios, we can create silk with a spectrum of strength and extensibility. </p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">h4 </ins>id="project_goals">Project Goals</<ins class="diffchange diffchange-inline">h4><p</ins>>For our project, we have chosen to start out with two different silk monomers, Major Ampullate Spidroin 1 (MaSp1) and 2 (MaSp2). The MaSp1 monomer contributes strength to spider silk while the MaSp2 contributes more flexibility.<sup>[4]</sup> As a proof of principle we hope to show that by assembling these two repeats into one protein at different ratios, we can create silk with a spectrum of strength and extensibility. </p> </div></td></tr>
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</table>Anuvedvermahttp://2014.igem.org/wiki/index.php?title=Team:UCLA/Project/Customizing_Silk&diff=357551&oldid=prevAnuvedverma at 21:42, 17 October 20142014-10-17T21:42:50Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p id="project_goals">For our project, we have chosen to start out with two different silk monomers, Major Ampullate Spidroin 1 (MaSp1) and 2 (MaSp2). The MaSp1 monomer contributes strength to spider silk while the MaSp2 contributes more flexibility.<sup>[4]</sup> As a proof of principle we hope to show that by assembling these two repeats into one protein at different ratios, we can create silk with a spectrum of strength and extensibility. </p> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p id="project_goals"<ins class="diffchange diffchange-inline">><h3>Project Goals</h3</ins>>For our project, we have chosen to start out with two different silk monomers, Major Ampullate Spidroin 1 (MaSp1) and 2 (MaSp2). The MaSp1 monomer contributes strength to spider silk while the MaSp2 contributes more flexibility.<sup>[4]</sup> As a proof of principle we hope to show that by assembling these two repeats into one protein at different ratios, we can create silk with a spectrum of strength and extensibility. </p> </div></td></tr>
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