Team:Bordeaux/Parts

From 2014.igem.org

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We decided to provide the registry with just a few parts but as documented as possible (in terms either of experiment or design and literature). These parts encode  proteins based on consensus sequences of natural proteins. They are synthetic proteins (not existing in nature), used in a context of "white biotechnology" as biomaterials with specific properties unravelled from the nature.
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<h1> Synthesis of the gene coding for the SLPs(BBa_K1317002)</h1>
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[[File:Bdx2014 resilin 2.jpg|center|300px]]<br>
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[https://2014.igem.org/Team:Bordeaux/Parts/BBa_K1317001 BBa_K1317001]<br>
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This part is the coding sequence for the resilin like polypeptide. This sequence was assembled from a consensus of the proresilin exon 1 from Drosophila melanogaster. This protein, in insects, allows resistance and elasticity used for jumping, flapping... The synthetic gene encodes a synthetic protein "resilin-like". The repeated aminoacids allow to retrieve these properties of resistance, resilience and elasticity, but with a minimal pattern of the original protein, which is more suited for downstream applications. It can be used to produce wire presenting these properties after wet-spinning.<br><br>
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[[File:Bdx2014 spider.jpg|center|300px]]<br>
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[https://2014.igem.org/Team:Bordeaux/Parts/BBa_K1317002 BBa_K1317002]<br>
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This part is the coding sequence for the silk like polypeptide. This sequence was assembled from a consensus of the MaSp1 from spiders. This protein, in spiders, allows resistance and elasticity used for spinning the webs. The synthetic gene encodes a synthetic protein "silk-like". The repeated aminoacids allow to retrieve these properties of resistance, resilience and elasticity, but with a minimal pattern of the original protein, which is more suited for downstream applications. It can be used to produce wire presenting these properties after wet-spinning.<br> <br>
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[[File:Bdx2014 elastin.jpg|center|300px]]<br>
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[https://2014.igem.org/Team:Bordeaux/Parts/BBa_K1317003 BBa_K1317003]<br>
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This part is made with a consensus of the natural elastin sequence. Elastin is a protein involved in maintaining shape and elasticity of several tissues like skin. It has a high elasticity and resilience as a polymer.
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In our project, the consensus sequence allows the use of a minimal pattern of the protein while keeping these particular properties. In an iGEM spirit the consensus itself can be used as a brick to vary length and nature of the polymers with our other biobricks (BBa_K1317001, BBa_K1317002).
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The basic length of a polymer is 20 monomers. The coding sequence can be repeated easily using our improvement of the biobrick assembly system to get rid of the Stop Codon. After variation of the length different properties are attributed to the part. For example it can be used as a fusion tag to purify easily any fuse protein thanks to the thermal cycling purification.
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ELP indeed have this particularity to solubilize at lower temperature. If it is used in a protein mixture (a lysate for example) after a few cycles of thermal cycling (from 4°C to 50°C), the pure fuse protein is obtained.
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This part can be used as a purification tag for easy, quick and column-free purification or as coding sequence for a polymer usable to get fiber with good elasticity and resilience. The polymer is biocompatible, biodegradable and could be used as a tool for surgery or other health applications.<br><br>
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<b>The part provided to the registry encodes for ELP20. Throughout all the experiments, if two copies are used it will be ELP40, and if three copies are used it will be ELP60.</b> <br> <br>
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[https://2014.igem.org/Team:Bordeaux/Parts/BBa_K1317004 BBa_K1317004]<br>
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The ELP presents specific properties: it precipitates at high temperature. This property can be used for thermal cycling purification for instance. By successive cycles of temperature, ELP can be purified from a mix of proteins. This special property has been used to create a composite part for the iGEM registry. It is a proof of concept of the use of this part as a tool for protein purification by fusing ELP to another protein. The protein used as an example is RFP, to ease vizualisation of the process.<br> <br>
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<h3>Initial strategy :<br></h3>
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[https://2014.igem.org/Team:Bordeaux/Parts/Assembly_improvement Improvement of the Biobrick standard assembly]<br>
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In order to get rid of the stop codon in a coding sequence, to fuse properly proteins, we proposed an improvement of the assembly system by using the restriction site NheI.<br>
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We tried to assemble the gene coding for the SLPs from 8 oligonucleotids with homolog regions with the Gibson Assembly. <br>
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First of all, consensus sequences for the spider silk were identified and our own protein was designed. Then, the nucleotidic sequence using the peptidic sequence was determined. We had to pay attention because our proteic sequence is made of repeted motifs.<br><br>
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</p>
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<center><img class="" src="https://static.igem.org/mediawiki/parts/9/9d/Bdx2014_SLP_synthesis_01.jpg" alt=""/><br>
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Table 1 : sequences of the 8 nucleotides<br><br></center>
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2 different methods were used with the Gibson Assembly1: in one step at 50°C or with cycles of denaturation at 95°C and annealing at 50°C (figure 1). The enzyme used was the Phusion® High Fidelity Polymerase.<br><br></p>
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<center><img class="" src="https://static.igem.org/mediawiki/parts/1/1d/Bdx2014_SLP_synthesis_02.jpg" alt=""/></center><br>
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Figure 1: Strategy of the Gibson Assembly to assemble the gene coding for the SLPs<br><br>
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<p>
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Our 8 oligo weren’t assembled with these 2 methods, so another method was used : the PCR-Fusion2.<br>
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This method is made of different steps using the Phusion® High Fidelity Polymerase (picture 2). In a first step fragments were joining two by two, then fragments 1-2 were joined to fragments 3-4 and a PCR is achieved using fragments 1 and 4 as primers. The same method was used for fragments 5-6 and 7-8.<br>
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Finally, fragments 1-2-3-4 were assembled to fragments 5-6-7-8 and a PCR was also achieved using the fragments 1 and 8 as primers.<br><br></p>
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<center><img class="" src="https://static.igem.org/mediawiki/parts/d/df/Bdx2014_SLP_synthesis04.png" alt=""/></center><br>Figure 2: PCR Fusion strategy to assemble the gene coding for the SLPs<br><br>
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<p>
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This method wasn’t successful because fragments 6 and 7 were unable to join. Therefore, new fragments were designed with a different homolog region. The fragment 8 that added only 2 nucleotids was suppressed and these 2  nucleotids were added on fragments 7. <br><br>
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<center><img class="" src="https://static.igem.org/mediawiki/parts/b/bc/Bdx2014_SLP_synthesis03.png" alt=""/></center><br>Table 2: Sequence of the new fragments<br><br>
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Then, a new strategy was used (picture 3). The two first steps are common but then, fragments 5-6 are joined to fragments 1-2-3-4 and the PCR is made with the fragments 1 and 6. Finally the fragment 7 is added and a PCR is also achieved. </p>
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<center><img class="" src="https://static.igem.org/mediawiki/parts/e/eb/Bdx2014_slp5.png" alt=""/></center><br>Fig 3 : Strategy to assemble the CDS for the SLPs using the new fragments 6 and 7<br><br>
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This method enable the assembly of the 7 fragments (picture 4). A fragment of 318 bp was expected on the electrophoresis gel.<br><br></p>
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<center><img class="" src="https://static.igem.org/mediawiki/parts/d/d6/Bdx2014_Slp6.png" alt=""/></center><br>Figure 4 : Gel electrophoresis on 3% agarose<br><br>
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<h3>Reference : </h3><br><br>
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[1] https://www.neb.com/tools-and-resources/feature-articles/gibson-assembly-building-a-synthetic-biology-toolset
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[2] Shevchuk N.A., Bryksin A.V., Nusinovich Y.A., Cabello F.C., Sutherland M. et Ladisch S. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously (2004) Nucleic Acids Res., 32(2), 19
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<h1>Part:BBa_K1317001 : CDS for resilin-like polypeptide (RLP)</h1>
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This part is the coding sequence for the resilin like polypeptide. This sequence was assembled from a consensus of the proresilin exon 1 from Drosophila melanogaster. This protein, in insects, allows resistance and elasticity used for jumping, flapping... The synthetic gene encodes a synthetic protein "resilin-like". The repeated aminoacids allow to retrieve these properties of resistance, resilience and elasticity, but with a minimal pattern of the original protein, which is more suited for downstream applications. It can be used to produce wire presenting these properties after wet-spinning.</p>
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<h3>Sequence and Features</h3>
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{{:Team:Bordeaux/Pied}}
{{:Team:Bordeaux/Pied}}

Latest revision as of 03:06, 18 October 2014

We decided to provide the registry with just a few parts but as documented as possible (in terms either of experiment or design and literature). These parts encode proteins based on consensus sequences of natural proteins. They are synthetic proteins (not existing in nature), used in a context of "white biotechnology" as biomaterials with specific properties unravelled from the nature.

Bdx2014 resilin 2.jpg

BBa_K1317001
This part is the coding sequence for the resilin like polypeptide. This sequence was assembled from a consensus of the proresilin exon 1 from Drosophila melanogaster. This protein, in insects, allows resistance and elasticity used for jumping, flapping... The synthetic gene encodes a synthetic protein "resilin-like". The repeated aminoacids allow to retrieve these properties of resistance, resilience and elasticity, but with a minimal pattern of the original protein, which is more suited for downstream applications. It can be used to produce wire presenting these properties after wet-spinning.

Bdx2014 spider.jpg

BBa_K1317002
This part is the coding sequence for the silk like polypeptide. This sequence was assembled from a consensus of the MaSp1 from spiders. This protein, in spiders, allows resistance and elasticity used for spinning the webs. The synthetic gene encodes a synthetic protein "silk-like". The repeated aminoacids allow to retrieve these properties of resistance, resilience and elasticity, but with a minimal pattern of the original protein, which is more suited for downstream applications. It can be used to produce wire presenting these properties after wet-spinning.

Bdx2014 elastin.jpg

BBa_K1317003
This part is made with a consensus of the natural elastin sequence. Elastin is a protein involved in maintaining shape and elasticity of several tissues like skin. It has a high elasticity and resilience as a polymer. In our project, the consensus sequence allows the use of a minimal pattern of the protein while keeping these particular properties. In an iGEM spirit the consensus itself can be used as a brick to vary length and nature of the polymers with our other biobricks (BBa_K1317001, BBa_K1317002). The basic length of a polymer is 20 monomers. The coding sequence can be repeated easily using our improvement of the biobrick assembly system to get rid of the Stop Codon. After variation of the length different properties are attributed to the part. For example it can be used as a fusion tag to purify easily any fuse protein thanks to the thermal cycling purification. ELP indeed have this particularity to solubilize at lower temperature. If it is used in a protein mixture (a lysate for example) after a few cycles of thermal cycling (from 4°C to 50°C), the pure fuse protein is obtained. This part can be used as a purification tag for easy, quick and column-free purification or as coding sequence for a polymer usable to get fiber with good elasticity and resilience. The polymer is biocompatible, biodegradable and could be used as a tool for surgery or other health applications.

The part provided to the registry encodes for ELP20. Throughout all the experiments, if two copies are used it will be ELP40, and if three copies are used it will be ELP60.

BBa_K1317004
The ELP presents specific properties: it precipitates at high temperature. This property can be used for thermal cycling purification for instance. By successive cycles of temperature, ELP can be purified from a mix of proteins. This special property has been used to create a composite part for the iGEM registry. It is a proof of concept of the use of this part as a tool for protein purification by fusing ELP to another protein. The protein used as an example is RFP, to ease vizualisation of the process.

Improvement of the Biobrick standard assembly
In order to get rid of the stop codon in a coding sequence, to fuse properly proteins, we proposed an improvement of the assembly system by using the restriction site NheI.

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