Difference between revisions of "Team:Bielefeld-CeBiTec/Project/toolbox/fusing"

 
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Fusing proteins is normally limited to the C‑ or N‑terminus of a protein.  
 
Fusing proteins is normally limited to the C‑ or N‑terminus of a protein.  
 
The incorporation of non-canonical amino acids that could be fused to each  
 
The incorporation of non-canonical amino acids that could be fused to each  
−
other or to surfaces enables several additional applications. This tool facilitates
+
other or to surfaces enables several additional applications. Examples are the
−
immobilization of proteins and improved stability of protein polymer networks.  
+
immobilization of proteins as well as improved stability of protein polymer networks.  
−
Furthermore, they lead to enhanced efficiency of pathways by combining enzymes of  
+
Furthermore, it leads to enhanced efficiency of pathways by combining enzymes of  
 
one pathway or for any other system where colocalization is beneficial.
 
one pathway or for any other system where colocalization is beneficial.
 
<br>
 
<br>
−
As proof of concept, we work on enhanced stability of a protein polymer. This networks  
+
As proof of concept, we work on enhanced stability of a protein polymer. These networks  
 
can be applied for different applications like modern biomaterials in medicine and industry  
 
can be applied for different applications like modern biomaterials in medicine and industry  
 
(Rnjak-Kovacina&nbsp;<i>et&nbsp;al.</i>,&nbsp;2011). The amino acids <i>N</i><sup>ε</sup>&#x2011;L&#x2011;cysteinyl&#x2011;L&#x2011;lysine (CL)  
 
(Rnjak-Kovacina&nbsp;<i>et&nbsp;al.</i>,&nbsp;2011). The amino acids <i>N</i><sup>ε</sup>&#x2011;L&#x2011;cysteinyl&#x2011;L&#x2011;lysine (CL)  
 
and <i>N</i><sup>γ</sup>&#x2011;2&#x2011;cyanobenzothiazol&#x2011;6&#x2011;yl&#x2011;L&#x2011;asparagine (CBT&#x2011;asparagine)  
 
and <i>N</i><sup>γ</sup>&#x2011;2&#x2011;cyanobenzothiazol&#x2011;6&#x2011;yl&#x2011;L&#x2011;asparagine (CBT&#x2011;asparagine)  
−
comprise key parts of this tool. Both amino acids can bind specificly to each other resulting in the formation  
+
comprise key parts of this tool. Both amino acids can bind specifically to each other resulting in the formation  
 
of a covalent bond between their side chains. We plan to use this covalent bond to increase the stability of  
 
of a covalent bond between their side chains. We plan to use this covalent bond to increase the stability of  
 
silk elastin like proteins (SELPs). The strengthened polymer network would be a perfect material to produce  
 
silk elastin like proteins (SELPs). The strengthened polymer network would be a perfect material to produce  
−
biological wound bindings which are very thin and they would be able to interact with the natural tissue matrix  
+
biological wound bindings which are very thin and would be able to interact with the natural tissue matrix  
 
(Boateng&nbsp;<i>et&nbsp;al.</i>,&nbsp;2008).
 
(Boateng&nbsp;<i>et&nbsp;al.</i>,&nbsp;2008).
 
</article>
 
</article>
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<article>
 
<article>
 
By synthesis of amino acids with side chains containing CBT and a 1,2&#x2011;aminothiol, polypeptides  
 
By synthesis of amino acids with side chains containing CBT and a 1,2&#x2011;aminothiol, polypeptides  
−
binding to each other should be produced. These amino acids are CL and CBT&#x2011;asparagine. The binding  
+
binding to each other should be produced. These amino acids are CL and CBT&#x2011;asparagine whose binding  
−
mechanism of both amino acids are shown in figure 2.
+
mechanism is shown in figure 2.
 
</article>
 
</article>
 
<!-- Mittleres zentriertes Bild -->
 
<!-- Mittleres zentriertes Bild -->
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<h3><i>N</i><sup>ε</sup>-L-cysteinyl-L-lysine</h3>
 
<h3><i>N</i><sup>ε</sup>-L-cysteinyl-L-lysine</h3>
 
<article>
 
<article>
−
We synthesized CL in our lab and provide the community with a validated protocol. Currently, we are
+
We synthesized CL and CBT&#x2011;asparagine in our lab. Additionally we are providing the community with a validated <a  
−
trying to synthesize CBT&#x2011;asparagine. Details of our synthesis protocol are described in the
+
href="https://static.igem.org/mediawiki/2017/a/a6/T--Bielefeld-CeBiTec--protocol_CBT-asparagine.pdf">protocol for the synthesis of CBT&#x2011;asparagine</a>.
−
<a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Notebook/Methods">method&nbsp;section</a>.
+
 
</article>
 
</article>
  
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chain of lysine so that CL contains a free 1,2-aminothiol group  
 
chain of lysine so that CL contains a free 1,2-aminothiol group  
 
(Nguyen&nbsp;<i>et&nbsp;al.</i>,&nbsp;2011). This is an important characteristic for the  
 
(Nguyen&nbsp;<i>et&nbsp;al.</i>,&nbsp;2011). This is an important characteristic for the  
−
specific binding between the CL and the CBT&#x2011;asparagine.  
+
specific binding between CL and CBT&#x2011;asparagine.  
 
<ul>
 
<ul>
 
<li>Name: <i>N</i><sup>ε</sup>-L-cysteinyl-L-lysine</li>
 
<li>Name: <i>N</i><sup>ε</sup>-L-cysteinyl-L-lysine</li>
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<article>
 
<article>
 
This specific binding can improve the stability of SELPs. These are linear polypeptides with repeats of silk and elastin consensus  
 
This specific binding can improve the stability of SELPs. These are linear polypeptides with repeats of silk and elastin consensus  
−
sequences. They show brought application and possibilities in medicine, tissue engineering and industry  
+
sequences. They show broad applications in medicine, tissue engineering and industry  
 
(Rnjak-Kovacina&nbsp;<i>et&nbsp;al.</i>,&nbsp;2011). The silk consensus sequence is GAGAGS and the elastin consensus sequence is  
 
(Rnjak-Kovacina&nbsp;<i>et&nbsp;al.</i>,&nbsp;2011). The silk consensus sequence is GAGAGS and the elastin consensus sequence is  
 
VPAVG. The consensus sequences can interact with each other and are able to form non-covalent hydrogen bonds. This results in a  
 
VPAVG. The consensus sequences can interact with each other and are able to form non-covalent hydrogen bonds. This results in a  
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/3/36/T--Bielefeld-CeBiTec--27-08-17-Schematic_SELP.png">
 
<img class="figure image" src="https://static.igem.org/mediawiki/2017/3/36/T--Bielefeld-CeBiTec--27-08-17-Schematic_SELP.png">
 
<p class="figure subtitle">
 
<p class="figure subtitle">
−
<b>Figure 5: Schematic structure of a SELP polymer network.</b><br>Silk consensus sequences are shown in green,  
+
<b>Figure 5: Schematic structure of a SELP polymer network.</b><br>Silk consensus sequences (GAGAGS) are shown in green,  
−
elastin consensus sequences are red and the blue lines show the hydrogen bonds of the consensus sequences.</p>
+
elastin consensus sequences (VPAVG) in red and the blue lines indicate the hydrogen bonds of the consensus sequences.</p>
 
</div>
 
</div>
 
<article>
 
<article>
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/b/b3/T--Bielefeld-CeBiTec--03-10-17-SELP_seq_Collins2013.png">
 
<img class="figure image" src="https://static.igem.org/mediawiki/2017/b/b3/T--Bielefeld-CeBiTec--03-10-17-SELP_seq_Collins2013.png">
 
<p class="figure subtitle">
 
<p class="figure subtitle">
−
<b>Figure 6: Schematic sequence of the SELP (Collins&nbsp;<i>et&nbsp;al.</i>,&nbsp;2013).</b><br>Silk consensus sequences  
+
<b>Figure 6: Schematic sequence of the SELP (Collins&nbsp;<i>et&nbsp;al.</i>,&nbsp;2013).</b><br>Silk consensus sequences (S)
−
are green and elastin consensus sequences are red.</p>
+
are shown in green and elastin consensus sequences (E) in red.</p>
 
</div>
 
</div>
  
 
<article>
 
<article>
−
By incorporation of CL and CBT&#x2011;asparagine between the silk and the elastin repeats, we receive a strengthened polymer network  
+
By incorporating CL and CBT&#x2011;asparagine between the silk and the elastin repeats, we receive a strengthened polymer network  
 
with covalent bonds (Figure 7).
 
with covalent bonds (Figure 7).
 
</article>
 
</article>
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/9e/T--Bielefeld-CeBiTec--27-08-17-SELP_ncAA.png">
 
<img class="figure image" src="https://static.igem.org/mediawiki/2017/9/9e/T--Bielefeld-CeBiTec--27-08-17-SELP_ncAA.png">
 
<p class="figure subtitle">
 
<p class="figure subtitle">
−
<b>Figure 7: Schematic structure of a SELP polymer network.</b>
+
<b>Figure 7: Schematic structure of a SELP polymer network including CL and CBT-asparagine.</b><br>CL and CBT-asparagine (purple) are introduced between the silk (green) and elastin (red) repeats.</p>
−
</p>
+
 
</div>
 
</div>
 
<h3>Recursive Directional Ligation by Plasmid Reconstruction (PRe-RDL)</h3>
 
<h3>Recursive Directional Ligation by Plasmid Reconstruction (PRe-RDL)</h3>
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The gene sequence for these SELPs has a high GC content and contains a high number of repeats leading to issues during synthesis.  
 
The gene sequence for these SELPs has a high GC content and contains a high number of repeats leading to issues during synthesis.  
 
Therefore, PRe-RDL can be applied to address this challenge. PRe-RDL uses three restriction sites of a parent plasmid which contains the  
 
Therefore, PRe-RDL can be applied to address this challenge. PRe-RDL uses three restriction sites of a parent plasmid which contains the  
−
gene of interest (goi) and the subsequently ligation of fragments of two different restricted parent plasmids. The first step involves  
+
gene of interest (goi) and the subsequent ligation of fragments of two different restricted parent plasmids. The first step involves  
 
the restriction of a parent plasmid at the 3'-end of the goi and in the backbone. The second step is the restriction of a parent plasmid  
 
the restriction of a parent plasmid at the 3'-end of the goi and in the backbone. The second step is the restriction of a parent plasmid  
 
at the 5'-end of the goi and at the same position of the backbone as in the first step. The final step is the ligation of both generated  
 
at the 5'-end of the goi and at the same position of the backbone as in the first step. The final step is the ligation of both generated  
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<p class="figure subtitle">
 
<p class="figure subtitle">
 
<b>Figure 8: Scheme of the PRe-RDL according to McDaniel&nbsp;<i>et&nbsp;al.</i>,&nbsp;2010 and applied to pSB1C3 containing one elastin consensus sequence.</b> The Pre-RDL
 
<b>Figure 8: Scheme of the PRe-RDL according to McDaniel&nbsp;<i>et&nbsp;al.</i>,&nbsp;2010 and applied to pSB1C3 containing one elastin consensus sequence.</b> The Pre-RDL
−
consists out of 3 steps, two different digestions (step 1 and 2) and one ligation (step 3).</p>
+
consists of 3 steps, two different digestions (step 1 and 2) and one ligation (step 3).</p>
 
</div>
 
</div>
 
<div class="article">
 
<div class="article">
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</div>
 
</div>
 
<div class="figure large">
 
<div class="figure large">
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<img class="figure image" src="https://static.igem.org/mediawiki/2017/5/5a/T--Bielefeld-CeBiTec--1-11-17_Design_elastin_silk.png">
+
<img class="figure image" src="https://static.igem.org/mediawiki/2017/f/f5/T--Bielefeld-CeBiTec--1-11-17_Design_elastin_silk.png">
 
<p class="figure subtitle">
 
<p class="figure subtitle">
−
<b>Figure 9: Design of <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201250">K2201250</a> and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201251">K2201251</a>.</b> </p>
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<b>Figure 9: Design of <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201250">BBa_K2201250</a> and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2201251">BBa_K2201251</a>.</b> </p>
 
</div>
 
</div>
 
<div class="article">
 
<div class="article">
 
Step 1 serves to create a fragment containing one part of the chloramphenicol resistance (CmR) and the whole monomer consensus sequence. To do so, the plasmid has to be digested  
 
Step 1 serves to create a fragment containing one part of the chloramphenicol resistance (CmR) and the whole monomer consensus sequence. To do so, the plasmid has to be digested  
−
by <i>Acu</i>I and <i>Bsp</i>EI. The other fragments which are resulting from this step contain the other part of the CmR and each one part of the origin of replication (ori).  
+
with <i>Acu</i>I and <i>Bsp</i>EI. The other fragments which are resulting from this step contain the other part of the CmR and each one part of the origin of replication (ori).  
 
This decreases the chance of religation of the three fragments originating from this step. For step 2, it is necessary to use <i>Bse</i>RI and <i>Bsp</i>EI. The result of this  
 
This decreases the chance of religation of the three fragments originating from this step. For step 2, it is necessary to use <i>Bse</i>RI and <i>Bsp</i>EI. The result of this  
 
digestion is one fragment containing one part of the CmR and the spacer between the prefix and the monomer consensus sequence and one fragment with the other part of the CmR,  
 
digestion is one fragment containing one part of the CmR and the spacer between the prefix and the monomer consensus sequence and one fragment with the other part of the CmR,  
−
the ori, and the monomer consensus squence. By ligation of both fragments of step 1 and 2 containing the monomer consensus sequence (step 3) you get pSB1C3 with two repeats of the  
+
the ori, and the monomer consensus sequence. By ligation of both fragments of step 1 and 2 containing the monomer consensus sequence (step 3) pSB1C3 with two repeats of the  
−
monomer consensus sequence. By repeating these steps it is possible to create repetitive sequences consisiting out of monomeric consenesus sequences. During the PRe-RDL it is possible
+
monomer consensus sequence results. By repeating these steps it is possible to create repetitive sequences consisting of monomeric consensus sequences. During the PRe-RDL it is also possible
−
to add codons like the amber codon using primers. After the PRe-RDL it is necessary to add a start and a stop codon.
+
to add codons like the amber codon using primers. After the PRe-RDL it is important to add a start and a stop codon.
 
</div>
 
</div>
  

Latest revision as of 00:43, 2 November 2017

Fusing

Short summary

Fusing proteins is normally limited to the C‑ or N‑terminus of a protein. The incorporation of non-canonical amino acids that could be fused to each other or to surfaces enables several additional applications. Examples are the immobilization of proteins as well as improved stability of protein polymer networks. Furthermore, it leads to enhanced efficiency of pathways by combining enzymes of one pathway or for any other system where colocalization is beneficial.
As proof of concept, we work on enhanced stability of a protein polymer. These networks can be applied for different applications like modern biomaterials in medicine and industry (Rnjak-Kovacina et al., 2011). The amino acids Nε‑L‑cysteinyl‑L‑lysine (CL) and Nγ‑2‑cyanobenzothiazol‑6‑yl‑L‑asparagine (CBT‑asparagine) comprise key parts of this tool. Both amino acids can bind specifically to each other resulting in the formation of a covalent bond between their side chains. We plan to use this covalent bond to increase the stability of silk elastin like proteins (SELPs). The strengthened polymer network would be a perfect material to produce biological wound bindings which are very thin and would be able to interact with the natural tissue matrix (Boateng et al., 2008).

Terminus independent fusion proteins

While terminus dependent binding systems for proteins are already in use, there are only a few systems for terminus independent binding systems. We want to expand the number of those systems. Our aim is to incorporate two non-canonical amino acids, which are able to build a specific bond to each other. According to the synthesis of D-luciferin for the firefly luciferase of Photinus pyralis, we decided to use the specific binding of 1,2‑aminothiols and the cyano group of cyanobenzothiazole (CBT). Figure 1 shows the biosynthesis of luciferin and the mechanism of the binding reaction of 1,2‑aminothiol and CBT.

Figure 1: Reaction of the 1,2‑aminothiol of cysteine and CBT to luciferin (Liang et al., 2010).

By synthesis of amino acids with side chains containing CBT and a 1,2‑aminothiol, polypeptides binding to each other should be produced. These amino acids are CL and CBT‑asparagine whose binding mechanism is shown in figure 2.

Figure 2: Specific binding reaction of CL and CBT-asparagine.

Nε-L-cysteinyl-L-lysine

CL is an amino acid consisting of cysteine and lysine. The cysteine was coupled to the side chain of lysine so that CL contains a free 1,2-aminothiol group (Nguyen et al., 2011). This is an important characteristic for the specific binding between CL and CBT‑asparagine.
  • Name: Nε-L-cysteinyl-L-lysine
  • Short: CL
  • Molecular Weight: 249.33 g mol-1
  • Storage: -20 – 4 °C
  • Function: Terminus independent binding system

Figure 3: Structure of CL.

Nγ-2-cyanobenzothiazol-6-yl-L-asparagine

Figure 4: Structure of CBT‑asparagine.

CBT‑asparagine is a completely novel amino acid, which we are synthesizing on our own. The synthesis is based on coupling the amino group of 6-Amino-CBT to the carboxyl group of the side chain of L-asparagine. The cyano group of the CBT enables the specific binding of the CBT‑asparagine to 1,2-aminothiols.
  • Name: Nγ‑2‑cyanobenzothiazol‑6‑yl‑L‑asparagine
  • Short: CBT‑asparagine
  • Molecular Weight: 290.30 g mol-1
  • Storage: -20 – 4 °C
  • Function: Terminus independent binding system

Silk Elastin like Proteins

This specific binding can improve the stability of SELPs. These are linear polypeptides with repeats of silk and elastin consensus sequences. They show broad applications in medicine, tissue engineering and industry (Rnjak-Kovacina et al., 2011). The silk consensus sequence is GAGAGS and the elastin consensus sequence is VPAVG. The consensus sequences can interact with each other and are able to form non-covalent hydrogen bonds. This results in a polymer network based on hydrogen bonds with a β-sheet structure. Figure 5 shows the schematic structure of a SELP polymer network.

Figure 5: Schematic structure of a SELP polymer network.
Silk consensus sequences (GAGAGS) are shown in green, elastin consensus sequences (VPAVG) in red and the blue lines indicate the hydrogen bonds of the consensus sequences.

According to the work of Collins et al. (2013), we decided to use a sequence with nine repeats of five repeats of the silk consensus sequence and nine repeats of the elastin consensus sequence (see figure 6).

Figure 6: Schematic sequence of the SELP (Collins et al., 2013).
Silk consensus sequences (S) are shown in green and elastin consensus sequences (E) in red.

By incorporating CL and CBT‑asparagine between the silk and the elastin repeats, we receive a strengthened polymer network with covalent bonds (Figure 7).

Figure 7: Schematic structure of a SELP polymer network including CL and CBT-asparagine.
CL and CBT-asparagine (purple) are introduced between the silk (green) and elastin (red) repeats.

Recursive Directional Ligation by Plasmid Reconstruction (PRe-RDL)

The gene sequence for these SELPs has a high GC content and contains a high number of repeats leading to issues during synthesis. Therefore, PRe-RDL can be applied to address this challenge. PRe-RDL uses three restriction sites of a parent plasmid which contains the gene of interest (goi) and the subsequent ligation of fragments of two different restricted parent plasmids. The first step involves the restriction of a parent plasmid at the 3'-end of the goi and in the backbone. The second step is the restriction of a parent plasmid at the 5'-end of the goi and at the same position of the backbone as in the first step. The final step is the ligation of both generated fragments containing the goi. The result is a plasmid with two copies of the goi (Figure 5).

Figure 8: Scheme of the PRe-RDL according to McDaniel et al., 2010 and applied to pSB1C3 containing one elastin consensus sequence. The Pre-RDL consists of 3 steps, two different digestions (step 1 and 2) and one ligation (step 3).

Applied to pSB1C3 containing the consensus sequences of monomers and the spacers between the BioBrick prefix and suffix shown in figure 9, it is possible to use the PRe-RDL in combination with the restriction enzymes, AcuI, BseRI, and BspEI, to build repetitive sequences of monomers.

Figure 9: Design of BBa_K2201250 and BBa_K2201251.

Step 1 serves to create a fragment containing one part of the chloramphenicol resistance (CmR) and the whole monomer consensus sequence. To do so, the plasmid has to be digested with AcuI and BspEI. The other fragments which are resulting from this step contain the other part of the CmR and each one part of the origin of replication (ori). This decreases the chance of religation of the three fragments originating from this step. For step 2, it is necessary to use BseRI and BspEI. The result of this digestion is one fragment containing one part of the CmR and the spacer between the prefix and the monomer consensus sequence and one fragment with the other part of the CmR, the ori, and the monomer consensus sequence. By ligation of both fragments of step 1 and 2 containing the monomer consensus sequence (step 3) pSB1C3 with two repeats of the monomer consensus sequence results. By repeating these steps it is possible to create repetitive sequences consisting of monomeric consensus sequences. During the PRe-RDL it is also possible to add codons like the amber codon using primers. After the PRe-RDL it is important to add a start and a stop codon.

References

Boateng, J., Matthews, K.H., Stevens, H.N.E., and Eccleston, G.M. (2008). Wound Healing Dressings and Drug Delivery Systems: A Review. J. Pharm. Sci. 97.

Collins, T., Azevedo-silva, J., Costa, A., Branca, F., Machado, R., and Casal, M. (2013). Batch production of a silk-elastin-like protein in E . coli BL21 ( DE3 ): key parameters for optimisation. Microb. Cell Fact. 12: 1–16.

Liang, G., Ren, H., and Rao, J. (2010). A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. Nat. Chem. 2: 54–60.

McDaniel, J.R., Mackay, J.A., Quiroz, F.G., and Chilkoti, A. (2010). Recursive Directional Ligation by Plasmid Reconstruction allows Rapid and Seamless Cloning of Oligomeric Genes. 11: 944–952.

Nguyen, D.P., Elliott, T., Holt, M., Muir, T.W., and Chin, J.W. (2011). Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation. J. Am. Chem. Soc. 133: 11418–11421.

Rnjak-Kovacina, J., Daamen, W.F., Pierna, M., Rodríguez-Cabello, J.C., and Weiss, A.S. (2011). Elastin Biopolymers. Compr. Biomater.: 329–346.