Difference between revisions of "Part:BBa K4905001"
 
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<partinfo>BBa_K4905001 short</partinfo>
 
<partinfo>BBa_K4905001 short</partinfo>
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<!-- The TU-Eindhoven 2023 team used this part for the formation of a hydrogel in e.coli. The repeating sequence of this part is (VPGIG)[60], also referred to as I, and (VPGAG)3(VPGGG)2[60], also referred to as A, which creates a hydrophilic and a hydrophobic part. It has been codon optimized for expression in E. coli cells. -->
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<h1>Information</h1>
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== Information ==  
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Elastin-like polypeptides (ELPs) are protein polymers derived from human tropoelastin. One of their key features is that they exhibit a phase separation that is often reversible whereby samples remain soluble below a transition temperature (T<sub>t</sub>) but form coacervates above T<sub>t</sub>. They have many possible applications in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators<sup>[2]</sup>.
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The general structure of polymeric ELPs is (VPGXG)n, where the monomeric unit is Val-Pro-Gly-X-Gly, and the "X" denotes a variable amino acid that can have consequences on the general properties of the ELP, such as the T<sub>t</sub>. Specifically, the hydrophilicity or hydrophobicity and the presence or absence of a charge on the guest residue play a great role in determining the T<sub>t</sub>. Also, the solubilization of the guest residue can affect the T<sub>t</sub>. The "n" denotes the number of monomeric units that comprise the polymer<sup>[1]</sup>. In general, these polymers are linear below the T<sub>t</sub>, but aggregate into spherical clumps above the T<sub>t</sub><sup>[3]</sup>. The repeating sequence for this specific part is (VPGIG)<sub>[60]</sub> and then (VPGAG)<sub>3</sub>(VPGGG)<sub>2</sub><sub>[12]</sub>.
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The TU-Eindhoven 2023 team used this part in a composite part for the formation of a hydrogel in E. coli. It has been codon optimized for expression in <i>E. coli</i> BL21 cells. The composite part is <a style="color:#F6B227" href="https://parts.igem.org/Part:BBa_K4905006"> BBa_K4905006 </a>.
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Elastin-like polypeptides (ELPs) are protein polymers derived from human tropoelastin. One of their key features is that they exhibit a phase separation that is often reversible whereby samples remain soluble below a transition temperature (Tt) but form coacervates above Tt. They have many possible applications in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators [2].
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<span class='h3bb'><h1>Sequence and Features</h1></span>
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<p> the following sequence was used to create this part. It was optimized for <i>E.coli</i> BL21 cells. The sequence was cloned into a pET24a(+) vector, this was multiplied in TOP10 competent cells. </p>
  
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The general structure of polymeric ELPs is (VPGXG)n, where the monomeric unit is Val-Pro-Gly-X-Gly, and the "X" denotes a variable amino acid that can have consequences on the general properties of the ELP, such as the transition temperature (Tt). Specifically, the hydrophilicity or hydrophobicity and the presence or absence of a charge on the guest residue play a great role in determining the Tt. Also, the solubilization of the guest residue can affect the Tt. The "n" denotes the number of monomeric units that comprise the polymer [1]. In general, these polymers are linear below the Tt, but aggregate into spherical clumps above the Tt [3].
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<partinfo>BBa_K4905001 SequenceAndFeatures</partinfo>
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The TU-Eindhoven 2023 team used this part in a composite part for the formation of a hydrogel in E. coli. The repeating sequence of this part is (VPGIG)[60], also referred to as I[60], and (VPGAG)3(VPGGG)2[60], also referred to as A[60], which creates a hydrophilic and a hydrophobic part. It has been codon optimized for expression in E. coli BL21 cells.
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<h1>Characterization</h1>
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== Characterization ==
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We ran an electrophoresis gel with the plasmid, digested with enzymes AcuI and BglI. The band of I[60]-A[60] is shown in slot 1. A 60 kb ladder (L) was used. Slot 2 was used for a different part.
 
We ran an electrophoresis gel with the plasmid, digested with enzymes AcuI and BglI. The band of I[60]-A[60] is shown in slot 1. A 60 kb ladder (L) was used. Slot 2 was used for a different part.
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# I[60]-A[60]: AcuI + BglI  
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# A[40]-I[60]: BseRI + BglI  
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<li>I[60]-A[60]: AcuI + BglI</li>
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# A[60]-I[60]: BseRI + BglI  
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<li>A[40]-I[60]: BseRI + BglI</li>
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<li>A[60]-I[60]: BseRI + BglI</li>
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[[File: bba-k4905001-characterization.png]]
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<figure><img src="https://static.igem.wiki/teams/4905/wiki/bba-k4905001/characterizationpart1.png" width="300px">
  
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<p><b>Figure 1 |</b> Electrophoresis gel with I[60]-A[60] in slot 1 </p>
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This digested DNA was used for the development of further composite parts.  
 
This digested DNA was used for the development of further composite parts.  
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===Usage and Biology===
 
  
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<h1>References</h1>
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<p>  
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== Sequence and Features ==
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<partinfo>BBa_K4905001 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4905001 parameters</partinfo>
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== References ==
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[1] Christensen, T., Hassouneh, W., Trabbic-Carlson, K., & Chilkoti, A. (2023). Predicting Transition Temperatures of Elastin-Like Polypeptide Fusion Proteins. https://doi.org/10.1021/bm400167h
 
[1] Christensen, T., Hassouneh, W., Trabbic-Carlson, K., & Chilkoti, A. (2023). Predicting Transition Temperatures of Elastin-Like Polypeptide Fusion Proteins. https://doi.org/10.1021/bm400167h
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[2] Despanie, J., Dhandhukia, J. P., Hamm-Alvarez, S. F., & MacKay, J. A. (2016). Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines. Journal of Controlled Release, 240, 93–108. https://doi.org/10.1016/j.jconrel.2015.11.010
 
[2] Despanie, J., Dhandhukia, J. P., Hamm-Alvarez, S. F., & MacKay, J. A. (2016). Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines. Journal of Controlled Release, 240, 93–108. https://doi.org/10.1016/j.jconrel.2015.11.010
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[3] Hassouneh, W., Christensen, T., & Chilkoti, A. (2010). Elastin-Like Polypeptides as a Purification Tag for Recombinant Proteins. Current Protocols in Protein Science, 61(1), 6.11.1-6.11.16. https://doi.org/10.1002/0471140864.PS0611S61
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[3] Hassouneh, W., Christensen, T., & Chilkoti, A. (2010). Elastin-Like Polypeptides as a Purification Tag for Recombinant Proteins. Current Protocols in Protein Science, 61(1), 6.11.1-6.11.16. https://doi.org/10.1002/0471140864.PS0611S61
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Latest revision as of 09:35, 6 October 2023


Elastin-Like polypeptide (VPGIG)[60]-(VPGAG)3(VPGGG)2[12]

Information

Elastin-like polypeptides (ELPs) are protein polymers derived from human tropoelastin. One of their key features is that they exhibit a phase separation that is often reversible whereby samples remain soluble below a transition temperature (Tt) but form coacervates above Tt. They have many possible applications in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators[2].

The general structure of polymeric ELPs is (VPGXG)n, where the monomeric unit is Val-Pro-Gly-X-Gly, and the "X" denotes a variable amino acid that can have consequences on the general properties of the ELP, such as the Tt. Specifically, the hydrophilicity or hydrophobicity and the presence or absence of a charge on the guest residue play a great role in determining the Tt. Also, the solubilization of the guest residue can affect the Tt. The "n" denotes the number of monomeric units that comprise the polymer[1]. In general, these polymers are linear below the Tt, but aggregate into spherical clumps above the Tt[3]. The repeating sequence for this specific part is (VPGIG)[60] and then (VPGAG)3(VPGGG)2[12].

The TU-Eindhoven 2023 team used this part in a composite part for the formation of a hydrogel in E. coli. It has been codon optimized for expression in E. coli BL21 cells. The composite part is BBa_K4905006 .

Sequence and Features

the following sequence was used to create this part. It was optimized for E.coli BL21 cells. The sequence was cloned into a pET24a(+) vector, this was multiplied in TOP10 competent cells.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1890
    Illegal XbaI site found at 7
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1890
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1890
    Illegal XhoI site found at 1907
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1890
    Illegal XbaI site found at 7
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1890
    Illegal XbaI site found at 7
    Illegal NgoMIV site found at 64
    Illegal NgoMIV site found at 244
    Illegal NgoMIV site found at 334
    Illegal NgoMIV site found at 514
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

We ran an electrophoresis gel with the plasmid, digested with enzymes AcuI and BglI. The band of I[60]-A[60] is shown in slot 1. A 60 kb ladder (L) was used. Slot 2 was used for a different part.

  1. I[60]-A[60]: AcuI + BglI
  2. A[40]-I[60]: BseRI + BglI
  3. A[60]-I[60]: BseRI + BglI

Figure 1 | Electrophoresis gel with I[60]-A[60] in slot 1

This digested DNA was used for the development of further composite parts.

References

[1] Christensen, T., Hassouneh, W., Trabbic-Carlson, K., & Chilkoti, A. (2023). Predicting Transition Temperatures of Elastin-Like Polypeptide Fusion Proteins. https://doi.org/10.1021/bm400167h

[2] Despanie, J., Dhandhukia, J. P., Hamm-Alvarez, S. F., & MacKay, J. A. (2016). Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines. Journal of Controlled Release, 240, 93–108. https://doi.org/10.1016/j.jconrel.2015.11.010

[3] Hassouneh, W., Christensen, T., & Chilkoti, A. (2010). Elastin-Like Polypeptides as a Purification Tag for Recombinant Proteins. Current Protocols in Protein Science, 61(1), 6.11.1-6.11.16. https://doi.org/10.1002/0471140864.PS0611S61