Difference between revisions of "Part:BBa K4905007"

 
 
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<partinfo>BBa_K4905007 short</partinfo>
 
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This part is made up of parts BBa_K4905004, BBa_K4905005, BBa_K4905001 and BBa_K4905002 . It was used by the Eindhoven 2023 team to form a hydrogel outside as well as inside e.coli BL21 cells.  
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<h1>Information</h1>
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This part is made up of the basic parts: Leucine zipper Z1 (<a href="https://parts.igem.org/Part:BBa_K4905004">BBa_K4905004</a>), Leucine zipper Z2 (<a href="https://parts.igem.org/Part:BBa_K4905005">BBa_K4905005</a>), Elastin-Like Polypeptide (ELP) sequence A[60]I[60] (<a href="https://parts.igem.org/Part:BBa_K4905001">BBa_K4905001</a>), and ELP sequence A[40]I[60] (<a href="https://parts.igem.org/Part:BBa_K4905002">BBa_K4905002</a>). This results in the sequence Z1-I[60]-A[100]-I[60]-Z2. With A[5] the sequence (VPGAG[3]VPGGG[2]), since there are five VPGXG repeats, and I the sequence (VPGIG). The numbers indicate the number of repeats of these sequences. This construct was used by the TU Eindhoven 2023 team to form a hydrogel outside as well as inside <i>E.coli</i> BL21 cells. A schematic overview of this is shown in figure 1.
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The construct consists of Elastin-like Polypeptides (ELPs) and two leucine zippers that have affinity for each other. ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature dependent. This allows them to be used in the formation of a reversible hydrogel.  
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<figure><img src="https://static.igem.wiki/teams/4905/wiki/partsconstructs/partsconstructs/part7nieuw.png" width="640px">
  
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<figcaption>
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<p><b>Figure 1 | </b>Schematic overview of the sequence of this construct. (VPGAG[3]VPGGG[2]) is from now on referred to as A[5] and VPGIG is referred to as I</p>
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<h2>General applications</h2>
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===Usage and Biology===
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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 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<sup>3</sup>.
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<h2>Construct design</h2>
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<span class='h3bb'>Sequence and Features</span>
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The construct consists of ELPs and two different leucine zippers that have affinity for each other. In general, ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature-dependent. They are made from a repeating VPGXG sequence, with X replaced by specific amino acids. This results in specific properties of the ELPs, especially related to the transition temperature Tt at which the ELPs will interact with each other on the hydrophobic sites<sup>2</sup>. When the temperature is below Tt, the water molecules surrounding the hydrophobic parts will go into the bulk water phase which gains the solvent entropy. This makes it possible to form interactions with other ELP molecules<sup>3</sup>.
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As shown in figure 2, this construct has a hydrophilic region in the middle (A[100]) and a hydrophobic region on each side of it (I[60]). On the ends the leucine zippers Z1 and Z2 are located for stronger interactions between the ELPs. Leucine zippers consist of a repeating unit that forms an alpha helix. Two leucine zippers together form ion pairs between the helices, which causes association<sup>1</sup>. These stronger and reversible interactions make them useful in the formation of a hydrogel at a specific Tt. In the end, the hydrogel is formed with electrostatic and hydrophobic interactions between the ELPs.
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<figure><img src="https://static.igem.wiki/teams/4905/wiki/parts/zippers-elps.png" width="400px">
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<p><b>Figure 2 | </b>Schematic representation of the composite part, an ELP with leucine zippers on the ends.</p>
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<span class='h3bb'><h1>Sequence and Features</h1></span>
 
<partinfo>BBa_K4905007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4905007 SequenceAndFeatures</partinfo>
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<h1>Results</h1>
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<h1>References</h1>
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<p>
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[1] Alber, T. (1992). Structure of the leucine zipper. Current Opinion in Genetics and Development, 2, 205–210
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[2] 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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[3] 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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===Functional Parameters===
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<partinfo>BBa_K4905007 parameters</partinfo>
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Latest revision as of 09:56, 6 October 2023


Elastin-Like Polypeptide Triblock with Leucine Zippers

Information

This part is made up of the basic parts: Leucine zipper Z1 (BBa_K4905004), Leucine zipper Z2 (BBa_K4905005), Elastin-Like Polypeptide (ELP) sequence A[60]I[60] (BBa_K4905001), and ELP sequence A[40]I[60] (BBa_K4905002). This results in the sequence Z1-I[60]-A[100]-I[60]-Z2. With A[5] the sequence (VPGAG[3]VPGGG[2]), since there are five VPGXG repeats, and I the sequence (VPGIG). The numbers indicate the number of repeats of these sequences. This construct was used by the TU Eindhoven 2023 team to form a hydrogel outside as well as inside E.coli BL21 cells. A schematic overview of this is shown in figure 1.

Figure 1 | Schematic overview of the sequence of this construct. (VPGAG[3]VPGGG[2]) is from now on referred to as A[5] and VPGIG is referred to as I

General applications

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 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 modulators3.

Construct design

The construct consists of ELPs and two different leucine zippers that have affinity for each other. In general, ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature-dependent. They are made from a repeating VPGXG sequence, with X replaced by specific amino acids. This results in specific properties of the ELPs, especially related to the transition temperature Tt at which the ELPs will interact with each other on the hydrophobic sites2. When the temperature is below Tt, the water molecules surrounding the hydrophobic parts will go into the bulk water phase which gains the solvent entropy. This makes it possible to form interactions with other ELP molecules3.

As shown in figure 2, this construct has a hydrophilic region in the middle (A[100]) and a hydrophobic region on each side of it (I[60]). On the ends the leucine zippers Z1 and Z2 are located for stronger interactions between the ELPs. Leucine zippers consist of a repeating unit that forms an alpha helix. Two leucine zippers together form ion pairs between the helices, which causes association1. These stronger and reversible interactions make them useful in the formation of a hydrogel at a specific Tt. In the end, the hydrogel is formed with electrostatic and hydrophobic interactions between the ELPs.

Figure 2 | Schematic representation of the composite part, an ELP with leucine zippers on the ends.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1982
    Illegal XbaI site found at 99
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1982
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1982
    Illegal XhoI site found at 1999
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1982
    Illegal XbaI site found at 99
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1982
    Illegal XbaI site found at 99
    Illegal NgoMIV site found at 156
    Illegal NgoMIV site found at 336
    Illegal NgoMIV site found at 426
    Illegal NgoMIV site found at 606
    Illegal NgoMIV site found at 2928
    Illegal NgoMIV site found at 3105
    Illegal NgoMIV site found at 3195
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

References

[1] Alber, T. (1992). Structure of the leucine zipper. Current Opinion in Genetics and Development, 2, 205–210

[2] 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

[3] 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