Difference between revisions of "Part:BBa K4905008"
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<partinfo>BBa_K4905008 short</partinfo>
 
<partinfo>BBa_K4905008 short</partinfo>
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==Information==
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This part is made up of the basic parts: two times Leucine zipper Z2 ([https://parts.igem.org/Part:BBa_K4905005 BBa_K4905005]), and two times Elastin-Like Polypeptide (ELP) sequence A[60]I[60] ([https://parts.igem.org/Part:BBa_K4905001 BBa_K4905001]). This results in the sequence Z2-I[60]-A[120]-I[60]-Z2. With A the sequence (VPGAG(3)VPGGG(2)) 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.
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<body>
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<h1>Information</h1>
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<p>
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This part is made up of the basic parts: two times Leucine zipper Z2 ([https://parts.igem.org/Part:BBa_K4905005 BBa_K4905005]), and two times Elastin-Like Polypeptide (ELP) sequence A[60]I[60] ([https://parts.igem.org/Part:BBa_K4905001 BBa_K4905001]). This results in the sequence Z2-I[60]-A[120]-I[60]-Z2. With A the sequence (VPGAG(3)VPGGG(2)) 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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</p>
  
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[[File:ELP.png|figSequence]]
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<figure><img src="https://static.igem.wiki/teams/4905/wiki/partsconstructs/partsconstructs/part8.png" width="640px">
  
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Figure 1: Schematic overview of the sequence of this construct. VPGAG(3)VPGGG(2) is from now on referred to as A and VPGIG is referred to as I.  
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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 and VPGIG is referred to as I. </p>
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</figcaption>
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</figure><be>
  
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=== General applications ===
 
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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 [3].
 
  
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=== Construct design ===
 
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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 [2]. 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 [3].
 
  
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<h2>General applications</h2>
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<p>
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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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</p>
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<h2>Construct design</h2>
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<p>
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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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</p>
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<p>
 
As shown in figure 2, this construct has a hydrophilic region in the middle (A[120]) and a hydrophobic region on each side of it (I[60]). On the ends the leucine zipper Z2 is 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 [1]. 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.  
 
As shown in figure 2, this construct has a hydrophilic region in the middle (A[120]) and a hydrophobic region on each side of it (I[60]). On the ends the leucine zipper Z2 is 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 [1]. 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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</p>
  
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<figure><img src="https://static.igem.wiki/teams/4905/wiki/bba-k4905006/construct-zipper-elp-wiki.png" width="300px">
  
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[[File:ELP.png|figELP]]
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<figcaption>
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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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</figure><be>
  
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Figure 2: Schematic representation of the composite part, an ELP with leucine zippers on the ends
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<p>
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As soon as the hydrogel is made inside <i>E.coli</i> BL21 cells, the cells are prevented from dividing. However, the cells remain functional. So they can still be used to express therapeutic agents, like Interleukin 10 in the TU Eindhoven 2023 teams project.
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</p>
  
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As soon as the hydrogel is made inside ''E.coli'' BL21 cells, the cells are prevented from dividing. However, the cells remain functional. So they can still be used to express therapeutic agents, like Interleukin 10 in the TU Eindhoven 2023 teams project.
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<h1>Sequence and Features<h1>
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== Sequence and Features ==
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<partinfo>BBa_K4905008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4905008 SequenceAndFeatures</partinfo>
  
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==Results==
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<h1>Results</h1>
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===Usage and Biology===
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<h1>References<h1>
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===Functional Parameters===
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<partinfo>BBa_K4905008 parameters</partinfo>
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Revision as of 10:07, 15 September 2023


Elastin-like polypeptide triblock with leucine zippers

Information

This part is made up of the basic parts: two times Leucine zipper Z2 ([https://parts.igem.org/Part:BBa_K4905005 BBa_K4905005]), and two times Elastin-Like Polypeptide (ELP) sequence A[60]I[60] ([https://parts.igem.org/Part:BBa_K4905001 BBa_K4905001]). This results in the sequence Z2-I[60]-A[120]-I[60]-Z2. With A the sequence (VPGAG(3)VPGGG(2)) 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 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[120]) and a hydrophobic region on each side of it (I[60]). On the ends the leucine zipper Z2 is 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 [1]. 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

As soon as the hydrogel is made inside E.coli BL21 cells, the cells are prevented from dividing. However, the cells remain functional. So they can still be used to express therapeutic agents, like Interleukin 10 in the TU Eindhoven 2023 teams project.

Sequence and Features<h1>

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1982
    Illegal EcoRI site found at 3908
    Illegal XbaI site found at 99
    Illegal XbaI site found at 2025
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1982
    Illegal EcoRI site found at 3908
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1982
    Illegal EcoRI site found at 3908
    Illegal XhoI site found at 1999
    Illegal XhoI site found at 3925
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1982
    Illegal EcoRI site found at 3908
    Illegal XbaI site found at 99
    Illegal XbaI site found at 2025
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1982
    Illegal EcoRI site found at 3908
    Illegal XbaI site found at 99
    Illegal XbaI site found at 2025
    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 2082
    Illegal NgoMIV site found at 2262
    Illegal NgoMIV site found at 2352
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

References