Difference between revisions of "Part:BBa K4905013"
 
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<partinfo>BBa_K4905013 short</partinfo>
 
<partinfo>BBa_K4905013 short</partinfo>
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==Information==
 
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This part is made up of the basic parts: Elastin-Like Polypeptide (ELP) sequence A[60]I[60] ([https://parts.igem.org/Part:BBa_K4905001 BBa_K4905001]), ELP sequence A[40]I[60] ([https://parts.igem.org/Part:BBa_K4905002 BBa_K4905002]), FRB ([https://parts.igem.org/Part:BBa_J18926 BBa_J18926]) and FKBP12 ([https://parts.igem.org/Part:BBa_K3610022 BBa_K3610022]). This results in the sequence FRB-I[60]-A[100]-I[60]-FKBP12. 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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[[File:ELP.png|figSequence]]
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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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<h1>Information</h1>
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<p>
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This part is made up of the basic parts: Elastin-Like Polypeptide (ELP) sequence A[60]I[60] (<a href="https://parts.igem.org/Part:BBa_K4905001">BBa_K4905001</a>), ELP sequence A[40]I[60] (<a href="https://parts.igem.org/Part:BBa_K4905002">BBa_K4905002</a>), FRB (<a href="https://parts.igem.org/Part:BBa_J18926">BBa_J18926</a>) and FKBP12 (<a href="https://parts.igem.org/Part:BBa_K3610022">BBa_K3610022</a>). This results in the sequence FRB-I[60]-A[100]-I[60]-FKBP12. 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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</p>
  
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=== General applications ===
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<figure><img src="https://static.igem.wiki/teams/4905/wiki/partsconstructs/partsconstructs/part13nieuw.png" width="640px">
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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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<figcaption>
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The construct consists of ELPs and FKBP12. 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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<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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</figure><be>
  
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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, an FRB and FKBP12 domain is located for stronger interactions between the ELPs. FKBP12 can form a complex with Rapamycin and the FKBP-rapamycin binding (FRB) domain. This mechanism is normally used in the mTOR pathway inside cells, which regulates cellular proliferation, protein synthesis, differentiation and survival, and lipid metabolism. In the case of a hydrogel, the addition of rapamycin to a cell with these ELPs can induce crosslinking between them to form a network (bron inobe en yandong liu). These stronger interactions make them useful in the formation of a hydrogel.
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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>2</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 FKBP12. 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>1</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>2</sup>.
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</p>
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<p>
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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, an FRB and FKBP12 domain is located for stronger interactions between the ELPs. FKBP12 can form a complex with Rapamycin and the FKBP-rapamycin binding (FRB) domain. This mechanism is normally used in the mTOR pathway inside cells, which regulates cellular proliferation, protein synthesis, differentiation and survival, and lipid metabolism. In the case of a hydrogel, the addition of rapamycin to a cell with these ELPs can induce crosslinking between them to form a network<sup>3,4</sup>. These stronger interactions make them useful in the formation of a hydrogel.
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[[File:ELP.png|figELP]]
 
  
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Figure 2: Schematic representation of the composite part, an ELP with leucine zippers on the ends
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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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<span class='h3bb'><h1>Sequence and Features</h1></span>
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<partinfo>BBa_K4905013 SequenceAndFeatures</partinfo>
  
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<h1>Results</h1>
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<h1>References</h1>
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===Usage and Biology===
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<p>
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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
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4905013 SequenceAndFeatures</partinfo>
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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
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<!-- Uncomment this to enable Functional Parameter display
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3. Inobe, T., & Nukina, N. (2016). Rapamycin-induced oligomer formation system of FRB–FKBP fusion proteins. Journal of Bioscience and Bioengineering, 122(1), 40–46. https://doi.org/10.1016/J.JBIOSC.2015.12.004
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===Functional Parameters===
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<partinfo>BBa_K4905013 parameters</partinfo>
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4. Liu, Y., Yang, F., Zou, S., & Qu, L. (2019). Rapamycin: A bacteria-derived immunosuppressant that has anti-atherosclerotic effects and its clinical application. Frontiers in Pharmacology, 9(JAN). https://doi.org/10.3389/FPHAR.2018.01520
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Latest revision as of 09:52, 6 October 2023


Elastin-Like Polypeptide Triblock with FKBP12 and FRB

Information

This part is made up of the basic parts: Elastin-Like Polypeptide (ELP) sequence A[60]I[60] (BBa_K4905001), ELP sequence A[40]I[60] (BBa_K4905002), FRB (BBa_J18926) and FKBP12 (BBa_K3610022). This results in the sequence FRB-I[60]-A[100]-I[60]-FKBP12. 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 modulators2.

Construct design

The construct consists of ELPs and FKBP12. 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 sites1. 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 molecules2.

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, an FRB and FKBP12 domain is located for stronger interactions between the ELPs. FKBP12 can form a complex with Rapamycin and the FKBP-rapamycin binding (FRB) domain. This mechanism is normally used in the mTOR pathway inside cells, which regulates cellular proliferation, protein synthesis, differentiation and survival, and lipid metabolism. In the case of a hydrogel, the addition of rapamycin to a cell with these ELPs can induce crosslinking between them to form a network3,4. These stronger interactions make them useful in the formation of a hydrogel.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2177
    Illegal XbaI site found at 294
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2177
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2177
    Illegal BglII site found at 225
    Illegal XhoI site found at 2194
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2177
    Illegal XbaI site found at 294
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2177
    Illegal XbaI site found at 294
    Illegal NgoMIV site found at 351
    Illegal NgoMIV site found at 531
    Illegal NgoMIV site found at 621
    Illegal NgoMIV site found at 801
    Illegal NgoMIV site found at 3123
    Illegal NgoMIV site found at 3300
    Illegal NgoMIV site found at 3390
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

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. Inobe, T., & Nukina, N. (2016). Rapamycin-induced oligomer formation system of FRB–FKBP fusion proteins. Journal of Bioscience and Bioengineering, 122(1), 40–46. https://doi.org/10.1016/J.JBIOSC.2015.12.004

4. Liu, Y., Yang, F., Zou, S., & Qu, L. (2019). Rapamycin: A bacteria-derived immunosuppressant that has anti-atherosclerotic effects and its clinical application. Frontiers in Pharmacology, 9(JAN). https://doi.org/10.3389/FPHAR.2018.01520