Difference between revisions of "Part:BBa K4905001"

Revision as of 13:11, 4 September 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 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].

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.

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.

I[60]-A[60]: AcuI + BglI
A[40]-I[60]: BseRI + BglI
A[60]-I[60]: BseRI + BglI

caption

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

Sequence and Features

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]

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

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4905001 short</partinfo>
-<!-- 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 and (VPGIG)60 which creates a hydrophilic and a hydrophobic part. It has been codon optimized for expression in e.coli cells. -->
+<!-- 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. -->
 == 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].
+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 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].
+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].
-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 and (VPGAG)60 which creates a hydrophilic and a hydrophobic part. It has been codon optimized for expression in e.coli bl21 cells.
+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.
 == Characterization ==
-We ran an electrophoresis gel with the plasmid, digested with enzymes AcuI and BglI. The band of A[60]I[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.
 #	I[60]-A[60]: AcuI + BglI
 #	A[40]-I[60]: BseRI + BglI