Team:Aalto-Helsinki/Parts
Basic Parts
This section lists all the basic parts that we have made and used during our project.
1] BBa_K2342000
DCD-1L, Dermcidin derived peptide
The part codes for the expression of dermcidin derived DCD-1L peptide. Dermcidin is a recently discovered
antimicrobial peptide (AMP) found in primates with no homology to other know AMPs. It is expressed in
a constitutive manner in eccrine sweat glands and secreted to epidermal surface as a part of the first line
of defense. Mature dermcidin precursor is 110 amino acids long, including a signal peptide. Once antimicrobial
peptide precursor is secreted with sweat to epidermal surface, 19 amino acid long signal peptide is cleaved,
and it goes under further proteolytic processing leading to several dermcidin derived peptides such as
DCD-1 and DCD-1L. DCD-1L is one of the most abundant forms of dermcidin derived peptides. DCD-1L is a 48 amino
acid long anionic peptide active against wide spectrum of bacteria including
Staphylococcus aureus,
Escherichia coli, and
Propionibacterium acnes.
For more details on the production and usage of this part please check the composite part page: BBa_K2342006, BBa_K2342007, BBa_K2342008.
2] BBa_K2342001
SUMO fusion tag, Smt3
Employing of Smt3 tag is beneficial in several aspects in expression of recombinant proteins. Ulp1 is
a robust enzyme that specifically facilitates cleavage of Smt3, leaving protein of interest unbound to the
tag. SUMO tag is known for its effect on solubility and preventing inclusion bodies of fusion peptide,
significantly easing purification step.
For more details on the production and usage of this part please check the composite part page: BBa_K2342006, BBa_K2342007, BBa_K2342008, BBa_K2342009.
3] BBa_K2342002
CBM, Cellulose Binding Domain
This part encodes for Cellulose binding domain (CBM3) from
Clostridium thermocellum. This part could be used with any other construct to bind to cellulose.
For more details on the production and usage of this part please check the composite part page: BBa_K2342007, BBa_K2342008.
4] BBa_K2342003
6x Histidine tag
6x Histidine tag is commonly used for purification of fusion proteins with Immobilized Metal Ion Affinity
Chromatography (IMAC) columns designed for histidine tagged proteins.
For more details on the production and usage of this part please check the composite part page: BBa_K2342006, BBa_K2342007, BBa_K2342008, BBa_K2342009.
5] BBa_K2342004
10 amino acid linker, 5X GS
10 amino acid long linker contains glycine and serine repeats.
6] BBa_K2342005
22 amino acid linker
22 amino acid long linker contains serine, alanine and glycine repeats.
For more details on the production and usage of this part please check the composite part page: BBa_K2342007, BBa_K2342008.
Composite Parts
This page lists all the composite parts that we have made during our project. We consider the first listed part our best part!
1] BBa_K2342006
DCD1L peptide linked to sumo fusion peptide with His6x tag.
This part comprises of a dermcidin derived antimicrobial peptide DCD-1L. This part allows expression of DCD-1L antimicrobial peptide in form of a fusion protein accompanied with His6x-Smt3 tag. The construct can be purified with commercially available and widely used affinity chromatography columns designed for His6x tag. Smt3 tag is used to keep antimicrobial peptide, DCD-1L, in inactive form by blocking its adhesion to phospholipid bilayer of the production host due to relatively large size of the tag. One major advantage of the fusion system used is that it facilitates easier detection of the peptide with a conventional method, SDS-PAGE. Also, SUMO tag is beneficial due to its effect on solubility of fusion peptide, significantly easing purification step. After purification, to cleave off His6x-Smt3 tag, Ulp1 enzyme that is known for its robust and specific proteolytic activity against SUMO fusion proteins, is used to obtain free DCD-1L antimicrobial peptide.
2] BBa_K2342007
DCD1L peptide linked to CBM with 22 AA linker (6XHis-Smt3_DCD1L_22Linker_CBM).
This part comprises of a dermcidin derived antimicrobial peptide DCD-1L linked to a Cellulose binding domain (CBM) with a 22 amino acid linker. Cellulose binding domain (CBM3), from Clostridium thermocellum, is used to immobilize DCD-1L on cellulose based materials. This part allows expression of DCD-1L antimicrobial peptide in form of a fusion protein accompanied with His6x-Smt3 tag on the N-terminus and CBM on C-terminus (6XHis-Smt3_DCD1L_22Linker_CBM) in E.coli without killing the host bacteria.
The construct can be purified with commercially available and widely used affinity chromatography columns designed for His6x tag. Smt3 tag is used to keep antimicrobial peptide, DCD-1L, in inactive form by blocking its adhesion to phospholipid bilayer of the production host due to relatively large size of the tag. One major advantage of the fusion system used is that it facilitated easier detection of the peptide with a conventional method, SDS-PAGE. Also, SUMO tag is beneficial due to its effect on solubility of fusion peptide significantly easing purification step. After purification, to cleave off His6x-Smt3 tag, Ulp1 enzyme that is known for its robust and specific proteolytic activity against SUMO fusion proteins, is used to obtain free DCD-1L-22 aa linker-CBM protein.
Preventing pathogen colonization on surfaces is crucial in order to prevent spreading of infectious diseases. Therefore, immobilization of antimicrobial peptides can be a good alternative to other bactericidal agents because of their wide antibiotic spectrum, given that immobilization is a known way of increasing stability and resilience of peptides.
For this purpose, we designed a construct containing a cellulose binding domain (CBM3), from Clostridium thermocellum, to immobilize DCD-1L on cellulose based materials. Our CBM constructs contain 22 amino acid long linker, to avoid interference with hexameric complex formation that leads to cell death. Cellulose is a medically safe, eco-friendly, and abundant material, already present or easily incorporated into many applications. What is more, versatile nature of cellulose materials from plastic-like hard materials to hydrogels expands application possibilities. Application range can be further increased through altering the part by changing DCD-1L to another AMP in this expression system .
3] BBa_K2342008
DCD1L peptide linked to a CBM with a 22 aa linker (His6x-Smt3_CBM_22Linker_DCD1L).
This part comprises of a dermcidin derived antimicrobial peptide DCD-1L linked to a Cellulose binding domain (CBM) with a 22 amino acid linker. Cellulose binding domain (CBM3), from Clostridium thermocellum, is used to immobilize DCD-1L on cellulose based materials. This part allows expression of DCD-1L antimicrobial peptide in form of a fusion protein accompanied with His6x-Smt3 tag on the N-terminus followed by CBM (His6x-Smt3_CBM_22Linker_DCD1L) in E.coli without killing the host bacteria.
The construct can be purified with commercially available and widely used affinity chromatography columns designed for His6x tag. Smt3 tag is used to keep antimicrobial peptide, DCD-1L, in inactive form by blocking its adhesion to phospholipid bilayer of the production host due to relatively large size of the tag. One major advantage of the fusion system used is that it facilitated easier detection of the peptide with a conventional method, SDS-PAGE. Also, SUMO tag is beneficial due to its effect on solubility of fusion peptide significantly easing purification step. After purification, to cleave off His6x-Smt3 tag, Ulp1 enzyme that is known for its robust and specific proteolytic activity against SUMO fusion proteins, is used to obtain free DCD-1L-22 aa linker CBM protein.
Preventing pathogen colonization on surfaces is crucial in order to prevent spreading of infectious diseases. Therefore, immobilization of antimicrobial peptides can be a good alternative to other bactericidal agents because of their wide antibiotic spectrum, given that immobilization is a known way of increasing stability and resilience of peptides.
For this purpose, we designed a construct containing a cellulose binding domain (CBM3) to immobilize DCD-1L on cellulose based materials. Since cellulose is a medically safe, eco-friendly, and abundant material, already present or can easily be incorporated in many applications. Furthermore, versatile nature of cellulose materials from plastic-like hard materials to hydrogels expands application possibilities. Application range can be further increased through altering the part by changing DCD-1L to another AMP in this expression system.
4] BBa_K2342009
LL-37 peptide linked to sumo fusion peptide with His6x (His6x-Smt3-LL-37).
This part allows expression of LL-37 antimicrobial peptide in the form of a fusion protein accompanied with His6x-Smt3 tag. The construct can be purified with commercially available and widely used affinity chromatography columns designed for His6x tag. Smt3 tag is used to keep antimicrobial peptide, LL37 in inactive form by blocking its adhesion to phospholipid bilayer of the production host due to relatively large size of the tag. One major advantage of the fusion system used is that it facilitated easier detection of the peptide with a conventional method, SDS-PAGE. Also, SUMO tag is beneficial due to its effect on solubility of fusion peptide significantly easing purification step. After purification, to cleave off His6x-Smt3 tag, Ulp1 enzyme that is known for its robust and specific proteolytic activity against SUMO fusion proteins, is used to obtain free LL-37 antimicrobial peptide.
