Antifreeze proteins (AFPs) are molecules with the unique property of binding and shaping ice crystals, preventing their growth into relatively large cell-lysing structures (Davies and Sykes 1997). They exhibit a property known as thermal hysteresis, whereby their activity decreases the freezing point of water without changing the melting point. This property allows organisms which produce AFPs to survive colder conditions. Additionally, AFPs have shown biolfilm inhibbiting properties, potetnially giving them uses in medical applications.
Codon optimized the sequence for E. coli and to minimize repetitive sequences; removed illegal restriction sites.
Taken from the fungus Typhula ishikariensis. Pubmed ID: 20030710
The Raghium inquisitor Anti Freeze Protein (RiAFP) is a 137 AA protein that prevents the growth of ice crystals. This protein is produced natively in the beetle Raghium inquisitor. (Kristiansen E, 2011) About Safety The RiAFP has no reported toxicity or hazardousness, but its important to remember to have the basic biosafety precautions using a biosecurity chamber when handling any transformed strains.
This part was designed by adding a 6xHis tag for affinity purification to the anti freeze protein from Raghium inquisitor. This part was codon optimized for its expression in E.coli.
Is produced natively in the beetle Raghium inquisitor. (Kristiansen E, 2011)
Taken from the longhorn beetle Rhagium inquisitor. Pubmed ID: 21078390
Antifreeze proteins (AFPs) are molecules with the unique property of binding and shaping ice crystals, preventing their growth into relatively large cell-lysing structures (Davies and Sykes 1997). They exhibit a property known as thermal hysteresis, whereby their activity decreases the freezing point of water without changing the melting point. This property allows organisms which produce AFPs to survive colder conditions. Additionally, AFPs have shown biolfilm inhibiting properties, potentially giving them uses in medical applications.
Taken from the rhizobacterium Pseudemonas putida GR12-2. Pubmed ID: 15317770
This part contains the constitutive promoter BBa_J23100, an RBS, the cell surface expression tag BclA, EpAFP, and a double terminator. It encodes an antifreeze protein that attaches to the cell surface.
Spliced together a cell surface expression tag and the antifreeze protein to test cell surface activity.
Composite of parts BBa_K1423005 and BBa_K1809028 without the YFP tag.
Non-Glycolated Antifreeze Protein (AFP) - 5-Repeated Segment
Nathan Barnett, Meghan Bowler, Kian Torabian
The standard non-glycolated antifreeze protein (AFP) from the Pseudopleuronecta americanus. Begins with a chain of C's and is followed by 5 rough repeats of "ANAAAAAALTA". The codons have been optimized for E. coli.
This specific sequence has the repeat 5 times. Studies show that increasing the number of repeats increases the antifreeze capabilities of the protein.
Comes from the white flounder, or Pseudopleuronecta americanus.
Taken from the longhorn sculpin Myoxocephalus octodecemspinosus. Pubmed ID: 11136728
Taken from Escherichia phage 121Q. NCBI accession: YP_009102201
Antifreeze protein, type I from Oceanibulbus indolifex HEL-45,
Antifreeze protein from Oceanibulbus indolifex
Genomic DNA, http://www.ebi.ac.uk/ena/data/view/EDQ05862
Non-Glycolated Antifreeze Protein (AFP) - 10-Repeated Segment
The standard non-glycolated antifreeze protein (AFP) from the Pseudopleuronecta americanus. Begins with a chain of C's and is followed by 10 rough repeats of "ANAAAAAALTA". The codons have been optimized for E. coli.
This sequence has 10 rough repeats of "ANAAAAAALTA", as compared to the natural peptide which has this sequence repeated roughly 5 times.
Based on the 5-repeated Segment AFP from the white flounder, Pseudopleuronecta americanus.
Taken from the mealworm beetle Tenebrio molitor. Pubmed ID: 17443386
Taken from the ocean pout Macrozarces americanus. Pubmed ID: 3840475
Insect antifreeze protein (RiAFP) Chain B
Codes for Chain B of the Rhagium inquisitor antifreeze protein - sequence optimized for human cell lines.
Codon optimization for human cell lines.
This is the coding region of a small antifreeze protein. It works by binding to the ice crystals as they form inside the cell, and by doing so prevents the ice crystals from enlarging and penetrating the cell membrane. We wanted to test if there would be a significant difference in the growth rate of moss, Physcomitrella patens, with the antifreeze gene, compared to the one without. This would be done at a low temperature to test the in-vivo function of the antifreeze protein.
We used codon optimization for moss so it would work better in Physcomitrella Patens.
The part is taken from a spruce budworm, Choristoneura fumiferana, living in areas with permafrost.
Malak Elbatarny, Joanna Semrau, Justine Ring, Morgan Litschko
This BioBrick is a fusion protein consisting of the ???E??? coil domain of our coiled-coil system fused in-frame to the C-terminus of a Type III AFP. Type III AFP (PDB fIle: 1AME) is a moderately active antifreeze protein from the ocean pout, with a small (7Kb), globular structure. The ???E??? coil refers to the coiled-coil with glutamic acid residues in the ???e??? and ???g??? positions of the helical wheel first used by Team Calgary 2013 (BBa_K1189011). There is a His-tag at the C-terminus of the ???E??? coil which provides an alternative purification method to ice-affinity. The fusion of the ???E??? coil to the Type III AFP will enable non-covalent, yet high affinity, specific attachment of AFPs to our protein scaffold and ???K??? coil BioBrick (BBa_K1831002). The interaction between the ???E??? and ???K??? coils involved oppositely charged residues, promoting the heterodimeric interaction of the coils that links AFPs to our scaffold. The addition of a His-tag to the C-terminus of the coil will allow for simple purification of the protein even for teams without access to an ice-affinity purification apparatus.
The TypeIII AFP was fused in-frame to the E-coil through a gly-ser linker. The gly-ser coding DNA used the specific codons for a BamHI site, which is a unique cut site not found in either the AFP, E-coil, or the pSB1C3 backbone. Thus if you require the part in a different backbone, simple confirmation on cloning by a single restriction digest is possible.
The TypeIII AFP (PDB: 1AME) was obtained from Dr. Peter Davies lab at Queen's University. The E-coil was a novel sequence based off the E-coil (BBa_K1189012) and K-coil (BBa_K1189013) pair designed by Team Calgary 2013
Taken from the Atlantic herring Clupea harengus. Pubmed ID: 9521729
Isolated from the part BBa_K652004 taken from the 2015 distribution plate 1, well 10C.
This part contains the constitutive promoter BBa_J23100, an RBS, the cell surface expression tag BclA, Ixodes capularis antifreeze glycoprotein (IAFGP), and a double terminator. It encodes an antifreeze protein that attaches to the cell surface.
Composite of parts BBa_K1423005 and BBa_K1809035 without the YFP tag.
Taken from the Antarctic bacterium Marinomonas primoryensis. Pubmed ID: 18095937
Insect antifreeze protein (RiAFP) Chain A
Amiel Stephen Paz, William Kindschuh, Vickram Gidwani
Codes for Chain A of the Rhagium inquisitor antifreeze protein - sequence optimized for human cell lines.
Sequenced from the Rhagium inquisitor genome.
Taken from carrot (Daucus carota). Pubmed ID: 9756474
Composite of parts BBa_J23119, BBa_B0015, BBa_B0034 and BBa_K652004.
Taken from the rye grass Lolium perenne. Pubmed ID: 21457707
Non-Glycolated Antifreeze Protein (AFP) - 1-Repeated Segment
The standard non-glycolated antifreeze protein (AFP) from the Pseudopleuronecta americanus. The codons have been optimized for E. coli.
This is a reduced segment of the 5-Repeated Segment AFP, with four "ANAAAAAALTA" taken out.
Comes from the white flounder, Pseudopleuronecta americanus.
Taken from the fish Brachyosis rostratus. Pubmed ID: 16754975
Justine Ring, Morgan Litschko, Malak Elbatarny, Joanna Semrau,
What: This BioBrick is a fusion protein consisting of the E-coil domain of our coiled-coil system fused in-frame to the C-terminus of a Type III AFP. Type III AFP (PDB fIle: 1AME) is a moderately active antifreeze protein from the ocean pout, with a small (7Kb), globular structure. The E-coil refers to the coiled-coil with glutamic acid residues in the ???e??? and ???g??? positions of the helical wheel first used by Team Calgary 2013 (BBa_K1189011). This protein has been shown to be purified effectively by ice-affinity. If a His-tag is required for your purposes, see (BBa_K1831001). Why: The fusion of the E-coil to the Type III AFP will enable non-covalent, yet high affinity, specific attachment of AFPs to our protein scaffold and ???K??? coil BioBrick (BBa_K1831002). The interaction between the ???E??? and ???K??? coils involved oppositely charged residues, promoting the heterodimeric interaction of the coils that links AFPs to our scaffold. There is no His-tag, but ice-affinity purification can be used. By including a His-tag on our K-coil fusion protein (our scaffold: BBa_K1831002), and not the AFP with E-coil, we have enabled simple separation of any non-interacting coiled-coil domains.
HeLa Optimized Type I Antifreeze Protein
We are designing a HeLa cell actuator that produces Type I antifreeze protein. The antifreeze protein we chose is naturally found in winter flounder serum and is a Type I AFP, known as AFP9. This amino acid sequence was published in a paper by Chao in 1996. It is very short, consisting of only 52 amino acids, in which there are three repeats of the same 9-amino acid section. The amino acid content is very Alanine-rich. Our designed part is a patchwork of components all chosen to maximize the output of our antifreeze protein. The CMV promoter, a constitutive gene that is expressed at a constant level, was used in order to encourage the maximum transcription of our target gene in the HeLa cells. The antifreeze protein sequence starts with a spacer, made up primarily of G???s and C???s to increase the guanine and cytosine content in our sequence and provide room for the ribosome to bind. This is followed by DNA2.0 Gene Designer software???s template kozak sequence, which is strong and promotes ribosome binding well. The kozak is then followed by the start codon, ATG, and the optimized antifreeze protein sequence. The protein sequence is followed by the stop codon and a terminator sequence, SV40 poly A, preset in the Gene Designer template. SV40 is used for "high level expression". The antibiotic resistance marker is the default ampicillin. The AFP has not been shown to be toxic to cells, so in order to produce as much of the protein as possible, the origin of replication strength is high.
We used the OPTIMIZER software with standard human codons at a guided random mix to generate the DNA sequence for the amino acid, then further manually modified some of the codons to eliminate repeated sequences.
The amino acid sequence for this protein was published in the paper "A natural variant of type I antifreeze protein with four ice-binding repeats is a particularly potent antifreeze", by Heman Chao, et al. in Protein Science, Vol 5, Issue 6, pages 1150???1156 in June 1996. We optimized the nucleotides for expression in human cells.
Taken from the spruce budworm Choristoneura fumiferana. Pubmed ID: 9306405