Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Karen Gomez, Ivy Nguyen, Yoo Hsiu Yeh
Date created: 2012-12-06 12:00:00
Date modified: 2016-01-28 12:19:58
HeLa Optimized Type I Antifreeze Protein
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_M36850_sequence (Version 1) |
Description
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.