Team:ACIBADEM ISTANBUL/Parts
Parts
Characterization of an existing part
Existing Part from the Registry of Standard Biological Parts:
Previous studies have shown that only the first 10 amino acids are required to capture the anti-venom activity of LTNF protein.
DNA sequence
CTG AAA GCG ATG GAT CCG ACC CCG CCG CTG
Amino acid sequence
L K A M D P T P P L
Improvement of previous part
Previous part submitted by our team in 2018, named as LTNF 2.0:
Our new part:
TGT GGC GGT GGA GGC TTG AAA GCA ATG GAC CCT ACC CCC CCC CTG TGG ATA AAA ACG GAG TCA CCA AGT ACG CCT TGT
Amino acid sequence
C GGGG LKAMDPTPPLWIKTESPSTP C
"Spacer":
Early modeling studies indicated that placing two Cysteine amino acids right next to the LTNF (Active site) sequence caused a great deal of strain, reducing its c-score as well as its activity. Four Glycine amino acids were found to greatly reduce the stress found between the first cysteine amino acid and the LTNF 10 active site, whilst also avoiding interfering with its biochemical properties, given its chemically inert and simplistic nature.
"Extension":
Adding a Cystiene bond to the end of the 10 amino acid LTNF sequence resulted in stress to the end part of the active site. As a way to combat this, we began adding the natural continuation of the LTNF 10 amino acids bit by bit, the highest c-score occurred when exactly an additional 10 amino acids (extensions) were added to the LTNF 10, striking a balance between the least stress and highest entropy levels.
Expression vector:
Our insert is cloned in pPIC9K instead of pPICZalphaA that we used last year. The expression vector that we used last year, pPICZalphaA, contains zeocin as a selective marker. As we aim to manufacture our end product, we conclude that using zeocin antibiotic is not cost-effective. Instead, we used pPIC9K expression vector which contains kanamycin and ampicillin as selective markers, which are much more cheaper than zeocin.
Validated parts:
Circularized C_LTNF_C in pPIC9K:The original coding sequence of the LTNF is inserted in a pPIC9K vector to express it in Pichia pastoris.
Two Cysteine amino acids have been added as the first (1) and last (26) amino acids in the part to form a disulphide bridge, thereby connecting the ends of the polypeptide chain together resulting in a more circularized structure. This process is called “circularization”.
C_LTNF_C without stop codon in pPIC9K:
In this construct, the insert has cysteine aminoacids and we excluded stop codon before the histidine cleavage site of the vector. By this way, we gain our protein with histidine tag, which will make easier to detect and purify our protein. This construct is used to compare the efficiency of His-tagged LTNF without Cysteine to His-tagged LTNF with two Cysteine residues.
In this construct, we excluded stop codon before the histidine cleavage site of the vector. By this way, we gain our protein with histidine tag, which will make easier to detect and purify our protein. This construct is used to compare the efficiency of His-tagged LTNF to His-tagged LTNF with two Cysteine residues.
Part Table
Please include a table of all the parts your team has made during your project on this page. Remember part characterization and measurement data must go on your team part pages on the Registry.