PART NAME /
NO.

SOURCE

KEY FEATURES

WHY IS IT SILVER?

Cytoplasmic-Winsulin

BBa_K2417003

N/A – Winsulin is self designed from literature and mammalian insulins

Novel single chain insulin analogue (Winsulin) with a 12AA linker (QRGGGSGGGQRR)

N-terminal 6x His Tag

4x GSS flexible linker

TEV protease cleavage site

Extended ribosome binding site

·      We validated our parts by showing that they work in producing insulin. We performed an ELISA assay testing for properly folded insulin, and detected the presence of Cytoplasmic Winsulin.

The results for the assay can be found here

PART NO.

PART NAME

SOURCE

DESCRIPTION

BBa_K2417004

Glycine Glycine Serine (x4) Linker Motif A

Designed

·      Linker motif used to provide spacer region

·      Used to expose affinity purification tags such as 6x His tag

·      Serine and glycine used as they are small and flexible

BBa_K2417006

Human Proinsulin Coding Sequence

Humans

·      Coding sequence for human proinsulin, sourced from NCIB

·      Consists of A, B and C chains, but C-peptide is cleaved out

·      Contains 3 disulphide bonds

·      Contains arginine residues adjacent to C-peptide to allow cleavage

BBa_K2417007

Winsulin - Single Chain Insulin Analogue

Designed

·      Consists of short linker peptide instead of C-peptide

·      Does not require cleavage therefore simplifies production procedures

·      Linker peptide raises pI of insulin analogue (long lasting after administration)

·      Increased thermostability (based on other single chain insulin analogues tested)

BBa_K2417008

6x Poly-Histidine Tag codon optimized for use in E. coli

Designed (optimized for use in Escherichia coli)

·      Codon optimized for use in E. coli

·      Used to immobilize ions on a chromatography affinity column (nickel, cobalt or copper)

·      Small, and easy to add to the ends of proteins of interest

BBa_K2417009

Extended ribosome binding site

T7 major capsid protein

·      High ribosomal recruitment efficiency

·      Sourced from the pET15b vectors

BBa_K2417010

Ecotin Periplasmic Signal Peptide

Escherichia coli

·      Small periplasmic, homodimeric protein

·      Upon fusion to N-terminus of protein will translocate protein to periplasm of E. coli

·      Periplasm is an oxidative environment ideal for protein folding, and simplifies extraction due to eliminating the need for purifying inclusion bodies from the cytoplasm

·      Periplasm has reduced proteases

BBa_K2417011

YncM Bacillus Secretion Peptide

Bacillus subtilis

·      Can be fused to the N-terminus of proteins to induce secretion to the surrounding media wheN proteins are expressed in Bacillus subtilis

·      Eliminates many steps in the purification process

BBa_K2417012

TEV Protease Cleavage Sequence

N/A

·      Sequence for which TEV protease is specific to

·      Can be added between protein of interest and peptides that require removal, such as purification tags

BBa_K2417013

Glycine Glycine Serine (x4) Linker

Motif B

Designed

·      Linker motif used to provide spacer region

·      Used to expose affinity purification tags such as 6x His tag

·      Serine and glycine used as they are small and flexible