PART NAME /
NO.

SOURCE

KEY FEATURES

WHY IS IT GOLD?

YncM-Winsulin

BBa_K2417005

Bacillus subtilis (YncM secretion tag)

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

YncM secretion tag to translocate protein into media

N-terminal 6x His Tag purification

4x GSS flexible linker

TEV protease cleavage site

Extended ribosome binding site

We demonstrated that it works:

·      We tested it in insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, proving its bioactivity

·      We showed via an ELISA assay that whilst the cell lysate did not contain Winsulin, the media in which the cells were growing did. Therefore the YncM secretion tag worked as expected

The results for these assays can be found here

Ecotin-Proinsulin

BBa_K2417000

E. Coli (Ecotin tag)

Human proinsulin

N-terminal Ecotin tag for targeting proteins to the periplasm

N-terminal 6x His tag

4x GGS flexible linker

trypsin protease cleavage site for His-tag and C-peptide cleavage

Extended ribosomal binding site

We improved previous parts:

BBa_M39904 and BBa_M1877 by:

completing their sequence

Adding a periplasmic transporter tag (for more efficient folding in an oxidative environment)

Adding an efficient ribosomal binding site

Adding an N-terminal His tag for purification

We demonstrated that it works:

·      By testing it insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, therefore proving its bioactivity

The results for these assays can be found here

Human (Human proinsulin)

Cytoplasmic-Proinsulin     BBa_K2417001

Human (Human proinsulin)

Human proinsulin

N-terminal 6x His tag

4x GGS flexible linker

Trypsin protease cleavage site for His-tag and C-peptide cleavage

Extended ribosomal binding site

We improved previous parts:

BBa_M39904 and BBa_M1877 by:

·      completing their sequence

·      Adding an efficient ribosomal binding site

·      Adding an N-terminal His tag for purification

We demonstrated that it works:

·      By testing it insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, therefore proving its bioactivity

The results for these assays 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