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