Team:Sydney Australia/Parts

GOLD

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

SILVER

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

BRONZE

PART NAME / NO.	SOURCE	KEY FEATURES	WHY IS IT BRONZE?
Ecotin-Winsulin BBa_K2417002	E. Coli (Ecotin Tag)	·      Novel single chain insulin analogue (Winsulin) with a 12AA linker (QRGGGSGGGQRR) ·      N-terminal Ecotin tag for targeting proteins to the periplasm ·      N-terminal 6x His Tag ·      4x GSS flexible linker ·      TEV protease cleavage site ·      Extended ribosome binding site	We submitted this DNA part to the registry, for other teams to use in the future
	N/A – Winsulin is self designed from literature and mammalian insulins