Team:Sydney Australia/Parts

GOLD

PART NAME / NO.	SOURCE	KEY FEATURES	WHAT IS IT GOLD?
{{:Team:Sydney_Australia/Templates/NavBar}} GOLD   PART NAME / NO.   SOURCE KEY FEATURES WHAT 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   GOLD   PART NAME / NO.   SOURCE KEY FEATURES WHAT 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   GOLD   PART NAME / NO.   SOURCE KEY FEATURES WHAT IS IT GOLD? YncM-Winsulin BBa_K2417005   {{:Team:Sydney_Australia/Templates/NavBar}} GOLD   PART NAME / NO.   SOURCE KEY FEATURES WHAT 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   GOLD   PART NAME / NO.   SOURCE KEY FEATURES WHAT 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   GOLD   PART NAME / NO.   SOURCE KEY FEATURES WHAT IS IT GOLD? {{:Team:Sydney_Australia/Templates/NavBar}} GOLD   PART NAME / NO.   SOURCE KEY FEATURES WHAT 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   GOLD   PART NAME / NO.   SOURCE KEY FEATURES WHAT 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   GOLD   PART NAME / NO.   SOURCE KEY FEATURES WHAT 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   YncM-Winsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417005">BBa_K2417005</a><o:p></o:p> </td> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:78.9pt"> <o:p> </o:p> Bacillus subtilis (YncM secretion tag)<o:p></o:p> <o:p> </o:p> </td> <td width="29%" style="width:29.62%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:78.9pt"> <o:p> </o:p> Novel short chain insulin analogue (Winsulin) with a 12AA linker (-QRGGGSGGGQRR-)<o:p></o:p> <o:p> </o:p> YncM secretion tag to translocate protein into media<o:p></o:p> <o:p> </o:p> N-terminal 6x His Tag purification<o:p></o:p> <o:p> </o:p> 4x GSS flexible linker<o:p></o:p> <o:p> </o:p> TEV protease cleavage site<o:p></o:p> <o:p> </o:p> Extended ribosome binding site<o:p></o:p> <o:p> </o:p> </td> <td width="41%" valign="top" style="width:41.56%;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:78.9pt"> <o:p> </o:p> <o:p> </o:p> We demonstrated that it works:<o:p></o:p> ·      We tested it in insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, proving its bioactivity <o:p></o:p> ·      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<o:p></o:p> <o:p> </o:p> The results for these assays can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> </td> </tr> <tr style="mso-yfti-irow:2;height:206.35pt"> <td width="15%" rowspan="2" style="width:15.92%;border:solid windowtext 1.0pt; border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> Ecotin-Proinsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417000">BBa_K2417000<o:p></o:p></a> <o:p> </o:p> </td> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> E. Coli (Ecotin tag)<o:p></o:p> </td> <td width="29%" rowspan="2" style="width:29.62%;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Human proinsulin<o:p></o:p> <o:p> </o:p> N-terminal Ecotin tag for targeting proteins to the periplasm<o:p></o:p> <o:p> </o:p> N-terminal 6x His tag<o:p></o:p> <o:p> </o:p> 4x GGS flexible linker<o:p></o:p> <o:p> </o:p> trypsin protease cleavage site for His-tag and C-peptide cleavage<o:p></o:p> <o:p> </o:p> Extended ribosomal binding site<o:p></o:p> <o:p> </o:p> </td> <td width="41%" rowspan="2" valign="top" style="width:41.56%;border-top:none; border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> <o:p></o:p> We improved previous parts:<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_M39904">BBa_M39904</a> and <a href="http://parts.igem.org/Part:BBa_M1877">BBa_M1877</a> by:<o:p></o:p> <o:p> </o:p> completing their sequence<o:p></o:p> <o:p> </o:p> Adding a periplasmic transporter tag (for more efficient folding in an oxidative environment)<o:p></o:p> <o:p> </o:p> Adding an efficient ribosomal binding site<o:p></o:p> <o:p> </o:p> Adding an N-terminal His tag for purification<o:p></o:p> <o:p> </o:p> We demonstrated that it works:<o:p></o:p> ·      By testing it insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, therefore proving its bioactivity<o:p></o:p> <o:p> </o:p> The results for these assays can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> </td> </tr> <tr style="mso-yfti-irow:3;height:72.85pt"> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:72.85pt"> Human (Human proinsulin)<o:p></o:p> </td> </tr> <tr style="mso-yfti-irow:4;mso-yfti-lastrow:yes;height:42.15pt"> <td width="15%" style="width:15.92%;border:solid windowtext 1.0pt;border-top: none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Cytoplasmic-Proinsulin     <a href="http://parts.igem.org/Part:BBa_K2417001">BBa_K2417001</a><o:p></o:p> </td> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> Human (Human proinsulin)<o:p></o:p> </td> <td width="29%" style="width:29.62%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> Human proinsulin<o:p></o:p> <o:p> </o:p> N-terminal 6x His tag<o:p></o:p> <o:p> </o:p> 4x GGS flexible linker<o:p></o:p> <o:p> </o:p> Trypsin protease cleavage site for His-tag and C-peptide cleavage<o:p></o:p> <o:p> </o:p> Extended ribosomal binding site<o:p></o:p> </td> <td width="41%" valign="top" style="width:41.56%;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> <o:p> </o:p> We improved previous parts:<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_M39904">BBa_M39904</a> and <a href="http://parts.igem.org/Part:BBa_M1877">BBa_M1877</a> by:<o:p></o:p> ·      completing their sequence<o:p></o:p> ·      Adding an efficient ribosomal binding site<o:p></o:p> ·      Adding an N-terminal His tag for purification<o:p></o:p> <o:p> </o:p> We demonstrated that it works:<o:p></o:p> ·      By testing it insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, therefore proving its bioactivity<o:p></o:p> <o:p> </o:p> The results for these assays can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> </td> </tr> </tbody></table> </div> </div> SILVER <tbody> </tbody> <o:p> </o:p> PART NAME /  NO.<o:p></o:p> <o:p> </o:p> SOURCE<o:p></o:p> KEY FEATURES<o:p></o:p> WHY IS IT SILVER?<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Cytoplasmic-Winsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417003">BBa_K2417003</a><o:p></o:p> <o:p> </o:p> N/A – Winsulin is self designed from literature and mammalian insulins<o:p></o:p> <o:p> </o:p> <o:p> </o:p> Novel single chain insulin analogue (Winsulin) with a 12AA linker (QRGGGSGGGQRR)<o:p></o:p> <o:p> </o:p> N-terminal 6x His Tag<o:p></o:p> <o:p> </o:p> 4x GSS flexible linker<o:p></o:p> <o:p> </o:p> TEV protease cleavage site<o:p></o:p> <o:p> </o:p> Extended ribosome binding site<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> ·      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.<o:p></o:p> <o:p> </o:p> <o:p> </o:p> The results for the assay can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> <o:p> </o:p> <o:p> </o:p> BRONZE <tbody> </tbody> <o:p> </o:p> PART NAME / NO.<o:p></o:p> <o:p> </o:p> SOURCE<o:p></o:p> KEY FEATURES<o:p></o:p> WHY IS IT BRONZE?<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Ecotin-Winsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417002">BBa_K2417002</a><o:p></o:p> <o:p> </o:p> E. Coli (Ecotin Tag)<o:p></o:p> <o:p> </o:p> <o:p> </o:p> Novel single chain insulin analogue (Winsulin) with a 12AA linker (QRGGGSGGGQRR)<o:p></o:p> <o:p> </o:p> N-terminal Ecotin tag for targeting proteins to the periplasm<o:p></o:p> <o:p> </o:p> N-terminal 6x His Tag<o:p></o:p> <o:p> </o:p> 4x GSS flexible linker<o:p></o:p> <o:p> </o:p> TEV protease cleavage site<o:p></o:p> <o:p> </o:p> Extended ribosome binding site<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> We submitted this DNA part to the registry, for other teams to use in the future<o:p></o:p> <o:p> </o:p> N/A – Winsulin is self designed from literature and mammalian insulins<o:p></o:p> <o:p> </o:p> </body> </html> Bacillus subtilis (YncM secretion tag)<o:p></o:p> <o:p> </o:p> </td> <td width="29%" style="width:29.62%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:78.9pt"> <o:p> </o:p> Novel short chain insulin analogue (Winsulin) with a 12AA linker (-QRGGGSGGGQRR-)<o:p></o:p> <o:p> </o:p> YncM secretion tag to translocate protein into media<o:p></o:p> <o:p> </o:p> N-terminal 6x His Tag purification<o:p></o:p> <o:p> </o:p> 4x GSS flexible linker<o:p></o:p> <o:p> </o:p> TEV protease cleavage site<o:p></o:p> <o:p> </o:p> Extended ribosome binding site<o:p></o:p> <o:p> </o:p> </td> <td width="41%" valign="top" style="width:41.56%;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:78.9pt"> <o:p> </o:p> <o:p> </o:p> We demonstrated that it works:<o:p></o:p> ·      We tested it in insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, proving its bioactivity <o:p></o:p> ·      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<o:p></o:p> <o:p> </o:p> The results for these assays can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> </td> </tr> <tr style="mso-yfti-irow:2;height:206.35pt"> <td width="15%" rowspan="2" style="width:15.92%;border:solid windowtext 1.0pt; border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> Ecotin-Proinsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417000">BBa_K2417000<o:p></o:p></a> <o:p> </o:p> </td> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> E. Coli (Ecotin tag)<o:p></o:p> </td> <td width="29%" rowspan="2" style="width:29.62%;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Human proinsulin<o:p></o:p> <o:p> </o:p> N-terminal Ecotin tag for targeting proteins to the periplasm<o:p></o:p> <o:p> </o:p> N-terminal 6x His tag<o:p></o:p> <o:p> </o:p> 4x GGS flexible linker<o:p></o:p> <o:p> </o:p> trypsin protease cleavage site for His-tag and C-peptide cleavage<o:p></o:p> <o:p> </o:p> Extended ribosomal binding site<o:p></o:p> <o:p> </o:p> </td> <td width="41%" rowspan="2" valign="top" style="width:41.56%;border-top:none; border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> <o:p></o:p> We improved previous parts:<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_M39904">BBa_M39904</a> and <a href="http://parts.igem.org/Part:BBa_M1877">BBa_M1877</a> by:<o:p></o:p> <o:p> </o:p> completing their sequence<o:p></o:p> <o:p> </o:p> Adding a periplasmic transporter tag (for more efficient folding in an oxidative environment)<o:p></o:p> <o:p> </o:p> Adding an efficient ribosomal binding site<o:p></o:p> <o:p> </o:p> Adding an N-terminal His tag for purification<o:p></o:p> <o:p> </o:p> We demonstrated that it works:<o:p></o:p> ·      By testing it insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, therefore proving its bioactivity<o:p></o:p> <o:p> </o:p> The results for these assays can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> </td> </tr> <tr style="mso-yfti-irow:3;height:72.85pt"> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:72.85pt"> Human (Human proinsulin)<o:p></o:p> </td> </tr> <tr style="mso-yfti-irow:4;mso-yfti-lastrow:yes;height:42.15pt"> <td width="15%" style="width:15.92%;border:solid windowtext 1.0pt;border-top: none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Cytoplasmic-Proinsulin     <a href="http://parts.igem.org/Part:BBa_K2417001">BBa_K2417001</a><o:p></o:p> </td> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> Human (Human proinsulin)<o:p></o:p> </td> <td width="29%" style="width:29.62%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> Human proinsulin<o:p></o:p> <o:p> </o:p> N-terminal 6x His tag<o:p></o:p> <o:p> </o:p> 4x GGS flexible linker<o:p></o:p> <o:p> </o:p> Trypsin protease cleavage site for His-tag and C-peptide cleavage<o:p></o:p> <o:p> </o:p> Extended ribosomal binding site<o:p></o:p> </td> <td width="41%" valign="top" style="width:41.56%;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> <o:p> </o:p> We improved previous parts:<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_M39904">BBa_M39904</a> and <a href="http://parts.igem.org/Part:BBa_M1877">BBa_M1877</a> by:<o:p></o:p> ·      completing their sequence<o:p></o:p> ·      Adding an efficient ribosomal binding site<o:p></o:p> ·      Adding an N-terminal His tag for purification<o:p></o:p> <o:p> </o:p> We demonstrated that it works:<o:p></o:p> ·      By testing it insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, therefore proving its bioactivity<o:p></o:p> <o:p> </o:p> The results for these assays can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> </td> </tr> </tbody></table> </div> </div> SILVER <tbody> </tbody> <o:p> </o:p> PART NAME /  NO.<o:p></o:p> <o:p> </o:p> SOURCE<o:p></o:p> KEY FEATURES<o:p></o:p> WHY IS IT SILVER?<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Cytoplasmic-Winsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417003">BBa_K2417003</a><o:p></o:p> <o:p> </o:p> N/A – Winsulin is self designed from literature and mammalian insulins<o:p></o:p> <o:p> </o:p> <o:p> </o:p> Novel single chain insulin analogue (Winsulin) with a 12AA linker (QRGGGSGGGQRR)<o:p></o:p> <o:p> </o:p> N-terminal 6x His Tag<o:p></o:p> <o:p> </o:p> 4x GSS flexible linker<o:p></o:p> <o:p> </o:p> TEV protease cleavage site<o:p></o:p> <o:p> </o:p> Extended ribosome binding site<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> ·      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.<o:p></o:p> <o:p> </o:p> <o:p> </o:p> The results for the assay can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> <o:p> </o:p> <o:p> </o:p> BRONZE <tbody> </tbody> <o:p> </o:p> PART NAME / NO.<o:p></o:p> <o:p> </o:p> SOURCE<o:p></o:p> KEY FEATURES<o:p></o:p> WHY IS IT BRONZE?<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Ecotin-Winsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417002">BBa_K2417002</a><o:p></o:p> <o:p> </o:p> E. Coli (Ecotin Tag)<o:p></o:p> <o:p> </o:p> <o:p> </o:p> Novel single chain insulin analogue (Winsulin) with a 12AA linker (QRGGGSGGGQRR)<o:p></o:p> <o:p> </o:p> N-terminal Ecotin tag for targeting proteins to the periplasm<o:p></o:p> <o:p> </o:p> N-terminal 6x His Tag<o:p></o:p> <o:p> </o:p> 4x GSS flexible linker<o:p></o:p> <o:p> </o:p> TEV protease cleavage site<o:p></o:p> <o:p> </o:p> Extended ribosome binding site<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> We submitted this DNA part to the registry, for other teams to use in the future<o:p></o:p> <o:p> </o:p> N/A – Winsulin is self designed from literature and mammalian insulins<o:p></o:p> <o:p> </o:p> </body> </html> YncM-Winsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417005">BBa_K2417005</a><o:p></o:p> </td> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:78.9pt"> <o:p> </o:p> Bacillus subtilis (YncM secretion tag)<o:p></o:p> <o:p> </o:p> </td> <td width="29%" style="width:29.62%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:78.9pt"> <o:p> </o:p> Novel short chain insulin analogue (Winsulin) with a 12AA linker (-QRGGGSGGGQRR-)<o:p></o:p> <o:p> </o:p> YncM secretion tag to translocate protein into media<o:p></o:p> <o:p> </o:p> N-terminal 6x His Tag purification<o:p></o:p> <o:p> </o:p> 4x GSS flexible linker<o:p></o:p> <o:p> </o:p> TEV protease cleavage site<o:p></o:p> <o:p> </o:p> Extended ribosome binding site<o:p></o:p> <o:p> </o:p> </td> <td width="41%" valign="top" style="width:41.56%;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:78.9pt"> <o:p> </o:p> <o:p> </o:p> We demonstrated that it works:<o:p></o:p> ·      We tested it in insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, proving its bioactivity <o:p></o:p> ·      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<o:p></o:p> <o:p> </o:p> The results for these assays can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> </td> </tr> <tr style="mso-yfti-irow:2;height:206.35pt"> <td width="15%" rowspan="2" style="width:15.92%;border:solid windowtext 1.0pt; border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> Ecotin-Proinsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417000">BBa_K2417000<o:p></o:p></a> <o:p> </o:p> </td> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> E. Coli (Ecotin tag)<o:p></o:p> </td> <td width="29%" rowspan="2" style="width:29.62%;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Human proinsulin<o:p></o:p> <o:p> </o:p> N-terminal Ecotin tag for targeting proteins to the periplasm<o:p></o:p> <o:p> </o:p> N-terminal 6x His tag<o:p></o:p> <o:p> </o:p> 4x GGS flexible linker<o:p></o:p> <o:p> </o:p> trypsin protease cleavage site for His-tag and C-peptide cleavage<o:p></o:p> <o:p> </o:p> Extended ribosomal binding site<o:p></o:p> <o:p> </o:p> </td> <td width="41%" rowspan="2" valign="top" style="width:41.56%;border-top:none; border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:206.35pt"> <o:p></o:p> We improved previous parts:<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_M39904">BBa_M39904</a> and <a href="http://parts.igem.org/Part:BBa_M1877">BBa_M1877</a> by:<o:p></o:p> <o:p> </o:p> completing their sequence<o:p></o:p> <o:p> </o:p> Adding a periplasmic transporter tag (for more efficient folding in an oxidative environment)<o:p></o:p> <o:p> </o:p> Adding an efficient ribosomal binding site<o:p></o:p> <o:p> </o:p> Adding an N-terminal His tag for purification<o:p></o:p> <o:p> </o:p> We demonstrated that it works:<o:p></o:p> ·      By testing it insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, therefore proving its bioactivity<o:p></o:p> <o:p> </o:p> The results for these assays can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> </td> </tr> <tr style="mso-yfti-irow:3;height:72.85pt"> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:72.85pt"> Human (Human proinsulin)<o:p></o:p> </td> </tr> <tr style="mso-yfti-irow:4;mso-yfti-lastrow:yes;height:42.15pt"> <td width="15%" style="width:15.92%;border:solid windowtext 1.0pt;border-top: none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Cytoplasmic-Proinsulin     <a href="http://parts.igem.org/Part:BBa_K2417001">BBa_K2417001</a><o:p></o:p> </td> <td width="12%" style="width:12.88%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> Human (Human proinsulin)<o:p></o:p> </td> <td width="29%" style="width:29.62%;border-top:none;border-left:none; border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> Human proinsulin<o:p></o:p> <o:p> </o:p> N-terminal 6x His tag<o:p></o:p> <o:p> </o:p> 4x GGS flexible linker<o:p></o:p> <o:p> </o:p> Trypsin protease cleavage site for His-tag and C-peptide cleavage<o:p></o:p> <o:p> </o:p> Extended ribosomal binding site<o:p></o:p> </td> <td width="41%" valign="top" style="width:41.56%;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt;height:42.15pt"> <o:p> </o:p> We improved previous parts:<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_M39904">BBa_M39904</a> and <a href="http://parts.igem.org/Part:BBa_M1877">BBa_M1877</a> by:<o:p></o:p> ·      completing their sequence<o:p></o:p> ·      Adding an efficient ribosomal binding site<o:p></o:p> ·      Adding an N-terminal His tag for purification<o:p></o:p> <o:p> </o:p> We demonstrated that it works:<o:p></o:p> ·      By testing it insulin-sensitive cell lines and saw an increase in glycogen synthesis and glucose oxidation above basal levels, therefore proving its bioactivity<o:p></o:p> <o:p> </o:p> The results for these assays can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> </td> </tr> </tbody></table> </div> </div> SILVER <tbody> </tbody> <o:p> </o:p> PART NAME /  NO.<o:p></o:p> <o:p> </o:p> SOURCE<o:p></o:p> KEY FEATURES<o:p></o:p> WHY IS IT SILVER?<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Cytoplasmic-Winsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417003">BBa_K2417003</a><o:p></o:p> <o:p> </o:p> N/A – Winsulin is self designed from literature and mammalian insulins<o:p></o:p> <o:p> </o:p> <o:p> </o:p> Novel single chain insulin analogue (Winsulin) with a 12AA linker (QRGGGSGGGQRR)<o:p></o:p> <o:p> </o:p> N-terminal 6x His Tag<o:p></o:p> <o:p> </o:p> 4x GSS flexible linker<o:p></o:p> <o:p> </o:p> TEV protease cleavage site<o:p></o:p> <o:p> </o:p> Extended ribosome binding site<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> ·      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.<o:p></o:p> <o:p> </o:p> <o:p> </o:p> The results for the assay can be found <a href="https://2017.igem.org/Team:Sydney_Australia/Results">here</a><o:p></o:p> <o:p> </o:p> <o:p> </o:p> BRONZE <tbody> </tbody> <o:p> </o:p> PART NAME / NO.<o:p></o:p> <o:p> </o:p> SOURCE<o:p></o:p> KEY FEATURES<o:p></o:p> WHY IS IT BRONZE?<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> Ecotin-Winsulin<o:p></o:p> <a href="http://parts.igem.org/Part:BBa_K2417002">BBa_K2417002</a><o:p></o:p> <o:p> </o:p> E. Coli (Ecotin Tag)<o:p></o:p> <o:p> </o:p> <o:p> </o:p> Novel single chain insulin analogue (Winsulin) with a 12AA linker (QRGGGSGGGQRR)<o:p></o:p> <o:p> </o:p> N-terminal Ecotin tag for targeting proteins to the periplasm<o:p></o:p> <o:p> </o:p> N-terminal 6x His Tag<o:p></o:p> <o:p> </o:p> 4x GSS flexible linker<o:p></o:p> <o:p> </o:p> TEV protease cleavage site<o:p></o:p> <o:p> </o:p> Extended ribosome binding site<o:p></o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> <o:p> </o:p> We submitted this DNA part to the registry, for other teams to use in the future<o:p></o:p> <o:p> </o:p> N/A – Winsulin is self designed from literature and mammalian insulins<o:p></o:p> <o:p> </o:p> </body> </html>