Team:Sydney Australia/Composite Part
COMPOSITE PARTS
All of our gold, silver and bronze winning parts are composite parts, consisting of a multitude of basic parts
The complete sequences and characteristics of these basic parts are linked on the Parts Registry pages of our composite parts which are linked in each table.
Best Composite Part
Our best compsite part is our Winsulin (BBa_K2417005) as this part is designed by us (with the help of Peter Arvan and his laboratory) and is also open-source. This part is a single chain insulin which means that there is no extra processing step that must occur to make it functional such as the need to cleave the c peptide in proinsulin to make it functional insulin. This simplifies the production of this insulin. Single-chained insulins are also considered to have greater stability compared to human insulin and other analogues and would therefore be preferable. The Winsulin is also fused to a YncM secretion tag. This tag means that it can be secreted out into the medium that the B.subtillis cells are growing in and would therefore eliminate the extra time consuming and costly step of needing to extract intracellular protein. The His-tag would then allow you to easily purify the Winsulin from the medium with ease.
For additional information regarding our part, click on the part hyperlink or the results.
GOLD
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PART NAME / |
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Novel short chain insulin
analogue (Winsulin) with a 12AA linker 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 |
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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 |
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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
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PART NAME / |
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Cytoplasmic-Winsulin |
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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
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PART NAME / |
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Ecotin-Winsulin |
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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 |
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N/A – Winsulin is self designed from literature and mammalian insulins |
