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Revision as of 11:49, 30 October 2017
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.
GOLD
|
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 |
|
|
|
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 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 / |
|
|
|
|
Cytoplasmic-Winsulin |
|
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 / |
|
|
|
|
Ecotin-Winsulin |
|
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 |
BASIC PARTS
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|
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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 |
|
|
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 |
|
|
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) |
|
|
6x Poly-Histidine Tag codon optimized for use in E. 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 |
|
|
|
T7 major capsid protein |
· High ribosomal recruitment efficiency · Sourced from the pET15b vectors |
|
|
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 |
|
|
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 |
|
|
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 |
|
|
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 |
