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<center><h4>All of our gold, silver and bronze winning parts are composite parts, consisting of a multitude of basic parts<br><br> The complete sequences and characteristics of these basic parts are linked on the Parts Registry pages of our composite parts which are <b>linked in each table.</b></h4></center>
 
<center><h4>All of our gold, silver and bronze winning parts are composite parts, consisting of a multitude of basic parts<br><br> The complete sequences and characteristics of these basic parts are linked on the Parts Registry pages of our composite parts which are <b>linked in each table.</b></h4></center>
 
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Revision as of 11:52, 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 /
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

 

BASIC PARTS


PART NO.

 


PART NAME


SOURCE


DESCRIPTION

 

 

BBa_K2417004

 

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

 

 

 

BBa_K2417006

 

 

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

 

 

BBa_K2417007

 

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)

 

 

BBa_K2417008

 

6x Poly-Histidine Tag codon optimized for use in E. coli


Designed (optimized for use in Escherichia 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



BBa_K2417009


Extended ribosome binding site

 

 

T7 major capsid protein

·      High ribosomal recruitment efficiency

·      Sourced from the pET15b vectors

 

 

BBa_K2417010

 

 

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

 



BBa_K2417011

 

 

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

 

 

BBa_K2417012

 

 

 

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

 

 

BBa_K2417013

 

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