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

 

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