Team:Sydney Australia/Design
Our Key Goals
The aim of the USYD iGEM 2017 team was to address the problem of insulin inaccessibility. The design of our insulin, and its means of expression, needed to look at five key characteristics:
Stability
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Single Chained
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Affordable
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Intellectual Property Issues
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Safety and Efficacy
Which is worse, that everyone has his price, or that the price is always so low.
Our Insulins:
In order to meet off all of these characteristics, we decided to use Human insulin and develop our very own Single Chain Insulin. Though we would have preferred to have a single insulin that holds all of these characteristics, at this point in time there isn't a single insulin fully categorised that meets all of these criteria.
Proinsulin
Sequence!
Characterisation!
Sequence Length:
pI:
Winsulin
Sequence!
Characterisation!
Sequence Length:
pI:
Our Expression Systems:
In order to maximise the yield of our insulin, while also reducing the post-expression purification methods currently undertaken by manufactuers, we will be trialling both of these constructs in three different expression systems. Two of these expression systems will be in BL21 E. coli, and the other will be in Bacillus subtilis.
E. coli Cytoplasmic Expression
E. coli Periplasmic Expression
Insulin accumulates in the cytoplasm under normal conditions within inclusion bodies, however will require downstream refolding in several chemical processes.
For this expression system, our gblocks will require an Ecotin fusion tag to redirect the protein to the bacterial periplasm for correct disulphide bond formation and folding. This, we hope, will reduce the number of chemical refolding steps that will be required for the Cytoplasmic Expression of insulin.
B. subtilis Excretory Expression
