Difference between revisions of "Team:Sydney Australia/Design"

Revision as of 08:28, 28 October 2017

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

For our project to work effectively, we must have a supply chain that’s not a cold chain, so that costs can be reduced. This will ultimately mean that the cost of these cold chains will not be passed onto the consumer. To achieve this, we hope to design an insulin that will not lose efficacy after being exposed to room temperature for long periods of time.

Single Chained

As a result of the difficult purification methods, Single Chain Insulins, or SCIs for short, have been developed with a small, C-peptide chain linker. This linker connects the A and B chains in such a way that the di-sulfide bonds form more favorably. We aim to develop our own single chain insulin to compare it’s simplicity.

Ease of Purification and Affordaility

We must also consider the impact of a difficult, costly manufacturing process on small scale manufacturing companies. This impact is too great to impose on this grass-roots organisations, so we have pursued to find a cheap, simple purification method which is able to produce high yields from a recombinant system.

Intellectual Property Issues

As a result of the way drugs are currently developed, all new inventions for therapies are protected by Intellectual Property Law through patents. These patents surrounding all currently prescribed and newly invented insulins has inspired our team to pursue a completely open source project.

Safety and Efficacy

Our insulin products must be of certifiable medical grade such that it can be approved for human use after stage IV clinical trials, or biosimilar clinical trials. Furthermore, it must also be as effective at least, as the other insulins on the market.

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

This expression system contains a YNCM fusion molecule that directs the protein to the extracellular space. We hope that this system will remove all of the cell lysis steps required with the E coli systems, and could potentially be optimised into a fed-batch system by Biofoundry and/or Open Insulin.

Our Six Constructs:

Cytoplasmic Proinsulin

Cytoplasmic Winsulin

Periplasmic Proinsulin

Periplasmic Winsulin

Secretory Proinsulin

Secretory Winsulin