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
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 at least as effective as the other insulins on the market.
Our Constructs
We designed our expression constructs in order to meet these goals. Click on each element of the construct to learn more about why we chose them:
Winsulin secreted by B. subtilis
BB prefix
RBS
YNCM Tag
His Tag
TEV
Winsulin
BB suffix
Proinsulin secreted by B. subtilis
BB prefix
RBS
YNCM Tag
His Tag
R
Proinsulin
BB suffix
Winsulin targeted to the periplasm of E. coli
BB prefix
RBS
Ecotin Tag
His Tag
TEV
Winsulin
BB suffix
Proinsulin targeted to the periplasm of E. coli
BB prefix
RBS
Ecotin Tag
His Tag
R
Proinsulin
BB suffix
Winsulin targeted to the cytoplasm of E. coli
BB prefix
RBS
His Tag
TEV
Winsulin
BB suffix
Proinsulin targeted to the cytoplasm of E. coli
BB prefix
RBS
His Tag
R
Proinsulin
BB suffix
iGEM BioBrick Prefix
Contains the restriction sites that are necessary for BioBrick compatibility including EcoRI, NotI & XbaI.
E. coli Extended Ribosome Binding Site
A derivative of the RBS found in gene 10 of the T7 bacteriophage, this 23 base pair sequence rich in A’s & T’s enhances ribosome binding to boost expression.
YncM Tag
The YNCM tag is a 12 amino acid sequence whose presence on the N-terminus of the protein targets it for secretion out of the cell into the surrounding media via the Sec pathway in Bacillus subtilis. YNCM was chosen because it was recently shown to be massively successful in targeting recombinant protein for secretion compared to a library of other signal peptides. Additionally, this was shown in B. subtilis strain WB600, which is the bacteria that our WB800 strain was derived from. So we expect that it should give us similar success in secretion of our constructs. (Guan et. al. 2016)
His Tag
We have included a tag comprised of 6 sequential histidines that form a vital aspect of our purification technique using affinity chromatography. Histidine’s high attraction to metal ions will cause the entire protein, insulin and all, to bind to a nickel column and separate it from the other proteins of the cell.
TEV Protease Cleavage Site
TEV is a sequence-specific cysteine protease derived from Tobacco Etch Virus. Because of its high specificity, it is commonly used for deliberate protein cleavage. In our project, we will use it to exclusively detach Winsulin from the nickel column, leaving the his tag and Ecotin/YNCM tags behind. This should provide us with a pure elution of Winsulin.
“R” Arginine Cleavage Site
Arginine acts as a recognition site for Trypsin Protease which we will use to specifically remove Proinsulin from the his tag and YNCM/Ecotin tag in a similar way to TEV. We have chosen to use Trypsin in these constructs because it allows us to further simplify the processing of proinsulin. Trypsin naturally cleaves the C-peptide from proinsulin which, following disulfide bond formation, leaves the active form of insulin. This is the way it works in our body, so we are confident that it will work here too.
iGEM BioBrick Suffix
Contains the restriction sites that are necessary for BioBrick compatibility including SpeI, NotI & PstI. We have also added an additional BamHI site at the terminus of our E. coli expressed constructs for ligation into pET-15b.
Ecotin Tag
Ecotin acts as a signal sequence to target the translated protein to the periplasm of the cell. There are a number of advantages that make it a good choice over other tags.
- Relatively low metabolic burden due to its small size
- No interaction with other proteins within the periplasm
- Is native in E. coli and contains a disulfide bond meaning it undergoes through an oxidative compartment that may assist in the formation of the disulfides in Proinsulin and Winsulin.
- It has already been shown to successfully target proinsulin to the periplasm (Malik et. al. 2007)
