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

Revision as of 10:25, 1 November 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 at least as effective as the other insulins on the market.

Our Insulin Analogue

Winsulin stuff

Our Constructs

We designed our expression constructs in order to test winsulin and proinsulin with multiple expression systems. Click on each element of the construct to learn more about why we chose it:

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)

Proinsulin

See our section above!

Winsulin

See our section above!

Our Vectors

Vector intro

E. Coli

pet16b justification

Figure 1. pET-15b was used for cytoplasmic and periplasmic expression of proinsulin and winsulin.

B. Subtilis

pUS258 is an integrative expression vector which undergoes homologous recombination to insert into the amyE gene of the Bacillus subtilis chromosome. It’s a relatively simple plasmid using the same IPTG induction system that we use across all of our expression systems.

Figure 2. pus258 was used for cytoplasmic and periplasmic expression of proinsulin and winsulin.

Our Expression Systems

In order to maximise the yield of our insulin, while also reducing the post-expression methods currently undertaken by manufacturers, we tested both of these constructs in three different expression systems. Two of these expression systems were in BL21 E. coli, and the other was in Bacillus subtilis.

E. Coli- Cytoplasm

Cytoplasmic Expression

Cytoplasmic expression is our recombinant protein production in its raw and simple form. With no tags attached to the fusion protein, both Winsulin and Proinsulin will accumulate within the cytoplasm to form aggregated inclusion bodies. Extraction will involve complete lysis of the cell and purification will require separating them from DNA, membranes and other proteins.

The reason to express in the cytoplasm is mainly to compare our other expression systems to where we will be able to see whether they are a viable means of production.

Figure 1. Details of E. Coli cytoplasmic expression of pro/winsulin

E. Coli- Periplasm

Periplasmic Expression

Potentially the biggest hurdle to overcome in efficiently producing recombinant insulin is having the three disulfide bonds in proinsulin and active insulin form correctly. This is because they require an oxidative environment that isn’t usually found in the cytoplasm of common protein factories like E. coli where proteins are naturally expressed.
Fortunately, the periplasmic compartment of gram negative bacteria, including E. coli is more oxidative than the cytoplasm and will therefore improve disulfide bond formation. So we are using the Ecotin tag to target proinsulin and Winsulin to the periplasm in the hope that they will fold correctly and form the correct disulfides.
This method also has the additional advantage that the periplasmic fraction can be extracted without lysing the entire cell, separating our insulin from cells DNA and the rest of the cytoplasmic “junk”.

Figure 2. Details of E. Coli periplasmic expression of pro/winsulin

B. Subtilis

Secretory Expression

Current methods of insulin production grow bacteria in huge vats. But bacteria like E. coli that only express the protein in the cytoplasm need to be lysed to extract it. Which means they need to drain hundreds of thousands of liters of culture to separate the cells from the media before lysing the cells and then purifying insulin from the DNA and other cellular proteins. Although we will be testing cytoplasmic E. coli expression similar to these methods, we wanted to come up with a way to efficiently separate our insulin from the cells and decided the best way would be have it secreted directly into the media. This is where Bacillus subtilis comes in! Bacillus has been a common tool for recombinant protein production for years and has been proven again and again that they are highly efficient secretion factories. The hope is that having the insulin separated from the cells in the media would mean that scaled up production could use a system where the media is constantly cycled out and the cells can continuously produce our insulin. As can be seen in Figure 3, expression using B. Subtilis can be very simple contributing to our goal to create an easily purifiable product.

Figure 3. Details of expression of pro/winsulin in B. Subtilis

Our Purification Process

Blurb about purification.

Assays

??

Additional Solutions

??

WB800 Bacillus Strain

One of the challenges that we found in the literature of producing recombinant insulin was its susceptibility to proteolytic decay. So to continue with the theme of maximising expression, it would make sense to reduce the effect of proteolytic decay.

Our idea: knockout proteases from an existing strain such as B. subtilis 168.

Our actual solution: find one that already exists.

And hence we discovered B. subtilis WB800. It is one of the more recent of a long line of protease deficient strains of Bacillus subtilis that have been optimised for recombinant protein secretion (Figure 5).

WB800 specifically has 8 proteases knocked that include both intracellular and membrane bound. The strain was kindly provided to us for experimental use by Professor Sui-Lam Wong of the University of Calgary, Canada.

Figure 5. WB800 protease knockout strain

pUS270

Although pUS258 appeared to work quite well as an expression vector, we noticed that it wasn't the most streamline process of cloning in our constructs nor transforming into Bacillus subtilis because of an MCS that was incompatible with the BioBrick cloning system, and the extra steps needed to ensure the correct integration of the plasmid into the chromosome.

So we decided to design a new compact, highly efficient replicative expression vector that would serve not just our own future experiments, but the whole iGEM community. Thus, pUS270 was born!

While we were too short on time to construct it ourselves, we are holding onto it for a future project that can be tested and eventually submitted to the registry.

Click on each feature for more information

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

Revision as of 10:25, 1 November 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

Single Chained

Ease of Purification and Affordaility

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 Insulin Analogue

Winsulin stuff

Our Constructs

We designed our expression constructs in order to test winsulin and proinsulin with multiple expression systems. Click on each element of the construct to learn more about why we chose it:

Winsulin secreted by B. subtilis

Proinsulin secreted by B. subtilis

Winsulin targeted to the periplasm of E. coli

Proinsulin targeted to the periplasm of E. coli

Winsulin targeted to the cytoplasm of E. coli

Proinsulin targeted to the cytoplasm of E. coli

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

His Tag

TEV Protease Cleavage Site

“R” Arginine Cleavage Site

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.

Proinsulin

See our section above!

Winsulin

See our section above!

Our Vectors

Vector intro

E. Coli

pet16b justification

B. Subtilis

pUS258 is an integrative expression vector which undergoes homologous recombination to insert into the amyE gene of the Bacillus subtilis chromosome. It’s a relatively simple plasmid using the same IPTG induction system that we use across all of our expression systems.

Our Expression Systems

E. Coli- Cytoplasm

Cytoplasmic Expression

E. Coli- Periplasm

Periplasmic Expression

B. Subtilis

Secretory Expression

Our Purification Process

Blurb about purification.

Assays

??

Additional Solutions

??

WB800 Bacillus Strain

pUS270

Lac Operon Repressor

Codes for the production of the repressor protein that binds to the Lac Operator to prevent expression until induction of the media with IPTG. We have designed pUS270 to have the LacI under the pVeg promoter for strong constitutive expression in Bacillus.

pIP501 Rep

This gene codes for the Replicator protein that pairs with the Bacillus OriV and is essential to promote the independent replication of pUS270 in Bacillus.

E. coli OriV

Taken from pSB1C3, having this origin of replication will make it easier to construct the plasmid using E. coli as well as improve cloning efficiency downstream. Chosen because of the high copy number of pSB1C3.

Multiple Cloning Site

Antibiotic Resistance

Codes for Neomycin Phosphotransferase II that gives transformants resistance to both Neomycin and Kanamycin. Allows selective screening of transformants

Antibiotic Resistance

This region allows independent replication of the vector within Bacillus at a high copy number to provide more copies of the recombinant gene to maximise expression.

Lac Operator

Consists of the target sequence for the lac repressor protein to contribute to the IPTG induction system.

SuperFold AmilCP-6