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

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<div class = "row" >
 
<div class = "row" >
 
<div class="para_container">
 
<div class="para_container">
−
<h2>Our Key Goals</h2>
+
<h20>Our Key Goals</h20>
 
<div class = "divider1"></div>
 
<div class = "divider1"></div>
 
<br>
 
<br>
  
−
<h4>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:</h4>
+
<h40>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:</h40>
 
</div>
 
</div>
 
</div>
 
</div>
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</div>
 
</div>
 
<div class="box1">
 
<div class="box1">
−
     <h3>Stability</h3>
+
     <h30>Stability</h30>
−
     <h4>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.</h4>
+
     <h40>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.</h40>
  
 
</div>
 
</div>
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</div>
 
</div>
 
<div class="box2">
 
<div class="box2">
−
     <h3>Single Chained</h3>
+
     <h30>Single Chained</h30>
−
     <h4>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.
+
     <h40>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.
−
</h4>
+
</h40>
  
 
</div>
 
</div>
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</div>
 
</div>
 
<div class="box3">
 
<div class="box3">
−
     <h3>Ease of Purification and Affordaility</h3>
+
     <h30>Ease of Purification and Affordaility</h30>
−
     <h4>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.   
+
     <h40>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.   
−
</h4>
+
</h40>
  
 
</div>
 
</div>
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</div>
 
</div>
 
<div class="box4">
 
<div class="box4">
−
     <h3>Intellectual Property Issues</h3>
+
     <h30>Intellectual Property Issues</h30>
−
     <h4>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.  
+
     <h40>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.  
−
</h4>
+
</h40>
  
 
</div>
 
</div>
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</div>
 
</div>
 
<div class="box5">
 
<div class="box5">
−
     <h3>Safety and Efficacy</h3>
+
     <h30>Safety and Efficacy</h30>
−
     <h4>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.  
+
     <h40>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.  
−
</h4>
+
</h40>
 
</div>
 
</div>
 
</div>
 
</div>
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<div class = "row" id="constructs-intro">
 
<div class = "row" id="constructs-intro">
 
<div class="para_container">
 
<div class="para_container">
−
<h2>Our Constructs</h2>
+
<h20>Our Constructs</h20>
 
<div class = "divider2"></div>
 
<div class = "divider2"></div>
 
<br>
 
<br>
−
<h4>We designed our expression constructs in order to meet these goals. Click on each element of the construct to learn more about <b>why</b> we chose them:</h4>
+
<h40>We designed our expression constructs in order to meet these goals. Click on each element of the construct to learn more about <b>why</b> we chose them:</h40>
 
</div>
 
</div>
 
</div>
 
</div>
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<div class="construct_1_caption">
 
<div class="construct_1_caption">
−
<h5> Winsulin secreted by B. subtilis </h5>
+
<h50> Winsulin secreted by B. subtilis </h50>
 
</div>
 
</div>
 
<div class="construct_1">
 
<div class="construct_1">
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<div class="filler"></div>
 
<div class="filler"></div>
 
<div class="construct_2_caption">
 
<div class="construct_2_caption">
−
<h5> Proinsulin secreted by B. subtilis </h5>
+
<h50> Proinsulin secreted by B. subtilis </h50>
 
</div>
 
</div>
 
<div class="construct_2">
 
<div class="construct_2">
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<div class="filler"></div>
 
<div class="filler"></div>
 
<div class="construct_3_caption">
 
<div class="construct_3_caption">
−
<h5> Winsulin targeted to the periplasm of E. coli </h5>
+
<h50> Winsulin targeted to the periplasm of E. coli </h50>
 
</div>
 
</div>
 
<div class="construct_3">
 
<div class="construct_3">
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<div class="filler"></div>
 
<div class="filler"></div>
 
<div class="construct_4_caption">
 
<div class="construct_4_caption">
−
<h5> Proinsulin targeted to the periplasm of E. coli </h5>
+
<h50> Proinsulin targeted to the periplasm of E. coli </h50>
 
</div>
 
</div>
 
<div class="construct_4">
 
<div class="construct_4">
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<div class="filler"></div>
 
<div class="filler"></div>
 
<div class="construct_5_caption">
 
<div class="construct_5_caption">
−
<h5> Winsulin targeted to the cytoplasm of E. coli </h5>
+
<h50> Winsulin targeted to the cytoplasm of E. coli </h50>
 
</div>
 
</div>
 
<div class="construct_5">
 
<div class="construct_5">
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<div class="filler"></div>
 
<div class="filler"></div>
 
<div class="construct_6_caption">
 
<div class="construct_6_caption">
−
<h5> Proinsulin targeted to the cytoplasm of E. coli </h5>
+
<h50> Proinsulin targeted to the cytoplasm of E. coli </h50>
 
</div>
 
</div>
 
<div class="construct_6">
 
<div class="construct_6">
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<div class="element_info_box_container">
 
<div class="element_info_box_container">
 
<div class="info_box BBprefix_info">
 
<div class="info_box BBprefix_info">
−
<h6>iGEM BioBrick Prefix</h6>
+
<h60>iGEM BioBrick Prefix</h60>
 
<br>
 
<br>
−
<h4>Contains the restriction sites that are necessary for BioBrick compatibility including EcoRI, NotI & XbaI.</h4>
+
<h40>Contains the restriction sites that are necessary for BioBrick compatibility including EcoRI, NotI & XbaI.</h40>
 
</div>
 
</div>
 
<div class="info_box RBS_info">
 
<div class="info_box RBS_info">
−
<h6>E. coli Extended Ribosome Binding Site</h6>
+
<h60>E. coli Extended Ribosome Binding Site</h60>
 
<br>
 
<br>
−
<h4>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.
+
<h40>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.
−
</h4>
+
</h40>
 
</div>
 
</div>
 
<div class="info_box YNCM_info">
 
<div class="info_box YNCM_info">
−
<h6>YncM Tag</h6>
+
<h60>YncM Tag</h60>
 
<br>
 
<br>
−
<h4>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)</h4>
+
<h40>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)</h40>
  
 
</div>
 
</div>
 
<div class="info_box HIS_info">
 
<div class="info_box HIS_info">
−
<h6>His Tag</h6>
+
<h60>His Tag</h60>
 
<br>
 
<br>
−
<h4>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.</h4>
+
<h40>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.</h40>
  
 
</div>
 
</div>
 
<div class="info_box TEV_info">
 
<div class="info_box TEV_info">
−
<h6>TEV Protease Cleavage Site</h6>
+
<h60>TEV Protease Cleavage Site</h60>
 
<br>
 
<br>
−
<h4>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.</h4>
+
<h40>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.</h40>
  
 
</div>
 
</div>
 
<div class="info_box ARG_info">
 
<div class="info_box ARG_info">
−
<h6>“R” Arginine Cleavage Site</h6>
+
<h60>“R” Arginine Cleavage Site</h60>
 
<br>
 
<br>
−
<h4>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. </h4>
+
<h40>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. </h40>
  
 
</div>
 
</div>
 
<div class="info_box BBsuffix_info">
 
<div class="info_box BBsuffix_info">
−
<h6>iGEM BioBrick Suffix</h6>
+
<h60>iGEM BioBrick Suffix</h60>
 
<br>
 
<br>
−
<h4>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.</h4>
+
<h40>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.</h40>
  
 
</div>
 
</div>
 
<div class="info_box ecotin_info">
 
<div class="info_box ecotin_info">
−
<h6>Ecotin Tag</h6>
+
<h60>Ecotin Tag</h60>
 
<br>
 
<br>
−
<h4>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.
+
<h40>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.
−
</h4>
+
</h40>
 
<ul>  
 
<ul>  
 
<li>Relatively low metabolic burden due to its small size
 
<li>Relatively low metabolic burden due to its small size

Revision as of 13:32, 30 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 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)