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

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<center><h4>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 <i>E. coli</i>, and the other will be in <i>Bacillus subtilis</i></h4></center>
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<center><h4>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 <i>E. coli</i>, and the other will be in <i>Bacillus subtilis.</i></h4></center>
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<center><h4><i>E. coli</i> Cytoplasmic Expression</h4></center>
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<center><h4><i>E. coli</i> Periplasmic Expression</h4></center>
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<center><h4>Insulin accumulates in the cytoplasm under normal conditions within inclusion bodies, however will require downstream refolding in several chemical processes.
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<center><h4>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. </h4></center>
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<center><h4><i>B. subtilis</i> Excretory Expression</h4></center>
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<center><h4>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 <i>E coli</i> systems, and could potentially be optimised into a feb-batch system by Biofoundry and/or Open Insulin.
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Revision as of 04:20, 9 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

Which is worse, that everyone has his price, or that the price is always so low.

Single Chained

I'm killing time while I wait for life to shower me with meaning and happiness.

Affordable

I'm killing time while I wait for life to shower me with meaning and happiness.

Intellectual Property Issues

I'm killing time while I wait for life to shower me with meaning and happiness.

Safety and Efficacy

Which is worse, that everyone has his price, or that the price is always so low.

Our Constructs:

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 feb-batch system by Biofoundry and/or Open Insulin.