Team:IIT Madras/Project

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<h2>Project Motivation</h2>
<h2>Project Motivation</h2>
On average, the world loses 2 people to cardiovascular complications every minute; Diabetes claims 4 times that number making it the 6th most common cause of death worldwide. India alone contributes 1 in every 25 of the diabetes cases every year. The World Health Organisation recently reported that up to 245 million people suffer from diabetes worldwide. It is estimated that diabetes consumes between 5 to 10% of the world's health care expenditure, and this is expected to go up to 12% in the next 20 years. There is, however, no known cure to this epidemic, and our only hope is to fall back to the old adage, 'Prevention is better than cure'. It has been known for a while that reducing the calories consumed in the form of sugar will help in the prevention of this disease, and is an essential part of living-with the disease. To date, seven different sweetening proteins have been identified and approved for consumption by the FDA. However, these proteins face several issues in production, separation and purification, making them an ideal candidate for further work, particularly focusing on expression and regulation.
On average, the world loses 2 people to cardiovascular complications every minute; Diabetes claims 4 times that number making it the 6th most common cause of death worldwide. India alone contributes 1 in every 25 of the diabetes cases every year. The World Health Organisation recently reported that up to 245 million people suffer from diabetes worldwide. It is estimated that diabetes consumes between 5 to 10% of the world's health care expenditure, and this is expected to go up to 12% in the next 20 years. There is, however, no known cure to this epidemic, and our only hope is to fall back to the old adage, 'Prevention is better than cure'. It has been known for a while that reducing the calories consumed in the form of sugar will help in the prevention of this disease, and is an essential part of living-with the disease. To date, seven different sweetening proteins have been identified and approved for consumption by the FDA. However, these proteins face several issues in production, separation and purification, making them an ideal candidate for further work, particularly focusing on expression and regulation.
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<h2>Ideation</h2>
<h2>Ideation</h2>
Our idea went through many levels of modification and rehashing before we were confident enough to state it in its entirety. Here is a brief summary of the entire process, one that took us a significant portion of two weeks.<br>
Our idea went through many levels of modification and rehashing before we were confident enough to state it in its entirety. Here is a brief summary of the entire process, one that took us a significant portion of two weeks.<br>

Revision as of 23:01, 27 October 2010

Click hiding> People

This is where the people stuff comes.

Contents

Project Details

Part Design

PCR Products

Legend
  • This color represents the portion to be added by Extension PCR / Inverse PCR / Assembly PCR.
  • This color represents sites that lie in the middle of the parts to be submitted. These are placed so that the promoters and the inserts can be switched out.
  • This represents an apparent restriction site, one that cannot be used due to the sequences' presence in an ORF
SCM
Gene of 295 bp to be synthesised by Assembly PCR / LCR.
Block 1
  1. Kpn1 | P170 | RBS | HindIII
  2. Kpn1 | NICE | RBS | HindIII
  3. HindIII| GFP | Terminator | BamH1
  4. Kpn1| Constitutive | RBS | GFP | Terminator | BamH1
Block 2
  1. Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1 | GFP | Terminator | BamH1 (by Inverse PCR) // Can't design a Primer this big.
  2. Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1
  3. Nhe1 | GFP | Terminator | BamH1 (Extension PCR from 1.4)
Block 3
  1. Kpn1 | Constitutive | RBS | HindIII
  2. HindIII | CRE-NLS | Terminator | Sac1
  3. Sac1 | Constitutive | RBS | loxP | Nhe1 | GFP | Terminator | loxP | Xho1 (by Inverse PCR + Extension PCR)
  4. Xho1 | RFP | Terminator | BamH1
Final Design
  1. Kpn1 | NICE | RBS | HindIII (as made in 1.1)
  2. HindIII | CRE-NLS | Terminator | Sac1 (as made in 3.2)
  3. Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 (by Inverse PCR + Extension PCR)
  4. Xho1 | SCM | Nhe1 | loxP | Terminator | BamH1
Part Submission
  1. EcoR1 | Not1 | Xba1 | NICE | RBS | Spe1 | Not1 | Pst1
  2. EcoR1 | Not1 | Xba1 | P170 | RBS | Spe1 | Not1 | Pst1
  3. EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1 | GFP | Terminator | BamH1 | Spe1 | Not1 | Pst1
  4. EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | CRE | Terminator | Sac1 | Constitutive | RBS | loxP | Sma1 | GFP | Terminator | loxP | Xho1 | RFP | Terminator | BamH1 | Spe1 | Not1 | Xba1
  5. EcoR1 | Not1 | Xba1 | Kpn1 | NICE | RBS | HindIII | CRE-NLS | Terminator | Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 | SCM | BamH1 | loxP | Terminator | Spe1 | Not1 | Pst1

Proof of Concept

Block 1 - To characterize promoters against a standard
  • P170 | GFP | Terminator
  • NICE | GFP | Terminator
  • Constitutive | GFP | Terminator
Block 2 - Test of export efficiency
  • Constitutive | GFP | Terminator
  • Constitutive | SP310mut2 | GFP | Terminator
Block 3 -To test CRE-loxP system
  • Constitutive | RBS | CRE | Terminator | Constitutive | RBS | loxP | GFP | Terminator | loxP | RFP | Terminator
Acid Tolerance Promoter (P170) | CRE | Terminator | loxP | Consitutive | GFP | Terminator | loxP | RFP | Terminator

Final design

NICE | CRE | Terminator | loxP | Acid Tolerance Inducer (P170) | Export tag | SCM | loxP | Terminator

The Experiments

Part 3

Results

 

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