Team:IIT Madras/Project

From 2010.igem.org

(Difference between revisions)
Line 39: Line 39:
:'''Legend'''
:'''Legend'''
-
:* <font style="color:red"> This color represents the portion to be added by Extension PCR / Inverse PCR / Assembly PCR.</font>
+
:* <font style="color:green"> This color represents the portion to be added by Extension PCR / Inverse PCR / Assembly PCR.</font>
-
:* <font style="color:blue"> 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.</font>
+
:* <font style="color:red"> 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.</font>
-
:* <font style="color:green">This represents an apparent restriction site, one that cannot be used due to the sequences' presence in an ORF</font>
+
:* <font style="color:blue">This represents an apparent restriction site, one that cannot be used due to the sequences' presence in an ORF</font>
</small>
</small>
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:'''Block 1'''
:'''Block 1'''
    
    
-
:# Kpn1 | P170 | RBS | HindIII
+
:# <font style="color:green">Kpn1</font> | P170 | <font style="color:green">RBS</font> | <font style="color:green">HindIII</font>
-
:# Kpn1 | NICE | RBS | HindIII
+
:# <font style="color:green">Kpn1</font> | NICE | <font style="color:green">RBS</font> | <font style="color:green">HindIII</font>
-
:# HindIII| GFP | Terminator | BamH1
+
:# <font style="color:green">HindIII</font>| GFP | Terminator | <font style="color:green">BamH1</font>
-
:# Kpn1| Constitutive | RBS | GFP | Terminator | BamH1
+
:# <font style="color:green">Kpn1</font>| Constitutive | RBS | GFP | Terminator | <font style="color:green">BamH1</font>
:'''Block 2'''
:'''Block 2'''
-
:# Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1 | GFP | Terminator | BamH1 (by Inverse PCR) // Can't design a Primer this big.
+
:# Kpn1 | Constitutive | RBS | <font style="color:green">HindIII</font> | <font style="color:green">SP310mut2</font> | <font style="color:green">Nhe1</font> | GFP | Terminator | BamH1 (by Inverse PCR) // Can't design a Primer this big.
-
:# Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1
+
:# <font style="color:green">Kpn1</font> | <font style="color:green">Constitutive</font> | <font style="color:green">RBS</font> | <font style="color:green">HindIII</font> | SP310mut2 | <font style="color:green">Nhe1</font>
-
:# Nhe1 | GFP | Terminator | BamH1 (Extension PCR from 1.4)
+
:# <font style="color:green">Nhe1</font> | GFP | Terminator | BamH1 (Extension PCR from 1.4)
:'''Block 3'''
:'''Block 3'''
-
:# Kpn1 | Constitutive | RBS | HindIII
+
:# <font style="color:green">Kpn1</font> | Constitutive | RBS | <font style="color:green">HindIII</font>
-
:# HindIII | CRE-NLS | Terminator | Sac1
+
:# <font style="color:green">HindIII</font> | CRE-NLS | <font style="color:green">Terminator</font> | <font style="color:green">Sac1</font>
-
:# Sac1 | Constitutive | RBS | loxP | Nhe1 | GFP | Terminator | loxP | Xho1 (by Inverse PCR + Extension PCR)
+
:# <font style="color:green">Sac1</font> | Constitutive | RBS | <font style="color:green">loxP</font> | <font style="color:green">Nhe1</font> | GFP | Terminator | <font style="color:green">loxP</font> | <font style="color:green">Xho1</font> (by Inverse PCR + Extension PCR)
-
:# Xho1 | RFP | Terminator | BamH1
+
:# <font style="color:green">Xho1</font> | RFP | Terminator | <font style="color:green">BamH1</font>
:'''Final Design'''
:'''Final Design'''
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:# Kpn1 | NICE | RBS | HindIII (as made in 1.1)
:# Kpn1 | NICE | RBS | HindIII (as made in 1.1)
:# HindIII | CRE-NLS | Terminator | Sac1 (as made in 3.2)
:# HindIII | CRE-NLS | Terminator | Sac1 (as made in 3.2)
-
:# Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 (by Inverse PCR + Extension PCR)
+
:# <font style="color:green">Sac1</font> | <font style="color:green">loxP</font> | P170 | <font style="color:green">RBS</font> | SP310mut2 | <font style="color:green">Xho1</font> (by Inverse PCR + Extension PCR)
-
:# Xho1 | SCM | Nhe1 | loxP | Terminator | BamH1
+
:# <font style="color:green">Xho1</font> | SCM | <font style="color:green">Nhe1</font> | <font style="color:green">loxP</font> | <font style="color:green">Terminator</font> | <font style="color:green">BamH1</font>
   
   
:'''Part Submission'''
:'''Part Submission'''
-
:# EcoR1 | Not1 | Xba1 | NICE | RBS | Spe1 | Not1 | Pst1
+
:# <font style="color:green">EcoR1 | Not1 | Xba1</font> | NICE | RBS | <font style="color:green">Spe1 | Not1 | Pst1</font>
-
:# EcoR1 | Not1 | Xba1 | P170 | RBS | Spe1 | Not1 | Pst1
+
:# <font style="color:green">EcoR1 | Not1 | Xba1</font> | P170 | RBS | <font style="color:green">Spe1 | Not1 | Pst1</font>
-
:# EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1 | GFP | Terminator | BamH1 | Spe1 | Not1 | Pst1
+
:# <font style="color:green">EcoR1 | Not1 | Xba1</font> | <font style="color:red">Kpn1</font> | Constitutive | RBS | <font style="color:red">HindIII</font> | SP310mut2 | <font style="color:red">Nhe1</font> | GFP | Terminator | <font style="color:red">BamH1</font> | <font style="color:green">Spe1 | Not1 | Pst1</font>
-
:# EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | CRE | Terminator | Sac1 | Constitutive | RBS | loxP | Sma1 | GFP | Terminator | loxP |       Xho1 | RFP | Terminator | BamH1 | Spe1 | Not1 | Xba1
+
:# <font style="color:green">EcoR1 | Not1 | Xba1</font> | <font style="color:red">Kpn1</font> | Constitutive | RBS | <font style="color:red">HindIII</font> | CRE | Terminator | <font style="color:red">Sac1</font> | Constitutive | RBS | loxP | <font style="color:red">Sma1</font> | GFP | Terminator | loxP | <font style="color:red">Xho1</font> | RFP | Terminator | <font style="color:red">BamH1</font> | <font style="color:green">Spe1 | Not1 | Xba1</font>
-
:# EcoR1 | Not1 | Xba1 | Kpn1 | NICE | RBS | HindIII | CRE-NLS | Terminator | Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 | SCM | BamH1 | loxP  | Terminator | Spe1 | Not1 | Pst1
+
:# <font style="color:green">EcoR1</font> | <font style="color:blue">Not1</font> | <font style="color:green">Xba1</font> | <font style="color:red">Kpn1</font> | NICE | RBS | <font style="color:red">HindIII</font> | CRE-NLS | Terminator | <font style="color:red">Sac1</font> | loxP | P170 | RBS | SP310mut2 | <font style="color:red">Xho1</font> | SCM | <font style="color:red">BamH1</font> | loxP  | Terminator | <font style="color:green">Spe1</font> | <font style="color:blue">Not1</font> | <font style="color:green">Pst1</font>
|}
|}
====Proof of Concept ====
====Proof of Concept ====
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=== Final design ===
=== Final design ===
{|
{|
-
<pre style="color:red">NICE | CRE | Terminator | loxP | Acid Tolerance Inducer (P170) | Export tag | SCM | loxP | Terminator</pre>
+
<big><big><pre style="color:red">NICE | CRE | Terminator | loxP | Acid Tolerance Inducer (P170) | Export tag | SCM | loxP | Terminator</pre></big></big>
   
   

Revision as of 10:01, 28 September 2010


Home Team Project Parts Submitted to the Registry Modeling Notebook Safety Gallery


Contents

Project Motivation

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.
We aim to use synthetic biology to engineer pro-biotic lactic acid bacteria used in the production of dairy products like yogurt, buttermilk and curds, to produce Monellin, a heat and pH stable sweetening protein. If we are successful in engineering Lactococcus lactis, a Gram positive bacteria to express and secrete Monellin, we will be able to produce dairy products low in poly-saccharide-based sweeteners, radically reducing the calorific content of these products. In order to be able to control the level of expression in this system, we plan to develop a regulatory system(s) that can be tweaked to suit varied purposes.

Project Abstract

We aim to use synthetic biology to engineer pro-biotic lactic acid bacteria to produce Monellin, a heat and pH stable sweetening protein. If we are successful in engineering Lactobacillus lactis, a Gram positive bacteria to express and secrete Monellin, we will be able to produce dairy products low in poly-saccharide-based sweeteners, radically reducing the calorific content of these products. In order to be able to control the level of expression in this system, we plan to develop a regulatory system(s) that simulates a logical AND gate in response to two bioloical inputs. We plan to use the decreasing pH during curdling and the addition of nicin as the two inputs. To achieve the AND gate we will be using the CRE gene in combination with loxP sites. By placing the loxP sites appropriately, we will create an expression system that will produce the Monellin in a window of conditions.

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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