Team:Imperial College London/Chemoinduction

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Chemoinduction feedback from todays session. Please delete as completed.<br>
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1) Add a few lines on the growth - why do we do this?
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2) Make it clear that the LacI promoter is different from ours - stick it under a Learn More tab.<br>
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<a href="https://static.igem.org/mediawiki/2009/5/5c/II09_MapIndicator_Chemoinduction.png" class="highslide" onclick="return hs.expand(this, config1)" title="When the population of cells increases to a sufficient density, module 1 is induced by IPTG addition"><img src="https://static.igem.org/mediawiki/2009/5/5c/II09_MapIndicator_Chemoinduction.png" alt="" title="Click to enlarge" width="75%"/></a>
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3) Genetic circuit needs to be standardised - see other modules.<br>
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4) The modeling does not correspond to the top - we won't have data to back this up. The 2 graphs are not the same. How do we sort this?<br>
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5) Write a description about the experiment and how we plan to get our results.<br>
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6) Matthieu calculated protein production in an excel file - PUT THIS IN! Table format with analysis. Nuri has this file...<br>
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7) Have a rationale section.<br>
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8) Add what teams can reuse from this module.<br>
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9) Have a conclusion of the page at the end - couple of lines.<br>
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=<font size='5'><b>Module Integration - Chemoinduction</b></font>=
=<font size='5'><b>Module Integration - Chemoinduction</b></font>=
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Module 2: Encapsulation
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Module 2 Timeline
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<br>
 
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=Chemoinduction=
=Chemoinduction=
==Overview==
==Overview==
[[Image:II09_NoIPTG_yesIPTG.jpg|350px|right]]
[[Image:II09_NoIPTG_yesIPTG.jpg|350px|right]]
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<b>Our system is 'kick started' by the addition of a compound, IPTG, which activates Module 1 of our system. This is achieved by putting the genes for Module 1 under control of a chemically inducible genetic component called a promoter.</b>
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Our system is 'kick started' by the addition of a compound, IPTG, which activates protein production (module 1) of our system. This is achieved by putting the genes for protein production (module 1) under control of a chemically inducible genetic part called a promoter.
==Rationale==
==Rationale==
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<b>In designing our system, we chose to use an inducible (switchable) promoter rather than a constitutive (always ON) promoter. This is to allow the cells to grow to a certain cell density before forcing them to produce protein. The production of protein can be a burden on the cell's resources, especially at the early stages of cell growth. Therefore, the culture should be allowed to grow to a certain density before the production of protein is induced. </b><br><br>
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In designing our system, we chose to use an inducible (switchable) promoter rather than a constitutive (always ON) promoter. This is to allow the cells to grow to a certain cell density before forcing them to produce protein. The production of protein can be a burden on the cell's resources, especially at the early stages of cell growth. Therefore, the culture should only be induced by the addition of IPTG once it has reached a critical cell density.<br><br>
<html><a href="https://2009.igem.org/Team:Imperial_College_London/Chemoinduction/Rationale"><img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html>&nbsp; <br><b><i> About our reasons for choosing chemical induction</i></b>
<html><a href="https://2009.igem.org/Team:Imperial_College_London/Chemoinduction/Rationale"><img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html>&nbsp; <br><b><i> About our reasons for choosing chemical induction</i></b>
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===The Lac Operon===
===The Lac Operon===
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The Lac Operon is a good example of a genetic operon (several genes under control of one promoter) that has been widely studied in the field of Genetics. <br>
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The Lac Operon is a prime example of a genetic operon (several genes under control of one promoter), and has been widely studied in the field of genetics. <br>
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The Lac Operon is a set of genes found in Escherichia Coli that control lactose metabolism in times when preferable sugars (ie glucose) are scarce. When glucose levels are high, the <i>E.coli</i> preferentially use this sugar. However, when glucose is not available, <i>E.coli</i> has a set of genes coding for the enzymes responsible for the metabolism of lactose. <br><br>
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We are making use of the same promoter as that found in the lac operon to control the activation of <b>Module 1</b><br><br>
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The Lac Operon is a set of genes found in <i>E. Coli</i> that control lactose metabolism in times when preferable sugars (ie. glucose) are scarce. When glucose levels are high, the <i>E.coli</i> feed on the glucose. However, when glucose is not available, <i>E.coli</i> has a set of genes coding for the enzymes responsible for the metabolism of the secondary carbon source, in this case, lactose. <br>
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In order to control the activation of <b>Module 1</b>, the team is using the same promoter as that found in the lac operon.<br><br>
<html><a href="https://2009.igem.org/Team:Imperial_College_London/Chemoinduction/LacOperon"><img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html>&nbsp; <b><i>About the lac operon</i></b>
<html><a href="https://2009.igem.org/Team:Imperial_College_London/Chemoinduction/LacOperon"><img style="vertical-align:bottom;" width=50px align="left" src="http://i691.photobucket.com/albums/vv271/dk806/II09Learnmore.png"></a></html>&nbsp; <b><i>About the lac operon</i></b>
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===Genetic Circuit===
 
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<b><i> These genetic circuits are the designs upon which the functionality of The E.ncapsulator is based. We will go through the genetic design of the system, module by module. If you are unfamiliar with the symbols used, then check out our [[Team:Imperial_College_London/Genetic_Circuit_Key |key]] to familiarise yourself with each circuit symbol and what it means. </b>
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===Genetic Circuit===
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These genetic circuits are the designs upon which the functionality of The E.ncapsulator is based. We will go through the genetic design of the system, module by module. If you are unfamiliar with the symbols used, then check out our [[Team:Imperial_College_London/Genetic_Circuit_Key |key]] to familiarise yourself with each circuit symbol and what it means.
<br><br>
<br><br>
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This diagram makes up part of the [[Team:Imperial_College_London/M1/Genetic |Module 1 genetic circuit]]</i>
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This diagram makes up part of the [[Team:Imperial_College_London/M1/Genetic |Module 1 genetic circuit]]
[[Image:II09_M1_Promoter.png|left|400px]]  
[[Image:II09_M1_Promoter.png|left|400px]]  
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==Results==
==Results==
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===Wetlab===
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===Wet Lab===
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[[Image:II09_IPTG_growth1.jpg|right|400px]]<br>
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Different concentrations of IPTG were added to a 96-well plate [link to assay] of e-coli Top 10 cells containing LacI-RFP genetic constructs. Here we measured the optical density of the cells over-time, for different concentrations of IPTG to monitor the effects in the culture. The IPTG growth curve results are plotted in the figure (ADD A FIGURE CAPTION TO IT: FIG1, FIG2 ETC).
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By visual assessment,the growth rate of the culture prior the saturation phase stays within a narrow range and does not change significantly with concentrations of [IPTG]. Therefore, we can conclude that [IPTG] in the concentration ranges we are using does not affect cellular growth and is non toxic. The effects of IPTG have been widely studied [SAY THAT WE ARE CONFIRMING SOMETHING WELL KNOWN ANYWAY BY OTHERS]<br>
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===Modelling===
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Different experiments were run using the chemoinduction module.
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Taking into account the indications from the wetlab, we can then draw a relationship between different input IPTG concentrations and the amount of polypeptide produced.  
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[[Image:II09_IPTG_growth1.jpg|right|300px]]
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Firstly, we did a preliminary test on the toxicity of IPTG, and whether this affected cell growth. We grew our cells in varying concentrations of IPTG and monitored the growth by measuring optical density.<br> IPTG, as expected, does not have a negative effect on cell growth.
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This provided us with a good starting point for our experiment. We then ligated the IPTG induced promoter to RFP, and tried to investigate the effects of IPTG on red flourescence. Unfortunately, we had problems with getting this construct to function, and we are still troubleshooting as of the wiki-freeze deadline.
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===Dry Lab===
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Taking into account the experimental data from the wet lab, we can then draw a relationship between different input IPTG concentrations and the amount of polypeptide produced.  
*When we introduce IPTG into the system, it temporarily removes LacI from the system. Hence, during this period of time, we produce the drug of interest.  
*When we introduce IPTG into the system, it temporarily removes LacI from the system. Hence, during this period of time, we produce the drug of interest.  
*When the effects of IPTG wear off, the system returns to equilibrium.  
*When the effects of IPTG wear off, the system returns to equilibrium.  
*The more IPTG we add in, the higher the amount of output protein.  
*The more IPTG we add in, the higher the amount of output protein.  
[[Image:II09_SIm_main_prot.jpg]]<br>
[[Image:II09_SIm_main_prot.jpg]]<br>
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DAVE:
 
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FROM THE BOTTOM ONWARDS WE MAY NEED TO SHORTEN OR ADD SOME STUFF TO LEARN MORE TABS.IVE COMMENTED IT OUT, NOT DELETED IT. ALSO ADD LEARN MORE TABS FOR WETLAB AND DRY LAB LIKE IN M3 OVERVIEW!
 
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==Conclusion==
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Chemoinduction is the most widely used means of inducing a promoter. By using this well characterised IPTG inducible promoter as the starting point for our system, we give the user full control as to when to kick-start the system.
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{{Imperial/09/Division}}
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===Project Tour===
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/Project_Overview"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/ProjectOverviewL.jpg"></a><a href="https://2009.igem.org/Team:Imperial_College_London/M1"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/Module1R.jpg"></a></center>
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===Temporal Control Contents===
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===Temporal Control: Chemoinduction===
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Chemical_Induction"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_chemicalinduction.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Autoinduction"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Drylabmainimage1.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Thermoinduction"><img style="vertical-align:bottom;" width="20%" src="http://i691.photobucket.com/albums/vv271/dk806/II09_Thermoinduction1.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Temporal_Control"><img style="vertical-align:bottom;"width="20%"src="http://i691.photobucket.com/albums/vv271/dk806/II09_Wetlabmainimage9.png"></a><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab/Autoinduction"><img style="vertical-align:bottom;" width="20%"  src="http://i691.photobucket.com/albums/vv271/dk806/II09_Drylabmainimage6.png"></a></center></html>
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<html><center><a href="https://2009.igem.org/Team:Imperial_College_London/Project_Overview"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/ProjectOverviewL.jpg"></a><a href="https://2009.igem.org/Team:Imperial_College_London/M1"><img width=150px src="http://i691.photobucket.com/albums/vv271/dk806/Module1R.jpg"></a></center>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Chemical_Induction"><b>Chemoinduction</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Autoinduction"><b>Autoinduction</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Temporal_Control/Thermoinduction"><b>Thermoinduction</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Wetlab/Results#Temporal_Control"><b>Wet Lab</b></a></center></td>
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<td width="20%"><center><a href="https://2009.igem.org/Team:Imperial_College_London/Drylab/Autoinduction"><b>Modelling</b></a></center></td></table></html>
{{Imperial/09/TemplateBottom}}
{{Imperial/09/TemplateBottom}}

Latest revision as of 03:48, 22 October 2009

Contents

Module Integration - Chemoinduction

Chemoinduction

Overview

II09 NoIPTG yesIPTG.jpg

Our system is 'kick started' by the addition of a compound, IPTG, which activates protein production (module 1) of our system. This is achieved by putting the genes for protein production (module 1) under control of a chemically inducible genetic part called a promoter.

Rationale

In designing our system, we chose to use an inducible (switchable) promoter rather than a constitutive (always ON) promoter. This is to allow the cells to grow to a certain cell density before forcing them to produce protein. The production of protein can be a burden on the cell's resources, especially at the early stages of cell growth. Therefore, the culture should only be induced by the addition of IPTG once it has reached a critical cell density.

 
About our reasons for choosing chemical induction

Theory

The Lac Operon

The Lac Operon is a prime example of a genetic operon (several genes under control of one promoter), and has been widely studied in the field of genetics.

The Lac Operon is a set of genes found in E. Coli that control lactose metabolism in times when preferable sugars (ie. glucose) are scarce. When glucose levels are high, the E.coli feed on the glucose. However, when glucose is not available, E.coli has a set of genes coding for the enzymes responsible for the metabolism of the secondary carbon source, in this case, lactose.

In order to control the activation of Module 1, the team is using the same promoter as that found in the lac operon.

  About the lac operon


Genetic Circuit

These genetic circuits are the designs upon which the functionality of The E.ncapsulator is based. We will go through the genetic design of the system, module by module. If you are unfamiliar with the symbols used, then check out our key to familiarise yourself with each circuit symbol and what it means.

This diagram makes up part of the Module 1 genetic circuit

II09 M1 Promoter.png


Under normal circumstances (without IPTG), the Lac inhibitor (LacI) is constitutively present in the system. This means that the promoter is repressed, and blocks the expression of the downstream genes. When IPTG is added, it binds to LacI and cancels the repression on the promoter. This allows for the expression of the downstream genes, and therefore for the start of Module 1.


Induction with IPTG

II09 IPTG.png

IPTG is an allolactose analogue. Allolactose is the lactose metabolite that triggers the transcription of the lactose operon in natural systems. Allolactose and IPTG activate transcription of the lac operon by binding to the LacI protein. When such binding occurs, the LacI protein changes confirmation, which renders it unable to bind to the operator region. This allows the RNA polymerase to continue reading beyond the operator and transcribe the downstream genes, thus removing the inhibition.

Results

Wet Lab

Different experiments were run using the chemoinduction module.

II09 IPTG growth1.jpg

Firstly, we did a preliminary test on the toxicity of IPTG, and whether this affected cell growth. We grew our cells in varying concentrations of IPTG and monitored the growth by measuring optical density.
IPTG, as expected, does not have a negative effect on cell growth.

This provided us with a good starting point for our experiment. We then ligated the IPTG induced promoter to RFP, and tried to investigate the effects of IPTG on red flourescence. Unfortunately, we had problems with getting this construct to function, and we are still troubleshooting as of the wiki-freeze deadline.

Dry Lab

Taking into account the experimental data from the wet lab, we can then draw a relationship between different input IPTG concentrations and the amount of polypeptide produced.

  • When we introduce IPTG into the system, it temporarily removes LacI from the system. Hence, during this period of time, we produce the drug of interest.
  • When the effects of IPTG wear off, the system returns to equilibrium.
  • The more IPTG we add in, the higher the amount of output protein.

II09 SIm main prot.jpg

Conclusion

Chemoinduction is the most widely used means of inducing a promoter. By using this well characterised IPTG inducible promoter as the starting point for our system, we give the user full control as to when to kick-start the system.

Project Tour



Temporal Control Contents

Chemoinduction
Autoinduction
Thermoinduction
Wet Lab
Modelling

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