Team:IIT Madras

From 2008.igem.org

(Difference between revisions)
(→Overview)
 
(One intermediate revision not shown)
Line 9: Line 9:
-
=Overview=
+
=StressKit=
{|align=center width=80%
{|align=center width=80%
 +
|rowspan=2|''A BioBrick library of Lac-repressed &sigma;<sup>24</sup>, &sigma;<sup>28</sup>, &sigma;<sup>32</sup> and &sigma;<sup>38</sup> promoters for Escherichia coli''
-
|rowspan=2| ''Engineering Biology''. The next quantum leap in understanding biological systems will be driven by attempts to engineer living organisms. This effort brings with it it's own fresh set of challenges and problems. iGem serves as the perfect testbed for these class of problems, often termed as synthetic biology.
+
Regulated gene expression is an essential part of the synthetic biologist's toolkit. Bacteria have evolved 'generalized stress response systems' which generate genome-wide changes in gene expression in response to globally-integrated information. Specific types of stress upregulate specific 'alternative sigma factors', which activate transcription by binding to nucleotide signatures at the -10 and -35 boxes of their cognate promoters. We set out to design, construct, and validate a library of sigma-dependent promoters for Escherichia coli, with the following design specifications: the promoters must conform to the BioBrick standard; they must be modular so they can be used multiply in devices; and they must be LacI repressed but sigma-dependent, off by default but behaving like native sigma-dependent promoters in the presence of IPTG. All our promoters are based on the LacO promoter of Lutz and Bujard, containing two LacI binding sites, but with -10 and -35 boxes modified to bind alternative sigma factors. We generated four hybrid promoters for each of the following:
 +
* &sigma;<sup>24</sup>: unfolded-protein response
 +
* &sigma;<sup>28</sup> flagellar biosynthesis
 +
* &sigma;<sup>32</sup> heat-shock response
 +
* &sigma;<sup>38</sup> stationary-phase expression
-
After a few months of brainstorming, team IIT Madras puts forward this question, ''Is it possible to make bacteria respond to physical changes in a customized way?''
+
We then cloned these promoters upstream of a YFP expression construct (BBa_E0430). We are currently characterizing the library of promoters against a standard control, the unmodified Lutz-Bujard LacO promoter, using spectrophotometry and fluorescence microscopy.
-
First, ''...physical changes''. Why do we stress on physical changes? For the most part, we've seen synthetic circuits designed to be chemically induced via a limited bag of reagents. These chemical induction techniques form the basis for almost all biological engineering but we aim to expand the horizon by bringing in an almost untapped method of induction, ''physical changes''. This change would imply a departure from the ideal environmental conditions for a bacteria. In microbiology, this sort of altered environment is defined as a ''stress''. So in effect, our physical changes are equivalent to subjecting bacteria to ''stress''.
+
Take a look into our detailed design documents to know more about the project, the [[Team:IIT_Madras/Project|''StressKit'']].
-
Second, ''...respond in a customizable way''. We should be able to tap into this response that the bacteria may produce. In the context of genetic engineering, this implies the ability to express a gene based on these stresses as input signals. This entry-point into the response circuits of bacteria open a crucial window into the nature of environmental adaptation and allow us to launch our own genetic programs as appropriate.
+
Browse through the [[Team:IIT_Madras/Notebook|experiments notebook]] to read our experimental data.  
-
Having an environmentally switchable response can take genetic circuits to a much more intelligent and smart design stage where we don't need to externally monitor and induce genes based on our judgement/measurements of the entire ensemble's properties. This lets the genes to ''take care of themselves'' in a certain sense. They switch on and off on their own when appropriate. A smart response. Neither constitutive nor blindly induction based.
+
To know more about the team, IIT Madras and who exactly we are,[[Team:IIT_Madras/Team|click here]]!
-
 
+
-
With the problem statement defined as above, the team of 6 undergraduate students set out to provide a solution. The additional constraints to the problem were that to be conformant with the BioBrick standards and to maximize the regulation of such designed constructs to be of maximum utility to the end user.
+
-
 
+
-
Take a look into our detailed design documents to know more about the project, the bacterial [[Team:IIT_Madras/Project|''StressKit'']].
+
-
 
+
-
How successful was the approach? Browse through the [[Team:IIT_Madras/Notebook|experiments notebook]] to find out.
+
-
 
+
-
To know more about IIT Madras and who exactly we are,[[Team:IIT_Madras/Team|click here]]!
+
!align=right valign=top | [[Image:IITitbiotech.jpg|left|thumb|''Dept. of Biotechnology<br>IIT Madras]]
!align=right valign=top | [[Image:IITitbiotech.jpg|left|thumb|''Dept. of Biotechnology<br>IIT Madras]]

Latest revision as of 13:55, 29 September 2008

IITMstresskit.png


Home About Us Project Details Notebook


StressKit

A BioBrick library of Lac-repressed σ24, σ28, σ32 and σ38 promoters for Escherichia coli

Regulated gene expression is an essential part of the synthetic biologist's toolkit. Bacteria have evolved 'generalized stress response systems' which generate genome-wide changes in gene expression in response to globally-integrated information. Specific types of stress upregulate specific 'alternative sigma factors', which activate transcription by binding to nucleotide signatures at the -10 and -35 boxes of their cognate promoters. We set out to design, construct, and validate a library of sigma-dependent promoters for Escherichia coli, with the following design specifications: the promoters must conform to the BioBrick standard; they must be modular so they can be used multiply in devices; and they must be LacI repressed but sigma-dependent, off by default but behaving like native sigma-dependent promoters in the presence of IPTG. All our promoters are based on the LacO promoter of Lutz and Bujard, containing two LacI binding sites, but with -10 and -35 boxes modified to bind alternative sigma factors. We generated four hybrid promoters for each of the following:

  • σ24: unfolded-protein response
  • σ28 flagellar biosynthesis
  • σ32 heat-shock response
  • σ38 stationary-phase expression

We then cloned these promoters upstream of a YFP expression construct (BBa_E0430). We are currently characterizing the library of promoters against a standard control, the unmodified Lutz-Bujard LacO promoter, using spectrophotometry and fluorescence microscopy.

Take a look into our detailed design documents to know more about the project, the StressKit.

Browse through the experiments notebook to read our experimental data.

To know more about the team, IIT Madras and who exactly we are,click here!

Dept. of Biotechnology
IIT Madras
Retrieved from "http://2008.igem.org/Team:IIT_Madras"