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Team:IIT Madras/Project
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==Promoter Design== | ==Promoter Design== | ||
{|align=center width=80% | {|align=center width=80% | ||
| - | |The promoter sequences of a number of genes regulated by the different sigmas were compiled. Their mode of regulation was also looked into. Apart from being just selected for transcription by a | + | |The promoter sequences of a number of genes regulated by the different sigmas were compiled. Their mode of regulation was also looked into. Apart from being just selected for transcription by a &sigma factor, most of the genes are ''trans'' regulated with both repressors and activators. This poses an additional complexity in the isolation of a promoter segment as ''cis'' regulatory elements need to be incorporated. |
To resolve this, we looked into the kind of structural requirements for &sigma factor + RNA Polymerase to bind DNA. Activation is key for expression from genes under &sigma<sup>54</sup>. For the others, the pattern of RNA Polymerase binding is similar to that of &sigma<sup>70</sup>, which involves base specific interaction only in the -10 and -35 promoter region. | To resolve this, we looked into the kind of structural requirements for &sigma factor + RNA Polymerase to bind DNA. Activation is key for expression from genes under &sigma<sup>54</sup>. For the others, the pattern of RNA Polymerase binding is similar to that of &sigma<sup>70</sup>, which involves base specific interaction only in the -10 and -35 promoter region. | ||
!valign=top|[[Image:IITMpromoters.jpg|thumb|Design of promoters as a fusion between a lac and sigma dependent promoter]] | !valign=top|[[Image:IITMpromoters.jpg|thumb|Design of promoters as a fusion between a lac and sigma dependent promoter]] | ||
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Revision as of 06:09, 15 September 2008
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Introduction
After months of brainstorming and evaluating a large number of other ideas, we defined our problem statement as follows,
Is it possible to make bacteria respond to physical changes in a customizable way? We analyze the question piecewise. First, how do bacteria adapt to physical change in their environment. These physical parameters typically comprise of temperature, pH, osmolarity. An additional environmental stress that bacteria face is that of nutrient starvation. These stresses pose a challenge to the organisms survival. The starting point for the idea was when we came to know of temperature rise being entangled with DnaK levels, termed as a heat shock. How is the bacteria able to selectively switch on expression of a small subset of proteins based on a change in temperature? Based on recent literature reports, it's learnt that sigma 32, a subunit of the RNA Polymerase Holoenzyme twists into a functional conformation only after a rise in temperature. This sigma subunit confers promoter sequence based selectivity to RNA Polymerase. To deal with different environmental niches, bacteria employ a family of such sigma factors to express different sets of proteins to adapt to these conditions. Usually, the number of these sigma factors vary from one bacterial species to another, with the more exotic ones having 50+ kinds of sigma subunits. E.Coli has a family of 7 sigma subunits to partition it's genome into various programs. This division is as follows,
We used the database ecocyc.org to survey the genes being controlled by these different sigmas and how exactly are they induced. Identifying and isolating the promoters of these genes would be the next step. A library of promoters which would be expressed by these different sigmas would make for novel BioBricks. These new promoters would cover the different environmental stresses that E.Coli respond to. Being designed within the specifications set out by the parts registry, it would be extensible with all the existing devices. A point to appreciate is that these promoters, unlike pLac, are expressed without any external chemical for induction and yet are not constitutive. This sets it apart in a league of environmental context specific promoters without the need for external inducer. |  |
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Promoter Design
| The promoter sequences of a number of genes regulated by the different sigmas were compiled. Their mode of regulation was also looked into. Apart from being just selected for transcription by a &sigma factor, most of the genes are trans regulated with both repressors and activators. This poses an additional complexity in the isolation of a promoter segment as cis regulatory elements need to be incorporated.
To resolve this, we looked into the kind of structural requirements for &sigma factor + RNA Polymerase to bind DNA. Activation is key for expression from genes under &sigma54. For the others, the pattern of RNA Polymerase binding is similar to that of &sigma70, which involves base specific interaction only in the -10 and -35 promoter region. | |
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