Team:IIT Madras/Project

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Stress

Living organisms, like E.Coli, adapt and gear themselves to environmental changes. They are in the constant process of modifying their proteome based on the changing requirements. An interesting question arises as to how exactly does a bacteria gain awareness of its environment and what mechanisms do they use to deal with it?

The first step of gene expression is transcription, carried out by a protein called RNA Polymerase. This multi-subunit complex confers multiple particularities to the process of transcription allowing it to carry out different functional roles. One of the subunits, the sigma subunit, lends sequence specificity to the complex for recognizing relevant parts of DNA, called promoter regions. Only on the successful binding of this sigma subunit with a compatible promoter sequence does the process of transcription initiation occur.

Bacteria make use of multiple sigma subunits to partition its genome into different programs. E.Coli, for example, organizes its genome using 6 sigma factors which help the organism survive in different environmental niches. It's also observed that organisms which exhibit a greater diversity in their living conditions make use of a higher number of sigma subunits. This would provide for an adaptive advantage in settling into tougher and tougher niches.

The different sigma subunits present in E.Coli are as follows,

σ^70/D (rpoD):housekeeping genes during exponential growth
σ^54/N (rpoN):nitrogen starvation response
σ^38/S (rpoS):master regulator for a generalized stress response
σ^32/H (rpoH):unfolded protein response
σ^28/F (rpoF):flagellar biosynthesis
σ^24/E (rpoE):response to unfolded proteins in the cell envelope

Chosen Genes

Design of Promoters

Our design strategy for the promoters took into consideration the regulatory diversity of the promoters, employing the use of distal, proximal and trans regulatory elements. Also, an important point to consider is that these promoters are in an 'always-on' state by default. They are constitutively expressed subject to a few environmental conditions.

We overcome these constraints by experimenting with a new style of 'synthetic promoter' where we fuse sigma factor recognition sequences into a lac promoter. This takes away the 'always-on' issue with our promoters and also places them under the convenient regulation of IPTG. These hybrid/engineered promoters were designed in a way to preserve as much of the consensus sequence while maintaining the lac repressor binding sites.

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-== '''Overall project''' ==
-=== Background ===
+===Stress===
-Our project looks at a specific category of bacterial transcription factors. Sigma factors are prokaryotic transcription initiation factors that enable the specific binding of RNA polymerase to a variety of promoters. Escherichia coli sigma factors function to differentially express various sets of genes under different environmental conditions. Based on the feasibility of different promoters and their architectures, we've selected the following as ideal candidates for our project:
+Living organisms, like E.Coli, adapt and gear themselves to environmental changes. They are in the constant process of modifying their proteome based on the changing requirements. An interesting question arises as to how exactly does a bacteria gain awareness of its environment and what mechanisms do they use to deal with it?
-* sigma70 (rpoD):housekeeping genes during exponential growth
+The first step of gene expression is transcription, carried out by a protein called RNA Polymerase. This multi-subunit complex confers multiple particularities to the process of transcription allowing it to carry out different functional roles. One of the subunits, the sigma subunit, lends sequence specificity to the complex for recognizing relevant parts of DNA, called promoter regions. Only on the successful binding of this sigma subunit with a compatible promoter sequence does the process of transcription initiation occur.
-* sigma38/S (rpoS):master regulator for a generalized stress response
-* sigma32/H (rpoH):unfolded protein response
-* sigma28/F (rpoF):flagellar biosynthesis
-* sigma24/E (rpoE):response to unfolded proteins in the cell envelope
-=== Objectives ===
+Bacteria make use of multiple sigma subunits to partition its genome into different programs. E.Coli, for example, organizes its genome using 6 sigma factors which help the organism survive in different environmental niches. It's also observed that organisms which exhibit a greater diversity in their living conditions make use of a higher number of sigma subunits. This would provide for an adaptive advantage in settling into tougher and tougher niches.
-Our goal is to assemble a library of sigma-dependent and lac contorlled promoters, and submit these to the Registry of Standard Biological Parts. This "StressKit" can be used to control gene expression specifically during each phase of the bacterial growth curve and will also give users the ability to regulate gene expression using temperature, pH and other stress inducers as external cues. This modular approach, identifying and standardizing a set of functional parts so they may be used in diverse future applications, is one of the central themes of synthetic biology.
+The different sigma subunits present in E.Coli are as follows,
+* &sigma;<sup>70/D</sup> (rpoD):housekeeping genes during exponential growth
+* &sigma;<sup>54/N</sup> (rpoN):nitrogen starvation response
+* &sigma;<sup>38/S</sup> (rpoS):master regulator for a generalized stress response
+* &sigma;<sup>32/H</sup> (rpoH):unfolded protein response
+* &sigma;<sup>28/F</sup> (rpoF):flagellar biosynthesis
+* &sigma;<sup>24/E</sup> (rpoE):response to unfolded proteins in the cell envelope
-===  Methodology ===
+===Chosen Genes===
-Candidate promoters have been identified from literature searches and bioinformatics. We've chosen 4 special sigma factors listed above in addition to the houskeeping sigma 70 to include in our library. Our design is an attempt to engineer the regulation achievable with a ''lac'' promoter fused with the differential expression of the sigma factor dependent promoters. The custom promoters have recognition elements of the 2 classes of promoters pasted between each other in the least intrusive fashion. These promoters are also designed to conform to the Registry's ''BioBrick'' format (involving the removal of various restriction sites, and insertion into a standard plasmid backbone). We will then introduce these promoters into fluorescent expression systems and test their activity under various environmental stresses using fluorescence microscopy and spectroscopic techniques.
-== Project Details==
+===Design of Promoters===
+Our design strategy for the promoters took into consideration the regulatory diversity of the promoters, employing the use of distal, proximal and trans regulatory elements. Also, an important point to consider is that these promoters are in an 'always-on' state by default. They are constitutively expressed subject to a few environmental conditions.
-=== Project Design ===
+We overcome these constraints by experimenting with a new style of 'synthetic promoter' where we fuse sigma factor recognition sequences into a lac promoter. This takes away the 'always-on' issue with our promoters and also places them under the convenient regulation of IPTG. These hybrid/engineered promoters were designed in a way to preserve as much of the consensus sequence while maintaining the lac repressor binding sites.
-=== The Experiments ===
-=== Modelling ===
-== Results ==