Team:IIT Madras/Project

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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,

σ⁷⁰, rpoD: Houskeeping genes
σ⁵⁴, rpoN: Activated on nitrogen starvation
σ³⁸, rpoS: Master regulator for a generalized stress response
σ³², rpoH: Activated on increase in unfolded proteins
σ²⁸, rpoF: Initiates flagellar biosynthesis
σ²⁴, rpoE: Activated on rise of unfolded proteins in the cellular envelope
σ¹⁹, FecI: Activated on iron starvation

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.

Design of promoters as a fusion between a lac and sigma dependent promoter

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 \begin{math}\sigma\end{math} 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.

Design of promoters as a fusion between a lac and sigma dependent promoter

@@ Line 8: / Line 8: @@
 |}
-===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?
+==Introduction==
+{|align=center width=80%
+|After months of brainstorming and evaluating a large number of other ideas, we defined our problem statement as follows,
-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.
+<blockquote>''Is it possible to make bacteria respond to physical changes in a customizable way?''</blockquote>
-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.
+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.
-The different sigma subunits present in E.Coli are as follows,
+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.
-* &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
-===Chosen Genes===
+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,
-===Design of Promoters===
+# &sigma;<sup>70</sup>, <tt>rpoD</tt>: Houskeeping genes
-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.
+# &sigma;<sup>54</sup>, <tt>rpoN</tt>: Activated on nitrogen starvation
+# &sigma;<sup>38</sup>, <tt>rpoS</tt>: Master regulator for a generalized stress response
+# &sigma;<sup>32</sup>, <tt>rpoH</tt>: Activated on increase in unfolded proteins
+# &sigma;<sup>28</sup>, <tt>rpoF</tt>: Initiates flagellar biosynthesis
+# &sigma;<sup>24</sup>, <tt>rpoE</tt>: Activated on rise of unfolded proteins in the cellular envelope
+# &sigma;<sup>19</sup>, <tt>FecI</tt>: Activated on iron starvation
-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.
+We used the database <tt>ecocyc.org</tt> 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 <tt>pLac</tt>, 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.
+!valign=top|[[Image:IITMpromoters.jpg|thumb|Design of promoters as a fusion between a lac and sigma dependent promoter]]
+|}
+==Promoter Design==
+{|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 \begin{math}\sigma\end{math} 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.
+!valign=top|[[Image:IITMpromoters.jpg|thumb|Design of promoters as a fusion between a lac and sigma dependent promoter]]
+|}