Is it possible to make bacteria respond to physical changes in a customizable way?

First, we analyze the question piecewise. How exactly do bacteria adapt to physical changes in their environment? Typically, these physical parameters comprise of temperature, pH, salt concentration, oxidation potential and nutrient concentration. These stresses pose a challenge to the organisms' survival. The lead for the idea was when we came across heat shock and the response it elicits in lactococcus. This phenomenon of temperature rise being related to DnaK levels was exactly the kind of system we wanted to develop on.

How are bacteria able to selectively switch on expression of a small subset of proteins based on a change in temperature, or any other physical parameter? The very ability of bacteria to accomplish this points to a transduction mechanism. They must transduce physical changes into gene expression. In this light, it is interesting to know that σ 32, a subunit of the RNA Polymerase Holoenzyme, twists into a functional conformation only on a rise in temperature. Further, this σ subunit confers sequence selectivity to RNA Polymerase, directing it to different promoter sites. Without this σ factor, RNA Polymerase has a very indiscriminate binding behaviour.

To deal with different environmental niches, bacteria employ a family of such σ factors. They express different sets of proteins in an attempt to adapt to these varying conditions. The exact number of these factors changes from one species to another, with the more exotic ones having a larger number. This reflects on the degree of specialization of an organism to survive different conditions. For example, E. coli has 7 σ subunits while S. coelicolor has over 60.

E.Coli has a family of 7 σ subunits to partition it's genome into various programs. This division is as follows,

1. σ⁷⁰, rpoD: Houskeeping genes
2. σ⁵⁴, rpoN: Activated on nitrogen starvation
3. σ³⁸, rpoS: Master regulator for a generalized stress response
4. σ³², rpoH: Activated on increase in unfolded proteins
5. σ²⁸, rpoF: Initiates flagellar biosynthesis
6. σ²⁴, rpoE: Activated on rise of unfolded proteins in the cellular envelope
7. 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 responds 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.

Their mode of regulation was also looked into. Apart from being just selected for transcription by a σ factor, genes are further regulated by both repressors and enhancers. On many occasions, transcription isn't possible without the presence of these regulatory elements. This poses an additional complexity in the isolation of a promoter segment as cis regulatory elements need to be incorporated. We had to circumvent this problem as it would greatly increase the length of our BioBricks. But more importantly, the exact region of these cis acting elements are not well documented.

To resolve this, we looked into the structural requirements for σ factor + RNA Polymerase, the holoenzyme, to bind DNA. Broadly, the sigmas fall into either a σ⁷⁰ or a σ⁵⁴ structural family. The σ⁵⁴ family of σ subunits cannot start transcription without an activating element. This activator has to structurally alter σ⁵⁴. This rules out its incorporation in our StressKit.

For the remaining 6 σs, the binding pattern is similar to σ⁷⁰s. They involves base specific interactions only in the -10 and -35 box of the promoter, where +1 is taken to be the transcription start site. Under each category of σs, there are a number of genes (See TABLE) with different promoter sequences. These sequences were analyzed for a consensus sequence in the -10 and -35 region. This consensus sequence should give a good idea about the critical length of bases bound by the σ factor.

Now, we can enhance this promoter design by incorporating a easily accessible form of regulation; IPTG based induction. We took the pLac synthetic promoter designed by Bujard et al

UNDER CONSTRUCTION