Gas Clock

The proper timing, or 'clocking', of the various stages of signal processing is fundamental to a properly functioning cellular automaton. In computers, clocking is rarely an issue. However, bacterial populations are not normally synchronized. As such, we needed to introduce a system where each stage of information processing could be tethered to a global 'clock'.

Biological clocking has been attempted in the past and is actually a ubiquitous system in synthetic biology - activating a synthetic network via light or some chemical signal are some basic examples of inducible clocks. However a true clock, one that is entirely autonomous and does not rely on a human interface, has, to our knowledge, never been achieved. To approach this challenge, we asked ourselves: What kind of signal could be generated by a population of cells and perceived at the same concentration throughout the entire population so as to maintain synchronicity? In our opinion, gas best fulfilled these requirements.

How can E.coli produce gas?

This question was answered by everyone's favorite plant model organism - Arabidopsis thaliana. A. thaliana contains an anabolic pathway that uses the methionine cycle, more specifically S-Adenosyl Methionine (SAM) to generate Ethylene Gas.

Ethylene biosynthesis is driven by the presence of two important proteins. 1-aminocyclo-propane 1-carboxylic acid (ACC) Synthase converts SAM To ACC and ACC Oxidase oxidizes ACC to ethylene, Co2 and Cyanide (Sauter et al, 2013). As such, both genes were introduced into the part that we called the Ethylene Synthesis Cassette (ESC). However, as E. coli uses SAM for its own metabolic needs, we introduced a second copy of SAM Synthetase so as to not disrupt basal metabolic requirements of the cell. The absolute necessity of SAM Synthetase is still unconfirmed and the effect of its presence or absence on cell viability will be verified in future work.

How can E.coli perceive gas?

Another important feature that was taken from A. thaliana is its ability to perceive Ethylene Gas. In plants, Ethylene gas is a hormone used in many tissues, involved in fruit ripening, senescence and growth (Abeles et al, 1992). A. thaliana uses a family of protein receptors called ETRs (Ethylene receptors) to recognize the presence of Ethylene. These proteins have a transmembrane receptor domain that binds ethylene and a cytosolic histidine kinase domain that has an autophosphorylative function. Upon binding to ethylene, conformational changes allow the cytosolic histidine-kinase domain to autophosphorylate. This phosphorylation is then passed on to a second protein, CTR1, which enters a signal cascade to activate downstream genes (Chen et al, 2005).

The histidine-kinase domain is an important feature of this protein as it falls within a family of protein domains called the two-component histidine kinases. The prototypical system consists of a histidine protein kinase, containing a conserved kinase core, and a response regulator protein, containing a conserved regulatory domain. Extracellular stimuli are sensed by, and serve to modulate the activities of, the histidine kinase. The histidine kinase then transfers a phosphate group to the response regulator. This leads to the activation of some response that ultimately leads to the expression of some downstream gene (Stock et al, 2000).

Now, how can this relate to E. coli? Interestingly enough, this question was answered by a previous IGEM project: E. coli Sees Light. In this project, Tabor et al generated a fusion protein that used the phytochrome, Cph1, from cyanobacteria, and EnvZ from E. coli (Levskaya et al, 2005). EnvZ is the an osmoregulating protein that perceives changes in osmotic pressure in E. coli. It is involved in the activation of the major outer membrane proteins OmpF and OmpC. This protein is also a two component histidine kinase as it translates its perception of changes in osmotic pressure to gene expression. It accomplishes this through the transfer of a phosphate group to OmpR. Once phosphorylated, OmpR-P acts as a transcriptional activator of genes under the control of the OmpC and OmpF promoters. (Mizuno et al 1982).

Using a similar approach, we generated a fusion protein of ETR1 from A. thaliana and EnvZ, which we named the E. coli compatible, Ethylene Inducible Histidine Kinase (EEHK) This fusion would conceptually link ethylene reception to the expression of genes under the control of the OmpC promoter.

To generate our fusion, we applied a semi-rational approach (Salis et al, 2009) to generate three site-specific fusions. These three specific fusions have been generated but remain to be assayed for function. In addition, to maximize our chances of locating functional fusion points, we designed a high throughput system that would allow the generation of multiple fusions. Using Nested Deletion and blunt end ligation, we plan to generate many, random fusions to be tested. A GFP marker under the control of the OmpC promoter will be used in the function assay for both specific fusions and random fusions

Genetic Circuit

To turn ethylene synthesis and its perception into an autonomous clocking system, we generated an Ethylene Oscillator. By cloning the ethylene synthesis cassette downstream of the Tet Operon promoter, and cloning the TetR gene downstream of the OmpC promoter, we generated a system where gas synthesis would induce the expression of its own repressor, thus generating an ethylene gas oscillator. This system can effectively control the expression of two sets of genes - those under the control of Ethylene gas, expressed when gas concentration is high, and those under control of the Tet Repressor, expressed when gas concentration is low.

To verify the validity of this oscillator in silica, we wrote a simulation to observe the effect of varying several parameters under our control (promoter strength, protein half-life, etc - for more information see simulation