Population counter

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Principle of the system

Each cell in our population harbors a construct that when stimulated has a small chance of excising a terminator and expressing RFP. The proportion of red cells in the population is thus an accurate count of the number of input stimuli.
To obtain such a counter, we needed a system that upon induction would cause only a small and rather constant number of cells to change phenotype. Moreover, it had to be able to work on a genetic level to make this phenotype inherited and stored in the population’s “memory”. For this purpose we’ve chosen the integron system. Usually its structure includes a fragment that encodes an integrase (intI) and a recognition sequence (attI) into which the gene cassettes are incorporated. These gene cassettes can be situated elsewhere in the genome. They are flanked by specific sequences (attC) and when the integrase is expressed they are excised by site-specific recombination and integrated into the attI site.
For our system the integration into the attl site wasn’t necessary so we used the integron system without the attl site so that the integrase could only excise a fragment without further integrating it. The excision of this fragment causes its loss from the genome and, according to our design, changes the cell’s phenotype. The integron system isn’t very effective. The chance that upon induction the integrase will efficiently excise the fragment flanked by attC sites is relatively low. This makes excision a rare event, which is just perfect for our project : we want only a small proportion of cells to become red after each induction pulse.

Counter

Our counter consists of two parts. The first one is carried by a plasmid containing an Ara-inducible integrase IntI. The second part is either carried by a plasmid or integrated on the chromosome. It contains a constitutive promoter upstream of two recombination sites flanking a double terminator, and a reporter gene.

There is a terminator flanked by attC sites between the constitutive promoter and the RFP gene. At the initial state, the terminator blocks the transcription of RFP so no cell emits red fluorescence. The integrase gene is put downstream of a pBAD promoter and gets expressed when arabinose is added to the media. This results in excision of the terminator flanked by 2 homologous attC sites in some of the cells (excision is a statistically rare event). So these cells start producing RFP.

Another version of the counter uses tetracyclin resistance cassette as the reporter.

Timer

Our final construct can act both as a counter and a timer. In order to have the timer function, we added quorum sensing system to our basic design.
Quorum sensing is a system that permits the bacteria sense the cell density of other bacteria in the media. When bacteria express LuxI, it enzymatically produces AHL (acyl homoserine lactone) molecules that can then diffuse out of the cell and throughout the media. It can enter another cells and, if the cell expresses LuxR protein, can bind to LuxR.

If bacterial density is high enough, AHL concentration rises in the media and consequently in the cells until it reaches the threshold when the cytosolic LuxR-AHL complex induces the pLux promoter.

The LuxR gene is cloned downstream of the promoter and is expressed constitutively. The LuxI gene is expressed together with RFP only in cells where the terminator got excised. These cells start producing AHL which diffuses into the media and the other cells. The AHL molecules then bind to the LuxR protein in all cells of the culture. When AHL concentrations reach the threshold, they induce the pLux promoter which drives the expression of GFP in all cells.