Part:BBa_K774103

A serious problem encountered again and again by synthetic biologists is, that for a particular ligand specific promoters do not exist and that it is very difficult to construct a transcription factor that is specific to the required ligand. There are however often broad spectrum (non-specific) promoters for a ligand of interest. These promoters and their transcription factors will induce transcription initiation when exposed to (or not exposed to) the investigated ligand, but also when under influence of other similar ligands. Assuming competitive binding, this is an interesting effect which can be exploited to give specific and accurate concentrations of each of the inducing ligands. In its simplest form of the Multi-Sensor system, there are two different transcription factors, each of which will cause transcription when exposed to either one or both of two different competitive ligands. They both posses different active sites, which causes there to be a different bias in both of those for each ligand. This will lead to transcription rates of one of the promoters to indicate a ratio between the two different ligands (for example nitrates and nitrites) as seen in line 1 ("Figure 3."). Because the two different transcription factors have different binding efficiencies to the two ligands, the line of the other promoter will take a different angle (line 2). The point where these two lines cross, gives the precise concentration of both ligands even though the two different promoters are non-specific. This effect can be modelled for more than two substrates. Visually the system can be designed with each concentration being a different access on a graph (which can soon become hyper dimensional). When you add a third ligand it will result in the two lines in Figure 5. to become two planes which intersect along one line. This means that a third promoter and transcription factor is necessary (the same way that two promoters pinpoint any single point in two-dimensional space, three are needed to triangulate a point in three-dimensional space). Once the third promoter and transcription factor is added to the graph, the three planes created intersect at a single point which gives the specific concentration of each of the ligands (Figure 5:). When four ligands are used, a hyper dimensional graph of four spatial dimensions with four different plains, each pertaining to a single promoter and transcription factor, will all intersect at a single point. This point will give the specific concentration of each of the four ligands (and so on and so on). Functional construction There are a number of different ways in which this theory could be put into practice. One would involve each one of the different promoters selected to be be fused to a fluorescent protein and cloned into different cells. In addition a constitutively expressed fluorescent protein will be used to control for metabolic and cell mass differences. A homogenised sample, split between each of the culture media, containing fluorescent protein with different promoters and The expression of each fluorescent protein will be measured using fluorometer and the data is to be mathematically analysed (see below). This will give the exact concentrations of each of the ligands. Another possible way of constructing the Multi-sensor system, would be to ligate all promoter - fluorescent sequences into one plasmid and transform this into a single cell. At this point the constitutively synthesised fluorescent protein for control, is no longer necessary because the comparison is made between expressions of different proteins within the same cell. This system has advantages and disadvantages. Because each cell contains the all promoters necessary for the system, each single cell can give a reading for the chemical concentrations of the ligands investigated. This means in its direct vicinity, that each cell can become a single “ pixel”. This would allow can for an image of chemical concentrations throughout a sample. The useful applications are vast. For example, in environments where diffusion rate is low and chemical concentrations vary e.g. soil, the multi-sensor system would be able to give accurate and locally precise data. It enables exact measurements of concentrations via laser microscopy but an overview of a great amount of data would also be available with just a simple photograph. please find figures at http://2012.igem.org/Team:NRP-UEA-Norwich/TheoreticalProject