Difference between revisions of "Team:William and Mary/Binding Array"

Past iGEM teams have managed to create a dizzying number of unique transfer functions from their genetic circuits, and while altering the maximal expression level through ribosome binding sites (RBS) HYPERLINK is common among the iGEM community, there is oftentimes a need to change the threshold of a transfer function. That is, to move a transfer function horizontally. For the Circuit Control Toolbox to reach the stated goal of having orthogonal control over transfer functions we needed to find a way to orthogonally shift the threshold of a given arbitrary genetic circuit. In the end we managed to accomplish this by using molecular titration.

Molecular titration as the name implies is the process of titrating out molecules of transcription factor. That means, for some amount of transcription factor, a constant amount is taken away, such that for any given amount of transcription factor concentration, we are actually working with functionally less of said transcription factor (Figure 1).

Figure 1: Diagram showing the interactions between an activator transcription factor and decoy binding array, which as a molecular titrator. Note that the number of decoy binding sites impacts the equilibrium of free transcription factor, which in turn impacts the equilibrium of the amount bound to the promoter. Diagram adapted from Lee et al. 2012 (“A regulatory role for repeated decoy transcription factor binding sites in target gene expression”)

To accomplish this shift in E. coli we used decoy binding arrays, which are plasmids containing many repeated sequences of transcription factor binding sites, these repeated binding sites cause a large number of transcription factors to be bound to sites which produce no product, thus titrating them out, see Brewster et al. 2014 (“The Transcription Factor Titration Effect Dictates Levels of Gene Expression”). This causes a rightward shift in graph of transcription factor vs gene product. If we then graph a that same gene product versus a small molecule inducer for said transcription factor, then depending on the type of transcription factor we will either get a shift to the right (activator) or the the left (repressor). (See in depth example)

Say you have a transcription factor TetR, which binds to some promoter, say pTet, which effects the production of some gene, GFP then the concentration of that repressor is related to the production of that gene product. That is to say, for our circuit that you can go from [TetR] -> [GFP] or in a more measurable sense [TetR] -> Fluorescence (Figure 1.5)

Figure 1.5: Example circuit. pTet GFP is repressed by TetR, and this relationship is repressed by the introduction of the small molecule inducer aTC. Diagram made with pidgeon cad.

This makes sense, because the chance of the transcription factor being bound to a given promoter is based on the number of transcription factors as well as the number of unbound promoters. Since the number of promoters is more or less held constant by plasmid number, you can model production of the gene under the control of the promoter off of [Transcription Factor] alone. So you can use molecular titration to shift your transfer function by making any given amount of transcription factor be equivalent to a smaller amount.

Coming back to our example, since TetR is a repressor, as we increase [TetR] we get a corresponding decrease in fluorescence. If we then introduce a decoy binding array encoding a repeated number of tetO (TetR binding sites), say 85 of them, then for some [TetR], we actually have a working [TetR] of some amount less (Figure 2) That means at a given [TetR] in our circuit with the decoy binding array we have the fluorescence of a different lesser [TetR] of our original circuit. This is a shift along the x axis to the right.