Difference between revisions of "Part:BBa K2066550"

Latest revision as of 07:45, 19 October 2016

85x tetO Binding Array

Part containing 85 tetO (tetR binding) sites. This part is used to induce a rightward shift in a transfer function by way of molecular titration.

Usage and Biology

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 the graph of transcription factor vs. gene product. If we then graph 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). As you can see below (figure 2), we used a repressor as the graph was shifted to the left.

To test our ability to shift our example circuit, a pTet GFP and constitutive tetR, we obtained a plasmid containing 85x TetO repeats off of Addgene from Finney-Manchester et al. (2013) (“Harnessing mutagenic homologous recombination for targeted mutagenesis in vivo by TaGTEAM). We moved the segment containing the repeats to the Biobrick Backbone, and then transformed a reporter circuit containing pTet GFP and TetR (Bba_K2066053) on the high copy plasmid 1A3 either with or without the repeat array on the high copy 1C3 backbone. We induced both circuits with varying concentrations of aTC and then measured fluorescence using flow cytometry, which allowed us to get single cell level resolution.

Figure 2: Population level FACs data comparing the relative fluorescence of a pTet GFP and TetR reporter with and without a tetO binding array. While the data is noisy, it is clear that the inflection point of the circuit with the binding array has shifted to the left as expected. Additionally, both circuits experienced a decrease in fluorescence at higher aTC concentrations, this is likely due to the toxicity of aTC in high concentrations.

Sequence and Features

Functional Parameters