Team:William and Mary/Synthetic Enhancer
One of the fundamental constraints of an electrical engineering style way of thinking about genetic circuitry is that electrical circuits often include digital, often binary, states of expression. This kind of input-output relationship showing two discrete “on” and “off” states is represented by the illustrative gene expression curve in Figure 1. As one can see, although the binary interpretation is appropriate at the low and high extremes of inducer concentration, there is an intermediate region where the continuous, or analog nature of gene expression becomes important. A natural way to expand on the advantages of the digital-circuit paradigm while incorporating the non-binary characteristics of gene expression is to develop genes and circuits which can exhibit discrete, multi-step response profiles. These would allow circuits to exhibit not just “off” and “on” states, but a number of intermediate, discrete states to better facilitate both precision of circuit behavior and readout as well as the expansion of the possibilities in the computation that can be performed with genetic circuitry. Often people will use the low and high expression regions to represent the analogous expression levels to binary 0 and 1, respectively. However, Amit et. al. developed a method by which intermediate levels of gene expression can be obtained in a stable, non-transient manner. To do so, they created a synthetic enhancer suites, schematically shown in Figure 2, consisting of enhancer binding sites, an assembled σ54 promoter, and small cassettes between the enhancer and the promoter, which can house DNA binding proteins in different combinations. It is the looping of the DNA from the enhancer to the promoter that causes an interaction that allows for transcription of the output. Binding of repressor proteins, such as TetR, to the addition cassettes between the enhancer and promoter can affect the flexibility of DNA looping and thus make it thermodynamically more difficult for the interaction to take place and thus suppresses transcriptional initiation. This synthetic enhancer suite can not only increase the complexity and sensitivity of a circuit, but also allow for a multimodal response with addition of discrete sets of defined enhancer binding protein binding sites, TetR, that interact independently from the enhancer and promoter. The physical interaction between the assembled σ54 promoter complex (glnAp2) and enhancer sites causes the activation of transcription. Amit et. al. generated a genetic circuit regulated by the availability of NRI binding protein as well as the NRII2302 helper protein, which can phosphorylate the NRI protein and activate it as to allow it to bind to the enhancer region. Once this happens, the looping (shown in Figure 2) the DNA from the enhancer to the promoter allows for transcription of more NRI (positive feedback) as well as the fluorescent reporter. When repressor binding sites are placed in the spacer region between the enhancer and promoter, this allows for the regulation of output. The TetR binding makes the DNA looping harder and more rigid, resulting in less transcription of the fluorescent reporter. Multiple repressor binding sites can give rise to discrete number of TetR binding to the region at a given time, thus modulating the ability for the DNA to loop, ultimately giving rise to different states of expression depending on the availability of functional apo-repressor protein. NRII endogenously has phosphatase and kinase activity, and thus to control the phosphatase activity, the 3.300LG E.coli strain, which consists of a knocked down version of NRII2302, was used to decouple the circuit from a nitrogen dependent PII signal transduction pathway.
Synthetic Enhancer
Background
Figure 1 - Illustration of induction curve for generic gene expressionMechanism
Figure 2 - Adapted from Amit et. al. a. Cartoon representation of how the synthetic enhancer suite works. The DNA binding NRI-P hexamer has to bind to the enhancer binding sites and this complex can loop (shown on the right) and kinetically bind with the poised promoter to allow for the transcription of NRI and mCherry. The NRI production allows for positive feedback so as long as it gets phosphorylated by NRII2302 (expressed by pACT Tet helper plasmid), it can bind to the enhancer sequences and continue expression. Adding TetR repressor binding sites between the promoter and enhancer regions can weaker the probability of the looping event and reduce the expression of NRI and mCherry at a given bound state.
