Part:BBa_K5466011

lexA-VP16 activated promoter

Synthetic promoter composed of the core promoter region from Saccharomyces cerevisiae TDH3 promoter, preceded by 4 lexA operators. This part allows for powerful activation by transcription factors harboring LexA DBD fused to an activation domain such as herpes simplex VP16 AD, for example, BBa_K5466010.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Orthogonality

To avoid unpredictable interference with the functionality of synthetic systems, the concept of orthogonality is crucial. An orthogonal system should deliver its intended functions with minimal, or ideally no, cross-talk with the host organism. This means that the system's functionality should not be affected by the host organism, nor should it impact the host in any unintended ways.

Synthetic promotor

A synthetic promoter is an artificially designed DNA sequence that controls the initiation of transcription. It contains specific motifs that are recognized by transcription factors or RNA polymerase to regulate the expression of a downstream gene. Synthetic promoters can be tailored to respond to various signals or environmental conditions, offering a tunable level of gene expression in engineered biological systems.

Rantasalo et al., (2016) identified there are three key modules essential for controlling the strength of the promoter, each of which can be modified to adjust signal transduction strength and produce a wide range of expression levels. The first is the binding site (BS) module, which consists of the sTF-specific BS located in the upstream activating region of the output promoter. The number of BSs can affect transcriptional activity, similar to how native transcription factor binding sites function. The second is the activation domain (AD) module, representing the effector part of the sTF, specifically the transcription activation domain, which determines the strength of activation. The third is the core promoter (CP) module, which is necessary for the assembly of the general transcription machinery and the initiation of transcription.

In this promoter, the LexA binding site is the consensus sequence CTGTATATATATACAG, repeated 4 times. The number of repetitions can vary (1–8), and the higher the number of repetitions, the stronger the promoter will be.

The core promoter is from TDH3p, a constitutive promoter, but it can be changed to adjust its strength.

The activator domain of the transcription factor can also be varied to modulate its strength. In our project, it is from VP16.

By adjusting these three modules, the output can be fine-tuned, allowing for a diverse range of expression levels that can be tailored to specific needs.

References

Rantasalo, A., Czeizler, E., Virtanen, R., Rousu, J., Lähdesmäki, H., Penttilä, M., Jäntti, J., & Mojzita, D. (2016). Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae. PLoS ONE, 11(2), e0148320. https://doi.org/10.1371/journal.pone.0148320