Part:BBa_K3806014

Theophylline-binding aptazyme regulating lacZ expression (cRBS). With T7 promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3204
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2321
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Sensing small molecules is the foundation of many applications, ranging from disease diagnosis, prognosis, or treatment to detecting small pollutants in the environment. Synthetic genetic switches are a promising tool to detect and quantify small molecules. Conventional synthetic biology-based biosensors to detect small molecules are based on transcription factors. However, transcription factors are not easily reprogrammable with respect to ligand selectivity. On the contrary, antibody-based biosensors can be easily developed into new sensing capabilities. Unfortunately, these types of sensors are not suitable to detect low molecular weight compounds. RNA-based biosensors are an interesting alternative since they present remarkable flexibility to be engineered into sensing a wide range of analytes with high sensitivity and selectivity. In particular, aptazymes, ligand-regulated self-cleaving ribozymes, are of special interest as cleavage of the aptazyme can be coupled to regulated gene expression in vivo or in vitro. Moreover, newly developed methods such as DRIVER (de novo rapid in vitro evolution of RNA biosensors) enable rapid, automated, and multiplexed engineering of aptazymes sequences to diverse ligands.

The TU Delft 2021 team provides the iGEM community with a novel genetic switch construct (BBa_K3806014) in which an aptazyme sequence is fused to the lacZ reporter gene for converting ligand concentration to a colorimetric read-out. The construct is designed to be modular and serves as a template to engineer other ligand-specific genetic circuits by swapping the aptazyme domain. It was demonstrated that the designed genetic construct can be expressed in a cell-free system by using a known theophylline binding aptazyme [1].

Aptazyme-regulated gene expression mechanism

The aptazyme-regulated expression of lacZ depends on the accessibility of the ribosomal binding site (RBS) for translation initiation. This design choice was inspired by the study of Klauser & Hartig [2] in which the exposure of the RBS for translation initiation is controlled using small RNA switches. In BBa_K3806014, the RBS of lacZ is also sequestered by an antisense helix. After transcription of the DNA template comprising the aptazyme and fused reporter gene, the RBS is hidden in the stem of the aptazyme. When the aptazyme is stabilized upon binding of the ligand, the RBS remains entirely sequestered by its antisense strand, and translation is mostly prohibited. Cleavage of the aptazyme in the absence of the ligand liberates the RBS (Fig. 1). As a result, the ribosome can bind to the RBS and translate the downstream reporter gene to β-galactosidase protein.

Subsequently, β-galactosidase enables a colorimetric change by converting yellow substrate chlorophenol red-b-D-galactopyranoside (CPRG) to the red product chlorophenol red (CPR).

Fig. 1. Aptazyme-regulated gene expression mechanism. Binding of the ligand renders a catalytically inactive aptazyme, the RBS remains entirely sequestered by its antisense strand, repressing translation (left). In the absence of the ligand, self-cleavage of the aptazyme frees the RBS, resulting in the binding of the small ribosomal subunit and initiation of translation (right).