Part:BBa_K2904140

asRNA helping modular Adda riboswitch turn off gene expression

Design

Background of 2019 OUC-China's project——RiboLego

Thermodynamic switches are found being energetic equilibrium between their on- and off-state. If switching is triggered, the equilibrium distribution shifts towards the new energetically best conformation. This implies that thermodynamic switches can reversibly and repeatedly toggle between on- and off-states. In contrast, kinetic switches are trapped in one state, depending on whether the ligand was present at the time of folding. Because ligands are hard to be degraded, the state of the dynamic riboswitch is difficult to be changed without ligand washing. However, the ligand washing or medium replacement is almost impossible for many actual industrial application scenarios. So a new approach is expected to utilize to regulate the state of riboswitch.

The construction of this part

Antisense RNA is endogenous in E.coli that do not require heterologous proteins to function. Owing to its simple design principles, small size, and highly orthogonal behavior, the engineered genetic parts of asRNA has been incorporated into genetic circuits. So we used asRNA to regulate the state of riboswitch. Antisense RNA can be thought to consist of two regions: a target binding region (TBR) containing a sequence that is complementary to the target gene, and an Hfq binding site which allows for binding of the Hfq protein. Hfq is a native chaperone protein that mediates RNA interactions by binding to a particular RNA binding site on the asRNA molecule. In our work, the engineered MicF binding site (MicF M7.4) was used as Hfq binding site because it performed well with low off-target effect in previous studies. By using model to design artificial TBR, we hope to utilize asRNA to turn off Adda riboswitch. When targeting the RBS of Adda riboswitch, asRNA can close modular Adda riboswitch even in presence of 2-AP.

Result

A two-plasmid system was constructed to characterize the capability of asRNA to inactivate a riboswitch when ligand is present. One plasmid can transcribe modular Adda riboswitch system with sfGFP as reporter controlled by aTc-induced promoter, another can transcribe asRNA using the IPTG-induced Plac promoter. We attempted to inhibit the expression of sfGFP by targeting the modular Adda riboswitch with asRNA. By adding IPTG and aTc at the same time, we observed a significant decrease of sfGFP level compared with the group without asRNA induction.

Figure1: The intensity of sfGFP by microplate reader during the entire cultivation period. The blue group represents the recombinant strain transformed with modular riboswitch system and asRNA when adding aTc, 2-AP and IPTG at the same time. The red one represents the strain transformed with the modular riboswitch system when aTc and 2-AP is present.

After combining asRNA with modular Adda riboswitch containing Tuner A, we also want to verify whether it can be used to regulate the riboswitch including other Tuners. Tuner E, which can achieve the lowest expression level was selected to verify the effect of asRNA. The results by microplate reader indicated that asRNA can also decrease the expression.

Figure2:The intensity of sfGFP by microplate reader during the entire cultivation period. It can verify that asRNA can be used to inactivate the modular Adda riboswitch including Tuner E when 2-AP exists.

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]