Part:BBa_K2904092

sfGFP regulated by modular Four U riboswitch

Design

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

Due to context-dependent performance and limited dynamic range, the widespread application of riboswitches is currently restricted. By replacing its original ORF with a new one, the structure of an aptamer domain can be subtly disrupted, resulting in a loss of ligand response. So riboswitch is still not be considered as a ‘plug and play' device. To tackle these problems, our project focuses on a standardized design principle to be used for modular and tunable riboswitch. The modular riboswitch we defined consists of the original riboswitch, Stabilizer and Tuner. Stabilizer can protect the structure of riboswitch from damage while Tuner can reduce the expression probability of fusion protein and make improvement of riboswitch function.

The construction of this part

Riboswitches can furthermore be classified into thermodynamic and kinetic switches. Different from kinetic switches, thermodynamic switches can reversibly and repeatedly toggle between "on-" and "off-" states, depending on temperature. Thermodynamic switches are temperature-sensing RNA sequences in 5’UTR of their mRNAs. At low temperature, they can fold into the structure, blocking access of ribosome; at high temperature, it open conformation, increasing the efficiency of translation initiation.

After successfully creating modular riboswitch with our method on kinetic switches and demonstrate that Tuner can control the expression of a range of functional outputs, we started to build modular thermodynamic riboswitch to test the universality of our design principle. Four U is an RNA thermometer that can be used for temperature sensitive post-transcriptional regulation that initiates translation at 37°C. Fortunately, we found that OUC-China team had used Four U to successfully express RFP in 2015, which provided us with great convenience! So we selected the first 132bp of RFP as Stabilizer of Four U. Then we used Tuner A to change the exprssion level. The superfolder green fluorescent protein (sfGFP) is the reporter gene to verify our modular riboswitch.
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
  INCOMPATIBLE WITH RFC[1000]

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