Part:BBa_K2904055

aTc inducible sfGFP regulated by modular Adda riboswitch containing a different source Stabilizer

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

When it comes to Stabilizer, a necessary factor to consider is the source. In addition to blast, by which the original target gene is selected from the genome, we can also choose the gene working well under the riboswitch from the past study. To validate the soure of Stabilizer is changable, we employed the first 150 bases of GFP as Stabilizer of Adda riboswitch because it can regulate the expression of GFP normally. This part was modular Adda riboswitch containing the original riboswitch, Stabilizer and Tuner B.

Result

In order to confirm the selection principle of Stabilizer, we tested our system by microplate reader, which is used to reflect the intensity of sfGFP changing over time. The following chart shows the dynamic curve measured every two hours. It can prove that the modular Adda riboswitch containing a different source of Stabilizer can restore the structure of riboswitch and control the downstream gene expression during the whole cultivation period.

Figure1: The results of modular Adda riboswitch containing a different source of Stabilizer and Tuner B by microplate reader.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 422