Part:BBa_K2904001

Tuner B

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

We defined a Tuner element to include a repressing region, a RBS region and a coupled junction region. The repressing region is the reverse complement of a subsequence of the RBS region so that Tuner can form a hairpin with appropriate ∆G. The stop and start codon fused in the junction region. Ribosomes recruited by the upstream riboswitch can open up the hairpin of Tuner before dissociation at the stop codon in the junction region. Additional ribosomes can then assemble at the Tuner RBS and initiate translation at the first start codon of the introduced gene of interest. Therefore, Tuner can facilitate tuning of a riboswitch’s response and help GOI express normally. The following diagram shows the structure of Tuner B and we marked each region clearly.

Figure1: The structure of Tuner B.

Result

Modular Adda riboswitch

Using this part, we could engineer modular riboswitch. In order to validate the effect of Tuner B, we utilized Adda riboswitch, which can regulate the expression of adenosine deaminase by binding 2-aminopurine in Vibrio vulnificus. The first 150bp of adenosine deaminase was chosen as Stabilizer of Adda riboswitch because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. We used Tuner B to construct modular Adda riboswitch and sfGFPas the reporter gene to reflect output of our system.

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 Tuner B can enhance the function of riboswitch and help riboswitch control the downstream gene expression during the whole cultivation period.

Fig.1 The results of modular Adda riboswitch containing Tuner B by microplate reader.

The above result demonstrate that Tuner B can help achieve tunable and efficient gene regulation. Besides, we also designed Tuner A to E.[http://2019.igem.org/Team:OUC-China/Model The design process about Tuners is on this page!]

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]