Part:BBa_K2904050

aTc inducible sfGFP regulated by modular Adda riboswitch containing Tuner A

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

This part was used to validate our design principle of modular riboswitch. We employed activating 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 sfGFPas the reporter gene to reflect output of our system. Besides, Tuner A was inserted between Stabilizer and sfGFP. The modular Adda riboswitch containing the original riboswitch, Stabilizer and Tuner A was under control of the tetracycline promoter, which was induced by aTc.

Fig.3 The electrophoretic result of miniToe family test system.

200px|thumb|left|alt text We test our design principle in different riboswitches including three kinetic switches: Adda riboswitch, Btub riboswitch, cobalamin biosensor, and one thermodynamic switch: FourU riboswitch. What's more, three different kinds of GOI is used including sfGFP, YFP, and mRFP1. The good results show the high universality of our design principles. We hope our guideline can easily be appied by future teams.

we design a toolkit named miniToe family focused on translational regulation, which is composed of a RNA endoribonuclease, Csy4 and a RNA module (hairpin). In our project, the cleavage function of Csy4 releases a cis-repressive RNA module (crRNA, paired with RBS) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. A Ribosome Binding Site (RBS) is an RNA sequence to which ribosomes can bind and initiate translation.

The sfGFP was regulated by the Adda riboswitch containing the original Adda riboswitch, Stabilizer and Tuner A.

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 407