Part:BBa_K2904050
aTc inducible sfGFP regulated by modular Adda riboswitch containing Tuner A
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
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.
Result
The result by confocal microscopy
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We did three kinds of experiments to help us confirm the function of modular Adda riboswitch containing Tuner A.
For the sake of functional test, other 2 circuits are set, Adda-sfGFP and Adda-Stabilizer-sfGFP, which also were under control of the same promoter.By Confocal Microscopy Leica TCS SP8, it’s obvious that no fluorescence could be observed when the adenine riboswitch had sfGFP introduced directly. The direct fusion of sfGFP to Stabilizer yielded very clear inclusion bodies, manifested as distinct spots present at one pole of the cell which are formed by misfolded insoluble proteins. By comparison, the modular Adda riboswitch yielded soluble working protein since Tuner has the ability to insulation the target gene from Stabilizer.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 407
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