Difference between revisions of "Part:BBa K2904126"

Latest revision as of 12:13, 21 October 2019

EYFP 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 that our modular riboswitch will work with different proteins. 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. Instead of sfGFP, We used EYFPas the reporter gene to reflect output of our system. Besides, Tuner A was inserted between Stabilizer and EYFP.

Result

The modular Adda riboswitch was under control of the tetracycline promoter, which was induced by aTc. To test its effect, we used microplate reader to measure the fluorescence intensity of EYFP. The figure below selects the data when steady state is reached (at least two consecutive subsequent data points do not increase fluorescence). The results demonstrate that modular Adda riboswitch can achieve rational regulation of EYFP by responsive to different concentrations of 2-AP.

Figure1: The results of modular Adda riboswitch regulating the expressio of EYFP.

Besides this part, we also created a deviceto express sfGFP regulated by modular Adda riboswitch containing Tuner A. It can work well!

Sequence and Features