Method for Screening and Rationally Engineering Riboswitch Capable of Specifically Recognizing Doxycycline

A method for screening a riboswitch capable of specifically recognizing doxycycline, comprising the following steps:

• (1) constructing a doxycycline riboswitch library through an RNA aptamer for doxycycline;
• (2) constructing a riboswitch reporting platform by fusing SacB gene with EGFP gene;
• (3) constructing a plasmid library using the doxycycline riboswitch library from step (1) and the riboswitch reporting platform from step (2);
• (4) transforming the plasmid library from step (3) into Escherichia coli to be co-incubated with a substrate, and collecting high-fluorescent cells which are positive cells by flow cytometry;
• (5) cultivating the positive cells from step (4) by spreading on a plate, and picking white nonfluorescent single colonies which are positive colonies;
• and
• (6) cultivating the positive colonies from step (5) on a well plate, measuring a fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 520 nm, and measuring OD600 using a spectrophotometer, calculating a fluorescence value per unit cell, and screening out a cell with a higher fluorescence value per unit, which is the cell carrying the riboswitch capable of specifically recognizing doxycycline.