A New Method for Converting Aptamers to Riboswitches Using the Theophylline Riboswitch

Abstract

Fluorescence Activated Cell Sorting, or FACS, is a method that was used to convert the theophylline aptamer into a riboswitch. This method could theoretically be used to convert other discovered aptamers into riboswitches, however it is a costly method and is only available to those with access to these high‐tech, expensive machines. The theophylline riboswitch was previously discovered by implementing the theophylline aptamer, with random sequences linking it to the expression platform, into a specifically‐designed plasmid, and then using a FACS machine to sort the cells.We can structure a new system that would select only the correct sequence, containing the theophylline riboswitch out of a pool, without the use of a FACS machine. To do so, we place the pool of sequences containing the aptamer, linked to the Shine Dalgarno by eight randomized nucleotides, into a newly designed plasmid, placNEO, and transform the plasmid into bacteria cells. Then, the use of replica plating along with screening will select the cells that only contain the plasmid with the correct riboswitch sequence. By doing so, we confirm that this system is efficient in converting aptamers into riboswitches without the need for a FACS machine.After an aptamer has been successfully converted into its riboswitch, the system of ratiometric fluorescence will allow for testing of the riboswitch’s function. This is done by designing a plasmid, pTRFlac, that contains genes for red and green fluorescence proteins, mCherry and GFP respectively, on either side of the inserted riboswitch. A PCR product encoding for mCherry, the riboswitch, and GFP will be inserted downstream of the lactose operon in pUC18. Ratios of the fluorescence intensities of the two florescent proteins will provide the ability to measure the riboswitch’s function through fluorescence readings.Support or Funding InformationMonmouth University Summer Research Program ICFNJ