COMPARISON OF RIBOSWITCH REPORTER SYSTEMS FOR LIVE CELL IMAGING OF CYCLIC-DI-GMP DYNAMICS IN BACILLUS SUBTILIS POPULATIONS

Abstract

Fluorescent riboswitch reporters can be used in vivo to monitor metabolite dynamics. Previous work used a fluorescent yfp reporter based on a cyclic di-GMP responsive riboswitch from Bacillus licheniformis to monitor cyclic di-GMP levels in individual Bacillus subtilis cells. The previous study found that cell fates in Bacillus subtilis are not uniform in the presence of varying cyclic di-GMP levels. It is important to further develop tools that enable single-cell imaging in Gram-positive bacteria. Fluorogenic aptamers are single-stranded RNA molecules that have been evolved via in vitro selection to bind strongly and specifically to fluorophore molecules and emit a fluorescent signal. These fluorogenic aptamers can be used instead of fluorescent proteins in riboswitch reporter systems to provide a more dynamic read-out of metabolite dynamics in cells. However, relatively little work has been done to evaluate the use of these fluorogenic aptamers as reporter systems in Gram-positive bacteria. The objective of this project is to evaluate the use of four different fluorogenic aptamers (Mango-III, Broccoli, dimeric Broccoli, and SpinachII) instead of yfp in a cyclic di-GMP responsive riboswitch reporter system in Bacillus subtilis. All plasmids containing the riboswitch reporters were constructed and successfully transformed into E. coli cells. Subsequently, the cyclic di-GMP responsive riboswitch reporter systems were successfully transformed into B. subtilis WT PY79 and a 𝝙pdeH mutant. Future work involves evaluating their performance in vivo in B. subtilis via laser confocal and fluorescence microscopy.