Integrating synthetic biology approaches with patterned biofilm formation to investigate bacterial persistence in heterogeneous structures

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PI Name: Dacheng RenProposal Number: 1706061Bacteria produce non-growing subpopulations, known as persister cells, which are highly tolerant to a variety of stresses such as heavy metals, heat, and antimicrobials. Persister formation increases when a bacterial culture enters the stationary phase or when bacteria form a biofilm, a surface-attached multicellular structure with bacterial cells embedded in a matrix produced by the attached cells. Increasing evidence indicates that persister cells play important roles in biofilm associated stress tolerance. In this project the team will integrate synthetic biology with patterned biofilm formation to control persister formation in biofilms and use this new system to study the effects of control agents. The research in this project will be used as a topic for developing new modules and experiments for courses in bioengineering and chemical engineering curricula. Outreach activities are proposed to broaden the participation of K12 teachers and students, especially underrepresented groups (including veterans), in science and engineering. Activities include summer workshops and tailored student projects in local schools. Together, these integrated activities will help recruit and prepare students with knowledge and skills to meet the challenge of new technologies to better serve the society.Motivated by the critical knowledge gap in understanding bacterial persistence, the PIs will develop a novel experimental system with the capability to control persister formation and wake-up in biofilms using near-infrared-light. They will monitor bacterial persistence in real-time in patterned biofilms with minimized structural heterogeneity. Specifically, this team will engineer a near-infrared-light-controlled gene expression system to manipulate persistence by tuning the expression level of selected toxin and antitoxin genes. Persister formation and wakeup will be monitored using unstable green fluorescent protein under the control of the growth-rate-dependent promoter PrrnB1. With the biofilm morphology and persister formation well controlled, this system will bring an unprecedented opportunity to obtain new insights into bacterial persistence in biofilms. Gene expression profiles of biofilm persister cells and normal cells will be compared and the changes in gene expression during persister formation and wake-up will be revealed. The light-induced biofilms with large numbers of persister cells and control biofilms will then be used to investigate the effects of antibiotics on persisters and normal cells in biofilms. The concentrations of antibiotics at different depths of biofilms and in normal vs. persister cells will be characterized by using mass spectrometry, and by molecular simulation and calculation based on mass transfer principles. The experimental results and modeling data will be integrated to understand biofilm persistence and killing by specific agents. The findings from this study will improve our general knowledge of bacterial physiology and help develop more effective methods of microbial control in applications such as healthcare and biosecurity.