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

细菌产生不生长的亚群，被称为持久性细胞，它们对重金属、高温和抗菌剂等各种压力具有高度的耐受性。当细菌培养物进入固定阶段或细菌形成生物膜时，细菌细胞嵌入附着细胞产生的基质中的表面附着的多细胞结构。越来越多的证据表明，持久性细胞在生物膜相关的胁迫耐受中起着重要作用。在这个项目中，该团队将把合成生物学与生物膜形成模式结合起来，控制生物膜中持久性的形成，并使用这个新系统来研究控制剂的效果。本课题的研究将作为生物工程和化学工程课程开发新模块和实验的主题。建议开展外展活动，以扩大K12教师和学生，特别是代表性不足的群体（包括退伍军人）在科学和工程方面的参与。活动包括在当地学校举办的暑期工作坊和量身定制的学生项目。这些综合活动将有助于招收和培养具备知识和技能的学生，以迎接新技术的挑战，更好地服务社会。在了解细菌持久性的关键知识差距的推动下，pi将开发一种新的实验系统，该系统具有使用近红外光控制生物膜中持久性形成和唤醒的能力。他们将实时监测细菌在具有最小结构异质性的图案生物膜中的持久性。具体来说，这个团队将设计一个近红外光控制的基因表达系统，通过调整选定的毒素和抗毒素基因的表达水平来操纵持久性。在生长速率依赖启动子PrrnB1的控制下，使用不稳定的绿色荧光蛋白监测持久性的形成和唤醒。随着生物膜形态和持久性形成的良好控制，该系统将为获得细菌在生物膜中的持久性提供前所未有的机会。比较生物膜持久性细胞和正常细胞的基因表达谱，揭示持久性细胞形成和觉醒过程中基因表达的变化。光诱导生物膜具有大量的持久细胞和对照生物膜，然后将用于研究抗生素对生物膜中持久细胞和正常细胞的影响。抗生素在生物膜的不同深度和正常细胞和持久性细胞中的浓度将通过质谱法和基于传质原理的分子模拟和计算来表征。实验结果和模型数据将被整合，以了解生物膜的持久性和特定药物的杀伤作用。这项研究的发现将提高我们对细菌生理学的一般认识，并有助于在医疗保健和生物安全等应用中开发更有效的微生物控制方法。

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.