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

将合成生物学方法与图案化生物膜形成相结合，研究异质结构中的细菌持久性

• 批准号: 1706061
• 负责人: Dacheng Ren
• 金额: $ 33万
• 依托单位: Syracuse University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2021-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1706061&HistoricalAwards=false
• 关键词: Integrating; synthetic; biology; approaches; patterned

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.

PI姓名：大成仁提案编号：1706061细菌产生不生长的亚群，称为持留细胞，其对多种应激如重金属、热和抗菌剂具有高度耐受性。当细菌培养物进入稳定期或当细菌形成生物膜时，持续形成增加，生物膜是一种表面附着的多细胞结构，其中细菌细胞嵌入由附着细胞产生的基质中。越来越多的证据表明，持留细胞在生物膜相关的胁迫耐受中发挥重要作用。 在这个项目中，该团队将合成生物学与模式化生物膜形成相结合，以控制生物膜中的持久性形成，并使用这个新系统来研究控制剂的影响。本计画之研究将作为生物工程与化学工程课程之新模组与实验之课题。建议开展外联活动，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）在科学和工程领域的参与。活动包括在当地学校举办暑期讲习班和定制学生项目。这些综合活动将有助于招募和培养具备知识和技能的学生，以应对新技术的挑战，从而更好地服务社会。由于在理解细菌持久性方面存在关键的知识差距，研究人员将开发一种新的实验系统，该系统能够使用近红外光控制生物膜中持久性的形成和唤醒。 他们将以最小的结构异质性实时监测图案化生物膜中的细菌持久性。具体来说，该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。在生长速率依赖性启动子PrrnB 1的控制下，使用不稳定的绿色荧光蛋白监测持久性形成和唤醒。随着生物膜形态和持久性形成的良好控制，该系统将带来前所未有的机会，以获得新的见解细菌在生物膜中的持久性。将比较生物膜持留细胞和正常细胞的基因表达谱，并揭示持留细胞形成和唤醒期间基因表达的变化。然后，利用含有大量持留细胞的光诱导生物膜和对照生物膜来研究抗生素对生物膜中持留细胞和正常细胞的影响。生物膜不同深度处以及正常细胞与持久细胞中抗生素的浓度将通过使用质谱法以及基于传质原理的分子模拟和计算来表征。实验结果和建模数据将被整合，以了解生物膜的持久性和特定试剂的杀伤。 这项研究的结果将提高我们对细菌生理学的一般知识，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。

项目成果

Development of the computational antibiotic screening platform (CLASP) to aid in the discovery of new antibiotics

• DOI: 10.1039/d0sm02035d
• 发表时间: 2021-03-14
• 期刊: SOFT MATTER
• 影响因子: 3.4
• 作者: Dai, Yinghui;Ma, Huilin;Nangia, Shikha
• 通讯作者: Nangia, Shikha

Magnetically driven active topography for long-term biofilm control

• DOI: 10.1038/s41467-020-16055-5
• 发表时间: 2020-05-05
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Gu, Huan;Lee, Sang Won;Ren, Dacheng
• 通讯作者: Ren, Dacheng

Bacterial Metabolism During Biofilm Growth Investigated by 13C Tracing

• DOI: 10.3389/fmicb.2018.02657
• 发表时间: 2018-11
• 期刊: Frontiers in Microbiology
• 影响因子: 5.2
• 作者: Ni Wan;Hao Wang;C. K. Ng;Manisha Mukherjee;D. Ren;B. Cao;Yinjie J. Tang

Antibiotic Susceptibility of Escherichia coli Cells during Early-Stage Biofilm Formation

• DOI: 10.1128/jb.00034-19
• 发表时间: 2019-09-01
• 期刊: JOURNAL OF BACTERIOLOGY
• 影响因子: 3.2
• 作者: Gu, Huan;Lee, Sang Won;Ren, Dacheng
• 通讯作者: Ren, Dacheng