具有IV-A1型CRISPR-Cas活性细胞的细菌集落生长动态的实时成像。

microLife Pub Date : 2025-04-01 eCollection Date: 2025-01-01 DOI:10.1093/femsml/uqaf006
Selina Rust, Lennart Randau
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引用次数: 0

摘要

油橄榄假单胞菌的IV-A1型CRISPR-Cas系统在没有目标DNA降解的情况下提供对移动遗传元件的防御。在最近的研究中,具有IV-A1型CRISPR-Cas活性的大肠杆菌BL21-AI细胞表现出异质集落生长表型。在这里,我们开发了一种方便的智能手机自动遥控延时成像系统(SMARTIS),可以随着时间的推移监测细菌的生长。该系统的设计包括一个配备LED灯的定制成像盒,一个可调节的加热系统和一个可以使用免费的用户友好应用程序远程控制的智能手机。SMARTIS可以长期观察生长菌落,并用于分析表达IV-A1型CRISPR核糖核蛋白的大肠杆菌细胞的不同生长行为。我们的研究结果表明,菌落的异质性可以在最初生长的几个小时内出现。我们进一步研究了不同表达系统对细菌生长和CRISPR干扰活性的影响,并证明了所观察到的集落形成单位的异质性受到质粒设计和骨干身份的强烈影响。本研究强调了仔细评估异质菌落生长动力学的重要性,并描述了一种实时成像系统,该系统具有广泛的应用范围,超出了对细菌宿主中CRISPR-Cas活性的研究。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Real-time imaging of bacterial colony growth dynamics for cells with Type IV-A1 CRISPR-Cas activity.

The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans provides defense against mobile genetic elements in the absence of target DNA degradation. In recent studies, Escherichia coli BL21-AI cells with Type IV-A1 CRISPR-Cas activity displayed a heterogeneous colony growth phenotype. Here, we developed a convenient smartphone-mediated automatic remote-controlled time-lapse imaging system (SMARTIS), that enables monitoring of growing bacteria over time. The system's design includes a custom-built imaging box equipped with LED lights, an adjustable heating system and a smartphone that can be remotely controlled using freely available, user-friendly applications. SMARTIS allowed long-term observation of growing colonies and was utilized to analyze different growth behaviors of E. coli cells expressing Type IV-A1 CRISPR ribonucleoproteins. Our findings reveal that heterogeneity in colonies can emerge within hours of initial growth. We further examined the influence of different expression systems on bacterial growth and CRISPR interference activity and demonstrated that the observed heterogeneity of colony-forming units is strongly influenced by plasmid design and backbone identity. This study highlights the importance of careful assessment of heterogenous colony growth dynamics and describes a real-time imaging system with wide applications beyond the study of CRISPR-Cas activity in bacterial hosts.

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