Real-time imaging of bacterial colony growth dynamics for cells with Type IV-A1 CRISPR-Cas activity
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Oxford University Press
Abstract
The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans provides defense against mobile genetic elements in the absence of target DN A de gr adation. In recent studies, Esc heric hia coli BL21-AI cells with Type IV-A1 CRISPR-Cas acti vity displayed a heter ogeneous colony growth phenotype . Here , w e developed a convenient smartphone-mediated automatic remote-controlled time-lapse imaging system (SMARTIS), that ena b les monitoring of growing bacteria over time. The system’s design includes a custom-built imaging box equipped with LED lights, an adjusta b le heating system and a smartphone that can be r emotel y contr olled using fr eel y av aila b le, user-friendly applications. SMARTIS allowed long-term observation of growing colonies and was utilized to anal yze differ ent gr owth 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 interfer ence acti vity and demonstrated that the observ ed heter ogeneity of colony-forming units is str ongl y influenced by plasmid design and backbone identity. This study highlights the importance of careful assessment of heterogenous colony growth dynamics and describes a r eal-time ima ging system with wide applications beyond the study of CRISPR-Cas activity in bacterial hosts.
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Except where otherwised noted, this item's license is described as Attribution 4.0 International