XZ
Reviewer
Xiaofeng Zhou
  • Stanford University
Research fields
  • Microbiology
Extracellular RNA Isolation from Biofilm Matrix of Pseudomonas aeruginosa

Most bacteria in natural ecosystems form biofilms-a bacterial community, surrounded by a polymer matrix that consists mostly of exopolysaccharides, proteins, and nucleic acids. Extracellular RNA as a matrix component is involved in biofilm formation-the fact that was confirmed by direct detection of extracellular RNA in the biofilm matrix, and by an interruption of the biofilm's structure with RNases. Number of protocols describing isolation of RNA from biofilm matrix are limited and usually involve uncommon equipment and reagents. Here we describe simple method for extracellular RNA isolation from biofilm matrix using basic laboratory reagent and equipment. Key steps of the protocol include separation of matrix and bacterial cells with high ionic solution of NaCl, RNA precipitation with LiCl and clean up with option to use inexpensive column for plasmid DNA isolation rather than specialized RNA kits. Described protocol allows to isolate extracellular RNA suitable for further molecular biology procedures such as sequencing, RT-PCR and cloning in less than one day (excluding time for biofilm growing up).

A High-throughput Interbacterial Competition Platform
Authors:  Hsiao-Han Lin and Erh-Min Lai, date: 09/05/2020, view: 4164, Q&A: 0
Contact-dependent interbacterial competition is a common strategy used by bacteria to fight for their ecological niches. Interbacterial competition is monitored by a competition assay involving co-culturing the attacker and the recipient bacterial cells on agar, followed by recovery of the surviving recipient cells. Conventional interbacterial competition assays rely on serial dilution, plate spreading, and colony counting experiments for the readout. The high demand for time and labor in a competition assay limits its use for large-scale screening. However, a high-throughput interbacterial competition screening method is required to screen genetic factors involved in an interbacterial competition. Here, using Agrobacterium tumefaciens as an attacker and Escherichia coli as a recipient, we developed a robust, fast, efficient, and high-throughput type VI secretion system-dependent interbacterial competition screening platform. This system allows for 96 simultaneous competition assays without the need for serial dilution and plate spreading. Data analysis of this system relies on only direct and straightforward colony counting. This platform may be easily adapted to identify novel factors involved in any contact-dependent interbacterial competition systems.
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