Question

Need help to understand this the first two methods in the following paper:

Host Intestinal Signal-Promoted Biofilm Dispersal Induces Vibrio cholerae Colonization.
Amanda J. Hay, Jun Zhu Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Please interpret the methods section (Bacterial strains and growth conditions. and Biofilm formation assays).

Answer 1

Bacterial strains and growth conditions:

1.V. cholerae El Tor C6706 was used as the wild-type strain in this study.

2.Inframe deletions were constructed by cloning the regions flanking the target genes into suicide vector pWM91, containing a sacB counterselectable maker.

3.PvpsA-lacZ transcriptional fusions were generated by cloning thevpsA promoter into a plasmid containing a lacZ reporter .

4.The transcriptional lux reporter of the promoter region of tcpA has been described previously .

5.Strains were propagated in LB containing appropriate antibiotics at 37°C, unless otherwise noted.

Biofilm formation assays:

Assays to quantify biofilms were performed as previously described . A 1:100 dilution of overnight-grown culture of V. choleraewas inoculated in LB broth into 10- by 75-mm borosilicate glass tubes and incubated for 22 to 24 h at 22°C. Subsequently, the tubes were rinsed three times with phosphate-buffered saline (PBS) and then filled with crystal violet stain. After 5 min, excess stain was rinsed off with deionized water. The biofilm-associated crystal violet was solubilized in dimethylsulfoxide (DMSO), and the optical density at 570 nm (OD570) of the resulting suspension was measured. All experiments were performed at least three independent times, and samples were performed in triplicate.