After 16, 24, 32, or 40 h of incubation at 200 rpm at
room temperature, 500 μl of 1% (wt/vol) crystal violet
(CV) solution was added to each tube and incubated for
15 min. Tubes were rinsed three times with 5 ml of dis-
O and air dried. Biofilm formation was quanti-
fied by measuring the absorbance at 575 nm. Each assay
was performed four times.
Thin layer chromatography (TLC) analysis
Supernatants and pellicles were collected after 36 h of
growth in st atic LB media. Pellicles were treated with
100 μg/mL proteinase K for removal of cells. Cell-less
pellicles and supernatants were subjected to exopolysac-
charide extraction and hydrolysis with trifluoroacetic
acid as described previously . The resulting mono-
saccharides were dissolved in ddH
O in the co ncentra -
tion of 10 mg/ml, and 2 μl of the sample was spotted
onto TLC plates (silica gel 60 F
; Merck). After devel-
opment in butan-1-ol-acetone-water (4:5:1), the TLC
plates were dipped in the reagent aniline-diphenylamine
in acetone and incubated for 2 to 5 min at 100°C.
Additional file 1: Primers used in this study. File contains all primers
used in this study
This research was supported by Major State Basic Research Development
Program (973 Program: 2010CB833803) and National Natural Science
Foundation of China (30870032) to HG. This research was also supported by
Chinese Science Foundation for Distinguished Group (No.50321402) to YL.
This research was also supported by The U.S. Department of Energy under
the Genomics: GTL Program through Shewanella Federation, Office of
Biological and Environmental Research, Office of Science.
School of Minerals processing and Bioengineering, Central south University,
Changsha, 410083, PR China.
Institute for Environmental Genomics and
Department of Botany and Microbiology, University of Oklahoma, Norman,
Institute of Microbiology and College of Life Sciences, Zhejiang
University, Hangzhou, Zhejiang 310058, PR China.
YL carried out pellicle formation and characterization experiments and
drafted the manuscript. HG conceived of the study, and participated in its
design, and directed all experiments and coordination and drafted the
manuscript. JC carried out the mutagenesis experiments. YD and LW carried
out SSA biofilm and TLC assays. ZH participated in design of the study and
helped to draft the manuscript. XL and GQ participated in the design of the
study and helped to draft the manuscript. JZ conceived of the study, and
participated in its design and coordination and helped to draft the
manuscript. All authors read and approved the final manuscript.
Received: 29 May 2010 Accepted: 16 November 2010
Published: 16 November 2010
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