2.2 Further
Characterization of QQ
Activities of Selected
Bacteria
- Sonicator equipped with a microtip.
- Water bath.
- 20 mg/ml proteinase K, filter-sterilized, and stored at 20 C.
- 6 M HCl, filter-sterilized, and stored at 4C.
2.3 Identification of
QQ Enzymes from
Selected Bacteria
- Kit for genomic DNA extraction.
- Protein sequences of identified QQ enzymes collected from
literatures. - BLAST software: ncbi-blast-2.2.31þwin32.exe (download
fromftp://ftp.ncbi.nlm.nih.gov/blast/executables/blastþ/).
3 Methods
3.1 High-Throughput
Screening for QQ
Bacteria
The protocol of high-throughput screening for QQ bacteria is
shown in Fig.1.- Inoculate marine bacterial strains in 5 ml of Marine Broth 2216
(MB), and incubate the cultures at 28C, 170 rpm for 24 h.
Run at least two technical replicates (seeNote 3). - Transfer 178μl of each bacterial culture to a clean 1.5-ml tube.
- Add 2μl of C6-HSL stock solution and 20μl of PIPES stock
solution to the tube. Mix gently by pipetting and incubate the
reaction mixtures at 28C for 24 h under static condition. Use
MB supplemented with C6-HSL as a negative control and MB
supplemented with DMSO as a blank control (seeNote 4). - During the incubation of mixtures, inoculate the biosensor
strainA. tumefaciensA136 in 5 ml of AT minimal glucose
medium supplemented with 50 μg/ml spectinomycin and
Fig. 1The high-throughput method for identifying quorum quenching bacteria. The reaction step was carried
out in eight PCR tubes for easy subsequent centrifugation. 96-well plates were used in the detection step to
facilitate high-throughput measurements. Reproduced with permission from Copyright ©2013, Rights
Managed by Nature Publishing Group [9]
300 Kaihao Tang and Xiao-Hua Zhang