4 Notes
- The biosensorA. tumefaciensA136 (pCF218) (pCF372) lacks
the Ti plasmid and provides AHL response system, the AHL-
responsive transcription factor TraR (pCF218) and the TraR-
regulatedtraI-lacZ fusion genes (pCF372). The biosensor
A136 can detect a broad range of AHLs (C6 to C14-HSL).
A136 was routinely maintained on LB agar supplemented with
50 μg/ml spectinomycin and 4.5μg/ml tetracycline at 28C,
and was grown in AT minimal glucose medium for liquid X-gal
assays. - We routinely purchase C6-HSL from Cayman Chemical Com-
pany or Sigma-Aldrich. C6-HSL can be alternatively prepared
in methanol. However, it is difficult to precisely pipette C6-
HSL dissolved in methanol even pre-wetting the tip several
times with the solvent before actual pipetting. Because solvents
with high vapor pressure (e.g., chloroform and methanol) can
easily drip outside during pipetting due to the evaporation of
the solvent. Furthermore, DMSO did not show effect on bio-
sensor A136 responsing to AHLs. Therefore, we prepared C6-
HSL in DMSO. - Please use the optimal media and incubation conditions for
growth of candidate bacteria. Generally, the mentioned media
and incubation conditions are suitable for most of the cultiva-
ble marine bacteria. - C6-HSL can be substituted by other AHLs (3-oxo-C6-HSL,
C8-HSL, 3-oxo-C8-HSL, C10-HSL, 3-oxo-C10-HSL, C12-
HSL, 3-oxo-C12-HSL, C14-HSL and 3-oxo-C14-HSL).
Compared to C6-HSL (1 mM), the recommended concentra-
tion was 2 mM for C10-HSL, C12-HSL, and C14-HSL, and
200 μM for the rest of the AHLs. As mentioned previously,
AHL lactonase generally has a broad substrate specificity, while
AHL acylase has a preference for long-chain AHLs. Addition-
ally, we found AHL acylases seem more common than lacto-
nases in the ocean. Therefore, C6-HSL and C12-HSL are
recommended to be used in the primary screening for QQ
bacteria from marine bacterial isolates. - Make sure that the biosensor A136 is freshly streaked on LB
agar plate before inoculating in AT minimal glucose medium. If
the colonies on LB plate are too old, the value of OD 600 of
A136 after 12 h incubation will not reach 2 (Subheading3.1,
step 5). In this situation, incubation time can be extended up
to 24 h. Sometimes, biosensor A136 with this state may affect
the blue color development in 96-well plate. In this situation,
incubation time can be extended up to approximate 18 h (Sub-
heading 3.1, step 6). It should be noticed that excessive
306 Kaihao Tang and Xiao-Hua Zhang