Real-time measurement of quorum-sensing signal autoinducer 3OC6HSL by a FRET-based nanosensor
Chang Zhang, Bang-Ce Ye*
Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong RD 130, Shanghai 200237, China
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Construction of plasmids
Four restriction sites BamHI, EcoRI, SacI, and SalI were used to insert 3 genes to construct a FRET biosensor. CFP was inserted into the pET-28a(+) vector between the SacI and SalI sites. The second fluorescent protein was inserted into the pET-28a(+) vector between the BamHI and EcoRI sites; In the last step, LuxR was inserted into the pET-28a(+) vector between the EcoRI and SacI sites.
Supplementary Figure 1. Construction of plasmids
Supplementary Table 1. Primers used for constructing and engineering sensor proteins. Restriction sites are indicated in red.
Gene inserted / Primercfp / Forward: TAA GAGCTCATGGTGAGCAAGGGCGAGGA
Reverse: TAAGTCGAC CTTGTACAGCTCGTCCATGCCG
yfp / Forward: TAA GGATCC GTGAGCAAGGGCGAGGAGC
Reverse: TAAGAATTC CAGCTCGTCCATGCCGAGAG
luxR / Forward: taaGAATTCAAAAACATAAATGCCGACGACA
Reverse: taaGAGCTCATTTTTAAAGTATGGGCAATCAATT
Protein renaturation
Inclusion bodies containing AiiA protein were purified using the Ni-NTA Superflow Cartridge Handbook (Qiagen, Hilden, Germany). Purified AiiA was dialyzed against decreasing concentrations of urea (6 M–0 M) to attain active AiiA.
Supplementary Figure 2. Renaturation of AiiA protein and verification of renatured AiiA activity
CV026 was cultured in LB overnight, and then renatured AiiA (0.36 mg/mL) was mixed with 3OC6HSL dissolved in M9 for 90 min to complete enzymatic degradation. 3OC6HSL dissolved in M9 with (1 and 2) or without (3 and 4) degradation was added to CV026 overnight. Samples 3 and 4 become purple, indicating that CV026 had recovered the ability to produce the characteristic purple pigment violacein in the presence of 3OC6HSL. Samples 1 and 2 exhibited no color, indicating that renatured AiiA has degradation activity for AHL and that our renaturation procedure is effective.