Protocols for Test

Strains and Growth Media

E.coli Top10 was used for all the experiments and grown in Luria–Bertani (LB) medium or M9 minimal medium using glycerol as carbon source. Kanamycin (10 μg/mL), ampicillin (50 μg/mL) and chloramphenicol (170 μg/mL) were added as appropriate.

Five Kinds of Protocols

Protocol 1
E.coli was grown overnight in LB medium at 37 °C and then diluted 100-fold in fresh LB medium in 96-well plates (Corning Incorporated, 3599). Then each culture (200 μL) was induced for 12 hours at 30°C with inducers of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences).

Protocol 2
E.coli was grown overnight in LB medium at 37 °C and then diluted 100-fold in fresh LB medium in 96-well plates (Corning Incorporated, 3599). After 3 hours’ culture at 30 °C, each culture (200 μL) was induced for 7 hours with inducers of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences).

Protocol 3
E.coli was grown overnight in LB medium at 37 °C and then diluted 100-fold in fresh LB medium in 96-well plates (Corning Incorporated, 3599). After 6 hours’ culture at 30 °C, each culture (200 μL) was centrifuged at 4000 r.p.m. for 10 minutes and was suspended in 200 μL of fresh LB medium containing inducers of different concentrations for 4 hours. Then the fluorescence intensity of cultures was measured by microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences).

Protocol 4
E.coli was grown overnight in LB medium at 37 °C and then diluted 100-fold in fresh LB medium in 96-well plates (Corning Incorporated, 3599). Then each culture (200 μL) was induced for 12 hours at 37°C with inducers of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences).

Protocol 5
E.coli was grown overnight in LB medium at 37 °C and then diluted 100-fold in fresh M9 minimal medium in 96-well plates (Corning Incorporated, 3599). Then each culture (200 μL) was induced for 12 hours at 30°C with inducers of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences).


Protocol 6
E.coli was grown overnight in LB medium at 37 °C and then diluted 100-fold in fresh M9 minimal medium in microcentrifuge tubes (1.5 mL, Axygen). Then each culture (200 μL) was induced for 12 hours at 30°C with inducers of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences).

Four Types of Test

The kind and concentration of aromatic compounds are different in these four types of test.
Primary test
Primary test is aims to reveal whether a certain biosensor can be induced by the compounds mentioned in previous research papers.
Inducers (found in previous research papers) were added into the LB medium at 1000 μM (for nontoxic compounds) or 100 μM (for toxic compounds).

ON-OFF test
ON-OFF test functions to land aromatic compounds that can induce a certain biosensor.
78 kinds of aromatic compounds were added into the LB medium at 1000 μM (for nontoxic compounds), 100 μM (for toxic compounds) or 10μM (for benzene).(For detail of all the compounds, Click Here).

Dose-response Curve test
Dose-response Curve test is to deeply characterize the relationship between fluorescence intensity (or induction ratio) and the concentration of inducers.
Inducers found in previous on-off test were added respectively into the LB medium at concentration ranging from micro-molar to mili-molar.

Orthogonality test
Orthogonality test is to prove that a compound which is not an inducer will not influence the detection of inducers.
Two kinds of aromatic compounds (one is an inducer while the other isn’t) were added together into the LB medium at concentration ranging from micro-molar to mili-molar.

Two Methods for Fluorescence Intensity Measurement and Data Analysis

Microplate reader analysis
E.coli were harvested by centrifugation at 4000 r.p.m. for 10 minutes and were suspended in 200 μl of PBS (phosphate-buffered saline). OD600 and GFPuv fluorescence (excitation 485 nm and emission 515 nm) was measured by microplate reader (Thermo).
As for data analysis, OD 600 and fluorescence intensity of PBS measured the same way was subtracted as blank. Net fluorescence intensity of each well was normalized by OD600 of the same well to measure the expression level of GFP. The averages of net fluorescence intensity were obtained from three replicates performed on different 96-well plates.

Flow cytometry analysis
Flow cytometer data were obtained using an LSRFortessa flow cytometer (BD Biosciences). All the data were gated by forward and side scatter, and each data consists of at least 10,000 cells. The geometry mean fluorescence was calculated with FlowJo. The averages of means were obtained from three replicates performed on different 96-well plates.