Team:Jilin China/Notebook

Notebook

07/11/2017
Transformation: pET28a-CaO19 was transformed into Top10, while pET28a-cphA-1 and pET28a-tfdB-JLU were transformed into BL21.

07/12/2017
Miniprep: pET28a-CaO19, pET28a-cphA-1 and pET28a-TfdB-JLU

07/13/2017
Transformation: Plasmids of Interlab test were transformed into DH5α.

07/14/2017
Miniprep: pET28a-cphA-1 was extracted and then was digested to test.

07/15/2017
Miniprep: Plasmids of Interlab were extracted from bacteria DH5α and were digested to test.
Inoculation: pET28a-J23114-DmpR-GFP-Rluc, unloaded pET28a vector (mock)

07/17/2017
EGFP Measurement: EGFP intensity of pET28a-J23114-DmpR-GFP-Rluc or mock was measured with Biotek every hour up to 8 hrs.
Inoculation: pET28a-tfdB-JLU.

07/18/2017
Miniprep: Plasmids pET28a-J23114-DmpR-GFP-Rluc and pET28a-CaO19 were extracted and digested to test.
Induction: Bacteria with TfdB-JLU were induced by IPTG overnight at 18 ℃.
Sequencing: Plasmids pET28a-cphA-1 and pET28a-CaO19 were sent out for sequencing.

07/19/2017
Miniprep: Parts of plasmids of Interlab were extracted from bacteria DH5α and digested.
TfdB-JLU purification: Overnight induced cells were harvested and sonicated. Supernatant after centrifuging were purified by passing through Ni²⁺-NTA column.

07/24/2017
Miniprep: Plasmids pET28a-tfdB-JLU were extracted.

07/28/2017
Sequencing: The synthesized plasmid T-A （pET28a-toxin-antitoxin）was sent to sequence.
Inoculation: pET28a-J23114-DmpR-GFP-Rluc, mock

07/29/2017
EGFP Measurement: Absorbance 600 and EGFP intensity of pET28a-J23114-DmpR-GFP-Rluc and mock were measured with Biotek every two hours.
Inoculation: pET28a-T-A

08/04/2017
OD600 Measurement: Arabinose (Ara) was added into T-A bacteria when the OD600 was 0.3 and then split into two groups to measure OD600 every 2 hours.
Inoculation: pET28a-T-A

08/05/2017
OD600 Measurement: Bacteria containing T-A were split into two groups at OD600 around 0.3. IPTG and Ara were added at same time, or IPTG was added 6 hrs later after Ara adding. OD600 was measured every two hours in the whole procedure.
Inoculation: pET28a-J23114-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a vector

08/08/2017
Interlab Measurement: EGFP intensity were measured following the Interlab protocol.
EGFP Measurement: EGFP intensity of pET28a-Pr-DmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc and mock were measured with Biotek every 2 hrs until 8 hrs.

08/10/2017
Interlab Measurement: EGFP were measured following the Interlab protocol.
Inoculation: pET28a-J23114-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a vector, pET28a-T-A

08/11/2017
OD600 Measurement: Bacteria containing T-A were split into two groups at OD600 around 0.3. IPTG and Ara were added at same time, or IPTG was added 6 hrs later after Ara adding. OD600 was measured every two hours in the whole procedure.
EGFP measurement: Absorbance 600 and EGFP intensity of pET28a-J23114-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc and mock were measured with Biotek every two hours till 8 hrs after 24 hrs.
Plate streaking: Bacteria with pET28a-toxin-antitoxin were confirmed with the method of plate streaking in Ara,IPTG,IPTG and Ara medium respectively.
Inoculation: pET28a-J23114-DmpR-GFP-Rluc, mock, pET28a-Pr-DmpR-GFP-Rluc, pET28a vector, pET28a-T-A

08/12/2017
Interlab Measurement: EGFP were measured following the protocol.
OD600 Measurement: Bacteria containing T-A were split into two groups at OD600 around 0.3. IPTG and Ara were added at same time, or IPTG was added 6 hrs later after Ara adding. OD600 was measured every two hours in the whole procedure.
EGFP Measurement: OD600 and EGFP intensity of pET28a-J23114-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc and mock were measured with Biotek continually very two hours till 8 hrs after 24 hrs.
Miniprep: pET28a-J23114-DmpR-GFP-Rluc was extracted and digested to test.
Inoculation: pET28a vector, pET28a-T-A

08/14/2017
Inoculation: pET28a vector, pET28a-T-A
Induction: IPTG was added into bacteria containing pET28a-toxin-antitoxin when OD600 was 0.3.

08/15/2017
OD600 Measurement: Bacteria containing T-A were split into two groups at OD600 around 0.3. IPTG and Ara were added at same time, or IPTG was added 6 hrs later after Ara adding. OD600 was measured every two hours in the whole procedure.
Inoculation: pET28a vector, pET28a-T-A

08/17/2017
OD600 Measurement: Bacteria containing T-A were split into two groups at OD600 around 0.3. IPTG and Ara were added at same time, or IPTG was added 6 hrs later after Ara adding. OD600 was measured every two hours in the whole procedure.

08/18/2017
Miniprep: Plasmids pET28a-J23114-DmpR-GFP-Rluc and pET28a-toxin-antitoxin were extracted and digested.
Inoculation: pET28a-J23114-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, mock

08/20/2017
EGFP Measurement: EGFP intensity of pET28a-J23114-DmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc and mock were measured every 2 hrs.

08/24/2017
PCR: The antitoxin was amplified using pET28a-toxin-antitoxin as the template.
Forward primer: 5’-CCGGAATTCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’
Reverse primer: 5’-TTTTCTGCAGCGGCCGCTACTAGTATTAATTTTTCATTTCGGGCGTCGGATAAAC-3’
Gel extraction: Antitoxin DNA fragment in agarose gel was purified with kit.

08/25/2017
Digestion and Purification: Purified antitoxin fragment was digested with EcoR I and Pst I, followed with DNA purification by purification kit.
Ligation: EcoR I/Pst I-antitoxin was ligated with EcoR I/Pst I-digested pSB1C3.

08/26/2017
Transformation: The pSB1C3-antitoxin ligation product was transformed into trans5a cells.

08/27/2017
Colony selection: 10 colonies/plate were picked for overnight culture.

08/29/2017
Miniprep and Digestion: The pSB1C3-antitoxin were extracted with TIANprep Rapid Mini Plasmid Kit and digested with EcoR I and Pst I .
Sequencing: The plasmids were sent out for sequencing with primers VF2 and VR.
Inoculation: pET28a-Pr-PEKDmpR-GFP-Rluc, mock

09/01/2017
EGFP Measurement: EGFP intensity of pET28a-Pr-PEKDmpR-GFP-Rluc and mock was measured at 0, 2, 3.5 hrs.
PCR: The cphA-1 was amplified using pET28a-cphA-1 as the template.
Forward primer: 5’-CCGGAATTCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’
Reverse primer: 5’-TTTTCTGCAGCGGCCGCTACTAGTATTAATTTTTCATTTCGGGCGTCGGATAAAC-3
Gel extraction: cphA-1 DNA fragment in agarose gel was purified with kit.
Inoculation: pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a, pET28a-T-A

09/02/2017
Digestion and Purification: Purified cphA-1 fragment was digested with EcoR I and Pst I, followed with DNA purification by purification kit.
Ligation: EcoR I/PstI-cphA-1 was ligated with EcoR I/Pst I-digested pSB1C3.
Transformation: Plasmids pET28a-J23101-DmpR-GFP-Rluc and pET28a-J23107-DmpR-GFP-Rluc were transformed into BL21.
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and followed by 0.1, 0.5 or 1.0 mM IPTG respectively. Absorbance 600 were measured for the two groups every 2 hrs.
EGFP Measurement: EGFP intensity of pET28a-Pr-PEKDmpR-GFP-Rluc and mock were tested every 1 hour up to 24 hrs.

09/03/2017
Transformation: The pSB1C3-cphA-1 ligation product was transformed into trans5a cells.
Inoculation: pET28a-T-A

09/04/2017
Colony selection: 10 colonies/plate were picked for overnight culture.
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and then followed by 0.1, 0.5 or 1.0 mM IPTG respectively. Absorbance 600 of the two groups were measured every 2 hrs.
Inoculation: pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc, pET28a-T-A, pET28a vector

09/05/2017
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and then followed with 0.1, 0.5 or 1.0 mM IPTG respectively. Absorbance 600 were measured for the two groups every 2 hrs.
EGFP Measurement: EGFP intensity of pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc and mock were measured every hour until 24 hrs.
Miniprep and Digestion: The pSB1C3-cphA-1 were extracted with TIANprep Rapid Mini Plasmid Kit and digested with EcoR I and Pst I .

Sequencing: The plasmids were sent out for sequencing with primers VF2 and VR.
Inoculation: pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc, pET28a vector

09/07/2017
EGFP Measurement: EGFP intensity of pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc and mock were measured every hour until 24 hrs.
PCR: The CaO19 fragment was amplified using pET28a-CaO19 as the template.
Forward primer: 5’-CCGGAATTCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’
Reverse primer: 5'-TTTTCTGCAGCGGCCGCTACTAGTATTAATTTTTCATTTCGGGCGTCGGATAAAC-3’
Gel extraction: CaO19 DNA fragment in agarose gel was purified with kit.

09/08/2017
Digestion and Purification: Purified CaO19 fragment was digested with EcoR I and Pst I, followed with DNA purification by kit.
Ligation: EcoR I/PstI-CaO19 was ligated with EcoR I/Pst I-digested pSB1C3.

09/09/2017
Induction: 0, 0.05, 0.1 or 0.5% IPTG was added into T-A bacteria respectively when OD600 was 0.3.
Transformation: The pSB1C3-CaO19 ligation product was transformed into trans5a cells.
Inoculation: pET28a-T-A

09/10/2017
Induction: 0, 0.05, 0.1 or 0.5% IPTG was added into T-A bacteria respectively when OD600 was 0.3.
Colony selection: 10 colonies/plate were picked for overnight culture.
Miniprep and Digestion: The pSB1C3-CaO19 were extracted with TIANprep Rapid Mini Plasmid Kit and digested with EcoR I and Pst I.
Sequencing: The plasmids were sent out for sequencing with primers VF2 and VR.

09/12/2017
PCR I: The toxin was amplified using pET28a-toxin-antitoxin as the template.
Forward primer: 5’-CCGGAATTCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’
Reverse primer: 5’-TTTTCTGCAGCGGCCGCTACTAGTATTAATTTTTCATTTCGGGCGTCGGATAAAC-3’
PCR II: The antitoxin was amplified with primers using pET28a-toxin-antitoxin as the template.
PCR III: The DmpR was amplified using pET28a-J23107-DmpR-GFP-Rluc as the template.
Forward primer: 5’-CCGGAATTCGCGGCCGCTTCTAGATGCCGATCAAGTACAAGCCTGAAATCC-3’
Reverse primer: 5’-TTTTCTGCAGCGGCCGCTACTAGTATTA GCCTTCGATGCCGATTTTCTTCAGC-3’
Gel extraction: Toxin, antitoxin and DmpR DNA fragment in agarose gel were purified with kit respectively.

09/13/2017
Digestion and Purification: Purified toxin, antitoxin and DmpR fragment were digested with EcoR I and Pst I, followed with DNA purification by kit respectively.
Ligation: EcoR I/PstI-toxin, EcoR I/PstI-antitoxin and EcoR I/PstI-DmpR were ligated with EcoR I/Pst I-digested pSB1C3 respectively.
Inoculation: pET28a-T-A

09/14/2017
Transformation: The pSB1C3-toxinpSB，1C3-antitoxin and pSB1C3-DmpR ligation product were transformed into trans5a cells respectively.
Induction: 0, 0.05, 0.1 or 0.5% IPTG was added into T-A bacteria respectively when OD600 was 0.3.

09/15/2017
Colony selection: 8 colonies/plate were picked for overnight culture.
Inoculation: pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc, pET28a vector

09/16/2017
EGFP Measurement: EGFP intensity of pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc and mock were measured every hour until 24 hrs.
EGFP measurement: Plasmids of Interlab were measured EGFP following protocol.
Luciferase analysis: Bacteria containing Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc or mock were inoculated and cultured. 16 hrs later, phenols were added. 3 hrs or 6 hrs later, renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 of the corresponding luciferase analysis samples were measured.
Miniprep and Digestion: The pSB1C3-toxin, pSB1C3-antitoxin and pSB1C3-DmpR were extracted with TIANprep Rapid Mini Plasmid Kit and digested with EcoR I and Pst I respectively.

Sequencing: The plasmids were sent out for sequencing with primers VF2 and VR.
Inoculation: pET28a-tfdB-JLU

09/18/2017
PCR: The toxin and antitoxin fragments were amplified with respective primers using pET28a-toxin-antitoxin as the template.
Gel extraction: Antitoxin and toxin DNA fragments in agarose gel were purified with kit respectively.
Induction: Bacteria with TfdB-JLU were induced by IPTG overnight at 18℃.

09/19/2017
Digestion and Purification: Purified antitoxin and toxin fragments were digested with EcoR I and Pst I, followed with DNA purification by purification kit respectively.
Ligation: EcoR I/PstI-antitoxin and EcoR I/PstI-antitoxin were ligated with EcoR I/PstI-digested pSB1C3 respectively.
TfdB-JLU purification: Overnight induced cells were harvested and sonicated. Supernatant after centrifuging were purified by passing through Ni²⁺-NTA column.
Enzyme assay: Incubated TfdB-JLU with 2,4-DCP and measure enzyme activity.
Inoculation: Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc, mock

09/20/2017
Luciferase analysis: Bacteria containing Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpRr-GFP-Rluc or mock were inoculated and cultured. 16 hrs later, phenols were added. 3 hrs or 12 hrs, renilla luciferase was measured following the in vivo ViviRen kit.
Abs600 Measurement: Absorbance 600 of the corresponding luciferase analysis samples were tested.
Transformation: The pSB1C3-antitoxin and the pSB1C3-toxin ligation products were transformed into trans5a cells respectively.
Enzyme assay: TfdB-JLU was incubated with 3-CP, 4-CP, phenol, 2,3-DCP, 2,5-DCP, 2,6-DCP, 3,4-DCP, 3,5-DCP, 2,4,5-TCP and enzyme activity was measured.

09/21/2017
Colony selection: 10 colonies/plate were picked for overnight culture.

09/22/2017
Miniprep and Digestion: The pSB1C3-antitoxin and the pSB1C3-toxin were extracted with TIANprep Rapid Mini Plasmid Kitand digested with EcoR I and Pst I.
Sequencing: The plasmids were sent out for sequencing with primers VF2 and VR.
Inoculationg: pET28a-T-A

09/24/2017
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and then followed by 0.1, 0.5 or 1.0 mM IPTG respectively. Absorbance 600 of the two groups were measured every 2 hrs.
Inoculationg: Ctrl, TfdB-JLU, mock Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc

09/26/2017
Luciferase analysis: Bacteria containing Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc or mock were inoculated and cultured. 16 hrs later, phenols were added. 3 hrs or 6 hrs later, renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 of the corresponding luciferase analysis samples were tested.
Inoculation: Ctrl, Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc
Induction: Bacteria with TfdB-JLU were induced by IPTG overnight at 18℃.

10/01/2017
Luciferase analysis: Bacteria containing Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc or mock were inoculated and cultured. 16 hrs later, phenols were added. 3, 6 or 12 hrs later, renilla luciferase was measured following the in vivo ViviRen kit.
HPLC: Bacteria containing TfdB-JLU and mock were incubated with 1, 2 or 5 mM phenol in PBS for 2 hrs, the assay was done using 2 hrs incubation supernatant.
4-AAP assay: Bacteria containing TfdB-JLU and mock were incubated with 1, 2 or 5 mM phenol in PBS for 4 hrs, the assay was done with 4 hrs incubation supernatant.
Inoculation: Ctrl, pET28a-T-A, Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc

10/02/2017
Luciferase analysis: Bacteria containing Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc or mock were inoculated and cultured. 16 hrs later, phenols were added. 3, 6, or 12 hrs later, renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 were tested for the corresponding luciferase analysis samples.
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and then following with 0.1, 0.5 or 1.0 mM IPTG respectively. Absorbance 600 were measured for the two groups every 2 hrs.
Inoculation: pET28a-TfdB-JLU, mock, Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc
PCR: The DmpR fragment was amplified with primers using J23107-DmpR-GFP-Rluc as the template.
Gel extraction: DmpR DNA fragment in agarose gel was purified with kit respectively.
Ligation: Purified DmpR fragement was ligated with pGEM-T Easy Vector.

10/04/2017
EGFP measurement: EGFP intensity of pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc and mock were measured every one hour until 24 hrs.
Inoculation: pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc, mock, pET28a-T-A
Transformation: The pGEM-T Easy-DmpR ligation product was transformed into trans5a cells.

10/05/2017
EGFP measurement: EGFP intensity of pET28a-Pr-PEKDmpR-GFP-Rluc, pET28a-Pr-DmpR-GFP-Rluc, pET28a-J23114-DmpR-GFP-Rluc, pET28a-J23101-DmpR-GFP-Rluc, pET28a-J23107-DmpR-GFP-Rluc and mock were measured every one hour until 24 hrs.
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and then following with 0.1, 0.5 or 1.0 mM IPTG respectively. Absorbance 600 of the two groups were measured every 2 hrs.
Inoculation: Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, mock
Inoculation: Bacteria with TfdB-JLU were induced by IPTG overnight at 18℃.
Colony selection: 10 colonies/plate were picked for overnight culture.

10/07/2017
Luciferase analysis: Bacteria containing Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc or mock were inoculated and cultured. 16 hrs later, 1mM or 2mM phenols were added. 3, 6 or 12 hrs later, renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 of the corresponding luciferase analysis samples were tested.
HPLC: Bacteria containing TfdB-JLU and mock were incubated with 1, 2 or 5 mM phenol in PBS for 4 hrs, the assay was done using 4 hrs incubation supernatant.
4-AAP assay: Bacteria containing TfdB-JLU and mock were incubated with 1, 2 or 5 mM phenol in PBS for 8 hrs, the assay was done with 8 hrs incubation supernatant.
Inoculation: pET28a-T-A
Miniprep and Digestion: The pGEM-T Easy-DmpR was extracted with TIANprep Rapid Mini Plasmid Kit and digested with EcoR I.
Digestion: One of the identified pGEM-T Easy-DmpR plasmid was digested with EcoR I and Pst I.

10/08/2017
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and then following with 0.1, 0.5 or 1.0 mM IPTG respectively. Absorbance 600 of the two groups were measured every 2 hrs.
Inoculation: pET28a-T-A, Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, mock
Gel extration: DmpR DNA fragment (got from pGEM-T Easy-DmpR) in agarose gel was purified with kit respectively.
Ligation: Purified DmpR fragement was ligated with EcoR I/PstI-digested pSB1C3.

10/11/2017
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and then following with 0.1, 0.5 or 1.0 mM IPTG respectively. Absorbance 600 of the two groups were measured every 2 hrs.
Luciferase analysis: Bacteria containing Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc or mock were inoculated and cultured. 16 hrs later, 1mM or 2mM phenols were added. 3, 6 or 12 hrs later, renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 of the corresponding luciferase analysis samples were measured.
Inoculation: Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc, mock
Transformation: The pSB1C3-DmpR ligation product was transformed into trans5a cells.

10/14/2017
Luciferase analysis: Bacteria containing Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc or mock were inoculated and cultured. 16 hrs later, phenols were added. 3, 6 or 12 hrs later, renilla luciferase was measured following the in vivo ViviRen kit.
Inoculation: pET28a-T-A, pET28a-TfdB-JLU, mock
Colony selection: 10 colonies/plate were picked for overnight culture.

10/15/2017
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and then following with 0.1, 0.5 or 1.0 mM IPTG respectively. Absorbance 600 were measured of the two groups were measured every 2 hrs.
Sequencing: The pSB1C3-DmpR plasmids were sent out for sequencing with primers VF2 and VR.
Abs600 Measurement: 0.1% or 0.2% Ara was added into the T-A bacteria and then following with 0.1,0.5 or 1.0 mM IPTG respectively. Absorbance 600 of the two groups were measured every 2 hrs.
Inoculation: Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc, mock
Induction: Bacteria with TfdB-JLU were induced by IPTG overnight at 18℃.

10/16/2017
Luciferase analysis: Bacteria containing Pr-PEKDmpR-GFP-Rluc, Pr-DmpR-GFP-Rluc, J23114-DmpR-GFP-Rluc, J23101-DmpR-GFP-Rluc, J23107-DmpR-GFP-Rluc or mock were inoculated and cultured. 16 hrs later, phenols were added. 3, 6 or 12 hrs later, renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 of the corresponding luciferase analysis samples were measured.
HPLC: Bacteria containing TfdB-JLU or mock was incubated with 1, 2 or 5 mM phenol in PBS for 4 hrs, the assay was done with 4 hrs incubation supernatant.
4-AAP assay: Bacteria containing TfdB-JLU or mock was incubated with 1, 2 or 5 mM phenol in PBS for 12 hrs, the assay was done with 12 hrs incubation supernatant.
Induction: Ctrl, pET28a-J23107-DmpR-TfdB-JLU
Digestion: One of the identified pGEM-T Easy-DmpR plasmid (differ from 10.7) was digested with EcoR I and Pst I.

10/17/2017
Inoculation: pET28a-TfdB-JLU, ctrl
OD600 Measurement and phenol test: Bacteria with J23107-DmpR-TfdB-JLU or ctrl was transferred to conical flask. Phenol was added 2 hrs later. OD600 was measured at once after phenol's adding and subsequent every 2 hrs till 24 hrs. Phenol was tested 2, 6, 10 or 24 hrs after its adding with both HPLC method and 4-AAP assay.
Gel extraction: DmpR DNA fragment(got from pGEM-T Easy-DmpR) in agarose gel was purified with kit respectively.
Ligation: Purified DmpR fragement was ligated with EcoRI/PstI-digested pSB1C3.

10/18/2017
Induction: Bacteria with TfdB-JLU were induced by IPTG overnight at 18℃.
Transformation: The pSB1C3-DmpR ligation product was transformed into trans5a cells.

10/19/2017
HPLC: Bacteria containing TfdB-JLU or mock was incubated with 1, 2 or 5 mM phenol in PBS for 4 hrs, the assay was done with 4 hrs incubation supernatant.
HPLC: Purified enzyme TfdB-JLU or mock was incubated with 1, 2 or 5 mM phenol in PBS for 1 hrs, the assay was done with 1 hrs incubation supernatant.
4-AAP assay: Bacteria containing TfdB-JLU or mock was incubated with 1, 2 or 5 mM phenol in PBS for 12 hrs, the assay was done with 12 hrs.
Inoculation: pET28a-TfdB-JLU, ctrl, pET28a-J23107-DmpR-TfdB-JLU
Colony selection: 10 colonies/plate were picked for overnight culture.

10/20/2017
Miniprep and Digestion: The pSB1C3-DmpR was extracted with TIANprep Rapid Mini Plasmid Kit and digested with EcoRI+Pst I.
Sequencing: The pSB1C3-DmpR plasmids were sent out for sequencing with primers VF2 and VR.
Induction: Bacteria with TfdB-JLU were induced by IPTG overnight at 18℃.
OD600 Measurement and phenol test: Bacteria with J23107-DmpR-TfdB-JLU or ctrl was transferred to conical flask. Phenol was added 2 hrs later. OD600 was measured at once after phenol's adding and subsequent every 2 hrs till 24 hrs culture. Phenol was tested 2, 6, 10 or 24 hrs after its adding with HPLC method and 4-AAP assay.

10/21/2017
HPLC: Bacteria containing TfdB-JLU or mock was incubated with 1, 2 or 5 mM phenol in PBS for 4 hrs, the assay was done with 4 hrs incubation supernatant.
4-AAP assay: Bacteria containing TfdB-JLU or mock was incubated with 1, 2 or 5 mM phenol in PBS for 12 hrs, the assay was done with 12 hrs.
Inoculation: ctrl, pET28a-J23107-DmpR-TfdB-JLU

10/22/2017
OD600 Measurement and phenol test: Bacteria with J23107-DmpR-tfdB-JLU or ctrl was transferred to conical flask. Phenol was added 2 hrs later. OD600 was measured at once after phenol's adding and subsequent every 2 hrs till 24 hrs. Phenol was tested 2, 6, 10 or 24 hrs after its adding with HPLC method and 4-AAP assay.
Inoculation: ctrl, pET28a-J23107-DmpR-TfdB-JLU

10/23/2017
OD600 Measurement and phenol test: Bacteria with J23107-DmpR-tfdB-JLU or ctrl was transferred to conical flask. Phenol was added 2 hrs later. OD600 was measured at once after phenol's adding and subsequent every 2 hrs till 24 hrs. Phenol was tested 2, 6, 10 or 24 hrs after its adding with HPLC method and 4-AAP assay.

10/24/2017
Inoculation: ctrl, pET28a-J23107-DmpR-TfdB-JLU

10/25/2017
OD600 Measurement and phenol test: Bacteria with J23107-DmpR-TfdB-JLU or ctrl was transferred to conical flask. Phenol was added 2 hrs later. Abs600 was measured at once after phenol's adding and subsequent every 2 hrs till 24 hrs culture. Phenol was tested 2, 6, 10 or 24 hrs after its adding with both HPLC method and 4-AAP assay.