Team:Jilin China/Notebook

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

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.

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 37 ℃.
Sequencing: Plasmids pET28a-cphA-1 and pET28a-CaO19 were sent out for sequencing.

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

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

07/28/2017
Sequencing: the synthesized plasmid T-A （pET28a-toxin-antitoxin）was sent to sequence.

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.

08/04/2017
OD 600 Measurement: Arabinose (Ara) was added when the OD 600 was 0.3 of T-A bacteria and then split into two groups to measure OD 600 every 2 hours.

08/05/2017
OD 600 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. OD 600 was measured every two hours in the whole procedure.

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 culture.

08/10/2017
Interlab Measurement: EGFP were measured following the Interlab protocol.

08/11/2017
OD 600 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. OD 600 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 culture.
Plate streaking: Bacteria with pET28a-toxin-antitoxin were confirmed with the method of plate streaking in Ara、IPTG、IPTG and Ara medium respectively.

08/12/2017
Interlab Measurement: EGFP were measured following the protocol.
OD 600 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. OD 600 was measured every two hours in the whole procedure.
EGFP measurement: OD600and 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 culture.
Miniprep: pET28a-J23114-Dmpr-GFP-Rluc was extracted and digested to test.

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

08/15/2017
OD 600 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. OD 600 was measured every two hours in the whole procedure.

08/17/2017
OD 600 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. OD 600 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.

08/20/2017
EGFP measurement: EGFP intensity of plasmid pET28a-J23114-Dmpr-GFP-Rluc, pET28a-Pr-Dmpr-GFP-Rluc and mock were measured every 2h.

08/24/2017
PCR: The antitoxin was amplified with primers (Forward: 5’-CCGGAAT TCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’, Reverse: 5‘-TTTTCTGCAGCGGCCGCTACTAGTATTAATTTT TCATTTCGGGCGTCGGATAAAC-3’) using pET28a-toxin-antitoxin as the template.
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 Kitand digested with EcoR I and Pst I .
Sequencing: The plasmids were sent out for sequencing with primers VF2 and VR.

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 with primers (Forward: 5’-CCGGAAT TCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’, Reverse: 5‘-TTTTCTGCAGCGGCCGCTACTAGTATTAATTTT TCATTTCGGGCGTCGGATAAAC-3’) using pET28a-cphA-1 as the template.
Gel extraction: cphA-1 DNA fragment in agarose gel was purified with kit.

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 then 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.

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 were measured for the two groups every 2 hrs. Absorbance 600 were measured for the two groups every 2 hrs.

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 culture.
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.

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 culture.
PCR: The CaO19 fragment was amplified with primers (Forward: 5’-CCGGAATTCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’, Reverse: 5‘-TTTTCTGCAGCGGCCGCTACTAGTATTAAT TTTTCATTTCGGGCGTCGGATAAAC-3’) using pET28a-CaO19 as the template.
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.

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 with primers (Forward: 5’-CCGGAAT TCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’, Reverse: 5‘-TTTTCTGCAGCGGCCGCTACTAGTATTAATTTT TCATTTCGGGCGTCGGATAAAC-3’) using pET28a-toxin-antitoxin as the template. PCR II: The antitoxin was amplified with primers using pET28a-toxin-antitoxin as the template.
Gel extraction: Toxin and antitoxin DNA fragment in agarose gel were purified with kit respectively.

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

09/14/2017
Transformation: The pSB1C3-toxin and pSB1C3-toxin ligation product were transformed intotrans5a 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: 20 colonies/plate were picked for overnight culture.

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 culture.
EGFP measurement: Plasmids for 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 for 3 hrs or 6 hrs. The Renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 were tested for the corresponding luciferase analysis samples.
Miniprep and Digestion: The pSB1C3-toxin and pSB1C3-antitoxin 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.

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 fragment in agarose gel were purified with kit respectively.

09/19/2017
Digestion and Purification: Purified antitoxin and toxin fragment were digested with EcoR I and Pst I, followed with DNA purification by purification kit respectively.
Ligation: EcoRI/PstI-antitoxin and EcoRI/PstI-antitoxin were ligated with EcoRI/PstI-digested pSB1C3 respectively.

09/20/2017
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 for 3 hrs or 12 hrs. The Renilla luciferase was measured following the in vivo ViviRen kit.
Abs 600 measurement: Absorbance 600 were tested for the corresponding luciferase analysis samples.
Transformation: The pSB1C3-antitoxin and the pSB1C3-toxin ligation products were transformed intotrans5a cells respectively.

09/21/2017
Colony selection: 20 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.

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 were measured for the two groups every 2 hrs.

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 for 3 hrs or 6 hrs. The Renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 were tested for the corresponding luciferase analysis samples.

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 for 3, 6, or 12 hrs. The Renilla luciferase was measured following the in vivo ViviRen kit.

10/02/31
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 for 3, 6, or 12 hrs. The 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.

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.

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 were measured for the two groups every 2 hrs.

10/072017
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 for 3, 6, or 12 hrs. The Renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 were tested for the corresponding luciferase analysis samples.

10/082017
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.

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 were measured for the two groups 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 for 3, 6, or 12 hrs. The Renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 were tested for the corresponding luciferase analysis samples.

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 for 3, 6, or 12 hrs. The Renilla luciferase was measured following the in vivo ViviRen kit.

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 for the two groups every 2 hrs. 10/18/2017
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 for 3, 6, or 12 hrs. The Renilla luciferase was measured following the in vivo ViviRen kit. Absorbance 600 were tested for the corresponding luciferase analysis samples.