Difference between revisions of "Team:Jilin China/Notebook"

Revision as of 03:02, 27 October 2017

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.

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.

@@ Line 2: / Line 2: @@
 <html>
+<div class="banner"><img src="https://static.igem.org/mediawiki/2017/b/b1/T--Jilin_China--_sec_bg_t.jpg"></div>
-<div class="column full_size">
+<div class="thr_box">
+	<p>07/11/2017 <br />
-<h1>Notebook</h1>
+	<strong style="color: #229d73;">Transformation:</strong> pET28a-CaO19 was transformed into Top10, while pET28a-cphA-1and pET28a-tfdB-JLU were transformed into BL21.
-<p> Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.</p>
+	</p><p>
+/12/2017 <br />
-</div>
+	<strong style="color: #229d73;">Miniprep:</strong> pET28a-CaO19, pET28a-cphA-1 and pET28a-tfdB-JLU
-<div class="clear"></div>
+	</p><p>
+/13/2017<br />
+	<strong style="color: #229d73;">Transformation:</strong> Plasmids of Interlab test were transformed into DH5α.
-<div class="column half_size">
+	</p><p>
-<h5>What should this page have?</h5>
+/14/2017<br />
-<ul>
+	<strong style="color: #229d73;">Miniprep:</strong> pET28a-cphA-1 was extracted and then was digested to test.
-<li>Chronological notes of what your team is doing.</li>
+	</p><p>
-<li> Brief descriptions of daily important events.</li>
+/15/2017<br />
-<li>Pictures of your progress. </li>
+	<strong style="color: #229d73;">Miniprep:</strong> Plasmids of Interlab were extracted from bacteria DH5α and were digested to test..
-<li>Mention who participated in what task.</li>
+	</p><p>
-</ul>
+/17/2017<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> EGFP intensity of pET28a-J23114-Dmpr-GFP-Rluc or mock was measured with Biotek every hour up to 8 hrs.
-</div>
+	</p><p>
+/18/2017<br />
-<div class="column half_size">
+	<strong style="color: #229d73;">Miniprep:</strong> Plasmids pET28a-J23114-Dmpr-GFP-Rluc and pET28a-CaO19 were extracted and digested to test . <br />
-<h5>Inspiration</h5>
+	<strong style="color: #229d73;">Induction:</strong> Bacteria with tfdB-JLU were induced by IPTG overnight at 37 ℃.<br />
-<p>You can see what others teams have done to organize their notes:</p>
+	<strong style="color: #229d73;">Sequencing:</strong> Plasmids pET28a-cphA-1 and pET28a-CaO19 were sent out for sequencing.
+	</p><p>
-<ul>
+/19/2017<br />
-<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
+	<strong style="color: #229d73;">Miniprep:</strong> Parts of plasmids for Interlab were extracted from bacteria DH5α and digested.
-<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
+	</p><p>
-<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
+/24/2017<br />
-<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
+	<strong style="color: #229d73;">Miniprep:</strong> Plasmid pET28a-tfdB-JLU were extracted.
-</ul>
+	</p><p>
+/28/2017<br />
+	<strong style="color: #229d73;">Sequencing:</strong> the synthesized plasmid T-A （pET28a-toxin-antitoxin）was sent to sequence.
+	</p><p>
+/29/2017<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> Absorbance 600 and EGFP intensity of pET28a-J23114-Dmpr-GFP-Rluc and mock were measured with Biotek every two hours.
+	</p><p>
+/04/2017<br />
+	<strong style="color: #229d73;">OD 600 Measurement:</strong> 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.
+	</p><p>
+/05/2017<br />
+	<strong style="color: #229d73;">OD 600 Measurement:</strong> 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.
+	</p><p>
+/08/2017<br />
+	<strong style="color: #229d73;">Interlab Measurement:</strong> EGFP intensity were measured following the Interlab protocol.<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> 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.
+	</p><p>
+/10/2017<br />
+	<strong style="color: #229d73;">Interlab Measurement:</strong> EGFP were measured following the Interlab protocol.
+	</p><p>
+/11/2017<br />
+	<strong style="color: #229d73;">OD 600 Measurement:</strong> 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. <br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> 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. <br />
+	<strong style="color: #229d73;">Plate streaking:</strong> Bacteria with pET28a-toxin-antitoxin were confirmed with the method of plate streaking in Ara、IPTG、IPTG and Ara medium respectively.
+	</p><p>
+/12/2017<br />
+	<strong style="color: #229d73;">Interlab Measurement:</strong> EGFP were measured following the protocol.<br />
+	<strong style="color: #229d73;">OD 600 Measurement:</strong> 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.  <br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> 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. <br />
+	<strong style="color: #229d73;">Miniprep:</strong> pET28a-J23114-Dmpr-GFP-Rluc was extracted and digested to test.
+	</p><p>
+/14/2017<br />
+	<strong style="color: #229d73;">Induction:</strong> IPTG was added into bacteria containing pET28a-toxin-antitoxin when OD600 was 0.3.
+	</p><p>
+/15/2017<br />
+	<strong style="color: #229d73;">OD 600 Measurement:</strong> 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.
+	</p><p>
+/17/2017<br />
+	<strong style="color: #229d73;">OD 600 Measurement:</strong> 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.
+	</p><p>
+/18/2017<br />
+	<strong style="color: #229d73;">Miniprep:</strong> Plasmids pET28a-J23114-Dmpr-GFP-Rluc and pET28a-toxin-antitoxin were extracted and digested.
+	</p><p>
+/20/2017<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> EGFP intensity of plasmid pET28a-J23114-Dmpr-GFP-Rluc, pET28a-Pr-Dmpr-GFP-Rluc and mock were measured every 2h.
+	</p><p>
+/24/2017<br />
+	<strong style="color: #229d73;">PCR:</strong> The antitoxin was amplified with primers (Forward: 5’-CCGGAAT
+	TCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’, Reverse: 5‘-TTTTCTGCAGCGGCCGCTACTAGTATTAATTTT
+	TCATTTCGGGCGTCGGATAAAC-3’) using pET28a-toxin-antitoxin as the template.<br />
+	<strong style="color: #229d73;">Gel extraction:</strong> Antitoxin DNA fragment in agarose gel was purified with kit.
+	</p><p>
+/25/2017<br />
+	<strong style="color: #229d73;">Digestion and Purification:</strong> Purified antitoxin fragment was digested with EcoR I and Pst I, followed with DNA purification by purification kit.<br />
+	<strong style="color: #229d73;">Ligation:</strong> EcoR I/Pst I-antitoxin was ligated with EcoR I/Pst I-digested pSB1C3.
+	</p><p>
+/26/2017<br />
+	<strong style="color: #229d73;">Transformation:</strong> The pSB1C3-antitoxin ligation product was transformed into trans5a cells.
+	</p><p>
+/27/2017<br />
+	<strong style="color: #229d73;">Colony selection:</strong> 10 colonies/plate were picked for overnight culture.
+	</p><p>
+/29/2017<br />
+	<strong style="color: #229d73;">Miniprep and Digestion:</strong> The pSB1C3-antitoxin were extracted with TIANprep Rapid Mini Plasmid Kitand digested with EcoR I and Pst I .<br />
+	<strong style="color: #229d73;">Sequencing:</strong> The plasmids were sent out for sequencing with primers VF2 and VR.
+	</p><p>
+/01/2017<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> EGFP intensity of pET28a-Pr-PEKDmpr-GFP-Rluc and mock was measured at 0, 2, 3.5 hrs.<br />
+	<strong style="color: #229d73;">PCR:</strong> The cphA-1 was amplified with primers (Forward: 5’-CCGGAAT
+	TCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’, Reverse: 5‘-TTTTCTGCAGCGGCCGCTACTAGTATTAATTTT
+	TCATTTCGGGCGTCGGATAAAC-3’) using pET28a-cphA-1 as the template.<br />
+	<strong style="color: #229d73;">Gel extraction:</strong> cphA-1 DNA fragment in agarose gel was purified with kit.
+	</p><p>
+/02/2017<br />
+	<strong style="color: #229d73;">Digestion and Purification:</strong> Purified cphA-1 fragment was digested with EcoR I and Pst I, followed with DNA purification by purification kit.<br />
+	<strong style="color: #229d73;">Ligation:</strong> EcoR I/PstI-cphA-1 was ligated with EcoR I/Pst I-digested pSB1C3.<br />
+	<strong style="color: #229d73;">Transformation:</strong> Plasmids pET28a-J23101-Dmpr-GFP-Rluc and pET28a-J23107-Dmpr-GFP-Rluc were transformed into BL21. <br />
+	<strong style="color: #229d73;">Abs600 Measurement:</strong> 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.<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> EGFP intensity of pET28a-Pr-PEKDmpr-GFP-Rluc  and mock were tested every 1 hour up to 24 hrs.
+	</p><p>
+/03/2017<br />
+	<strong style="color: #229d73;">Transformation:</strong> The pSB1C3-cphA-1 ligation product was transformed into trans5a cells.
+	</p><p>
+/04/2017<br />
+	<strong style="color: #229d73;">Colony selection:</strong> 10 colonies/plate were picked for overnight culture.<br />
+	<strong style="color: #229d73;">Abs600 Measurement:</strong> 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.
+	</p><p>
+/05/2017<br />
+	<strong style="color: #229d73;">Abs600 Measurement:</strong> 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. <br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> 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.<br />
+	<strong style="color: #229d73;">Miniprep and Digestion:</strong> The pSB1C3-cphA-1 were extracted with TIANprep Rapid Mini Plasmid Kit and digested with EcoR I and Pst I .<br />
+	<img src="https://static.igem.org/mediawiki/2017/2/2a/T--Jilin_China--notebook1.png" /><br />
+	<strong style="color: #229d73;">Sequencing:</strong> The plasmids were sent out for sequencing with primers VF2 and VR.
+	</p><p>
+/07/2017 <br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> 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.<br />
+	<strong style="color: #229d73;">PCR:</strong> The CaO19 fragment was amplified with primers (Forward: 5’-CCGGAATTCGCGGCCGCTTCTAGATGTCAGACACACTCCCCGGGACAACAC-3’, Reverse: 5‘-TTTTCTGCAGCGGCCGCTACTAGTATTAAT
+	TTTTCATTTCGGGCGTCGGATAAAC-3’) using pET28a-CaO19 as the template.<br />
+	<strong style="color: #229d73;">Gel extraction:</strong> CaO19 DNA fragment in agarose gel was purified with kit.
+	</p><p>
+/08/2017<br />
+	<strong style="color: #229d73;">Digestion and Purification:</strong> Purified CaO19 fragment was digested with EcoR I and Pst I, followed with DNA purification by kit.<br />
+	<strong style="color: #229d73;">Ligation:</strong> EcoR I/PstI-CaO19 was ligated with EcoR I/Pst I-digested pSB1C3.
+	</p><p>
+/09/2017<br />
+	<strong style="color: #229d73;">Induction:</strong> 0, 0.05, 0.1 or 0.5% IPTG was added into T-A bacteria respectively when OD600 was 0.3.<br />
+	<strong style="color: #229d73;">Transformation:</strong> The pSB1C3-CaO19 ligation product was transformed into trans5a cells.
+	</p><p>
+/10/2017<br />
+	<strong style="color: #229d73;">Induction:</strong> 0, 0.05, 0.1 or 0.5% IPTG was added into T-A bacteria respectively when OD600 was 0.3.<br />
+	<strong style="color: #229d73;">Colony selection:</strong> 10 colonies/plate were picked for overnight culture.<br />
+	<strong style="color: #229d73;">Miniprep and Digestion:</strong> The pSB1C3-CaO19 were extracted with TIANprep Rapid Mini Plasmid Kit and digested with EcoR I and Pst I.<br />
+	<strong style="color: #229d73;">Sequencing:</strong> The plasmids were sent out for sequencing with primers VF2 and VR.
+	</p><p>
+/12/2017 <br />
+	<strong style="color: #229d73;">PCR I:</strong> 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.<br />
+	<strong style="color: #229d73;">Gel extraction:</strong> Toxin and antitoxin DNA fragment in agarose gel were purified with kit respectively.
+	</p><p>
+/13/2017<br />
+	<strong style="color: #229d73;">Digestion and Purification:</strong> Purified toxin and antitoxin fragment were digested with EcoR I and Pst I, followed with DNA purification by kit respectively.<br />
+	<strong style="color: #229d73;">Ligation:</strong> EcoR I/PstI-toxin and EcoR I/PstI-toxin were ligated with EcoR I/Pst I-digested pSB1C3 respectively.
+	</p><p>
+/14/2017<br />
+	<strong style="color: #229d73;">Transformation:</strong> The pSB1C3-toxin and pSB1C3-toxin ligation product were transformed intotrans5a cells respectively.<br />
+	<strong style="color: #229d73;">Induction:</strong> 0, 0.05, 0.1 or 0.5% IPTG was added into T-A bacteria respectively when OD600 was 0.3.
+	</p><p>
+/15/2017<br />
+	<strong style="color: #229d73;">Colony selection:</strong> 20 colonies/plate were picked for overnight culture.
+	</p><p>
+/16/2017<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> 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.<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> Plasmids for of Interlab were measured EGFP following protocol.<br />
+	<strong style="color: #229d73;">Luciferase analysis:</strong> 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.<br />
+	<strong style="color: #229d73;">Miniprep and Digestion:</strong> The pSB1C3-toxin and pSB1C3-antitoxin were extracted with TIANprep Rapid Mini Plasmid Kit and digested with EcoR I and Pst I respectively.<br />
+	<img src="https://static.igem.org/mediawiki/2017/3/3d/T--Jilin_China--notebook2.png" /><br />
+	<strong style="color: #229d73;">Sequencing:</strong> The plasmids were sent out for sequencing with primers VF2 and VR.
+	</p><p>
+/18/2017<br />
+	<strong style="color: #229d73;">PCR:</strong> The toxin and antitoxin fragments were amplified with respective primers using pET28a-toxin-antitoxin as the template.<br />
+	<strong style="color: #229d73;">Gel extraction:</strong> Antitoxin and toxin DNA fragment in agarose gel were purified with kit respectively.
+	</p><p>
+/19/2017<br />
+	<strong style="color: #229d73;">Digestion and Purification:</strong> Purified antitoxin and toxin fragment were digested with EcoR I and Pst I, followed with DNA purification by purification kit respectively.<br />
+	<strong style="color: #229d73;">Ligation:</strong> EcoRI/PstI-antitoxin and EcoRI/PstI-antitoxin were ligated with EcoRI/PstI-digested pSB1C3 respectively.
+	</p><p>
+/20/2017<br />
+	<strong style="color: #229d73;">Luciferase analysis:</strong> 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. <br />
+	<strong style="color: #229d73;">Abs 600 measurement:</strong> Absorbance 600 were tested for the corresponding luciferase analysis samples.<br />
+	<strong style="color: #229d73;">Transformation:</strong> The pSB1C3-antitoxin and the pSB1C3-toxin ligation products were transformed intotrans5a cells respectively.
+	</p><p>
+/21/2017<br />
+	<strong style="color: #229d73;">Colony selection:</strong> 20 colonies/plate were picked for overnight culture.
+	</p><p>
+/22/2017<br />
+	<strong style="color: #229d73;">Miniprep and Digestion:</strong> The pSB1C3-antitoxin and the pSB1C3-toxin were extracted with TIANprep Rapid Mini Plasmid Kitand digested with EcoR I and Pst I.<br />
+	<strong style="color: #229d73;">Sequencing:</strong> The plasmids were sent out for sequencing with primers VF2 and VR.
+	</p><p>
+/24/2017<br />
+	<strong style="color: #229d73;">Abs600 Measurement:</strong> 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.
+	</p><p>
+/26/2017<br />
+	<strong style="color: #229d73;">Luciferase analysis:</strong> 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.
+	</p><p>
+/01/2017<br />
+	<strong style="color: #229d73;">Luciferase analysis:</strong> 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.
+	</p><p>
+/02/31<br />
+	<strong style="color: #229d73;">Luciferase analysis:</strong> 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.<br />
+	<strong style="color: #229d73;">Abs600 Measurement:</strong> 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.
+	</p><p>
+/04/2017<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> 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.
+	</p><p>
+/05/2017<br />
+	<strong style="color: #229d73;">EGFP measurement:</strong> 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.<br />
+	<strong style="color: #229d73;">Abs600 Measurement:</strong> 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.
+	</p><p>
+/072017<br />
+	<strong style="color: #229d73;">Luciferase analysis:</strong> 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.
+	</p><p>
+/082017<br />
+	<strong style="color: #229d73;">Abs600 Measurement:</strong> 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.
+	</p><p>
+/11/2017<br />
+	<strong style="color: #229d73;">Abs600 Measurement:</strong> 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.<br />
+	<strong style="color: #229d73;">Luciferase analysis:</strong> 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.
+	</p><p>
+/14/2017<br />
+	<strong style="color: #229d73;">Luciferase analysis:</strong> 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.
+	</p><p>
+/15/2017<br />
+	<strong style="color: #229d73;">Abs600 Measurement:</strong> 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.
+/18/2017<br />
+	<strong style="color: #229d73;">Luciferase analysis:</strong> 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.
+	</p>
 </div>
 </html>