Revision as of 10:40, 13 October 2016

TEAM TIANJIN

Team Tianjin-Attribution

Notes for Bacterial Consortium

Week1(7/24/2016-7/30/2016)

Optimization of Culture Conditions

Jul.26th

Prepare M9 medium with TPA and culture Pseudomonas putida KT2440 at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀.

Jul.27th

1.Extract 10 ml LB medium to 5 test tubes and add 0μl, 50μl,, 100μl,, 250μl,, 500μl EG to them, respectively.
2.Add 5μl bacteria solution of Pseudomonas putida KT2440 to five test tubes above.
3.Culture them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀.

Jul.28th

Add 6g TPA, 2.9gNaOH and 1.2g glucose to M9 medium, and culture them in the improved M9 medium and M9 medium of Jul.26 at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Jul.30th

Cultured different bacteria in M9 medium with sodium terephthalate and different concentration of glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Week2(7/31/2016-8/6/2016)

Optimization of Culture Conditions

Jul.31th

Cultured Rhodococcus jostii RHA1 in M9 medium with sodium terephthalate and glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Aug.1th

Cultured Rhodococcus jostii RHA1 in W medium with sodium terephthalate and glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Aug.2th

Cultured Pseudomonas putida KT2440 in W medium with sodium terephthalate and glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Aug.3th

Cultured Pseudomonas putida KT2440 , Rhodococcus jostii RHA1 , Pseudomonas putida KT2440 and Rhodococcus jostii RHA1in W medium with sodium terephthalate and glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Aug.4th

Observed the growth of 1-day pre-cultured bacteria in LB at 30℃:

All above were kept culturing for one more day and were checked on the next day. The next step was to explore the optimal condition for each bacterium and to co-culture each teo of them to see whether the consortium could work well.

The most probable pairs:
1. P.putida KT2440 + R.jostii RHA1;
2. B.subtilis 168 + R.jostii RHA1;
3. B.subtills 168 + P.putida KT2440;
4. R.jostii RHA1 + Y.lipolytia;
5. P.putida KT2440 + Y.lipolytia;
6. E.coli(RFP) + Y.lipolytia.

Aug.5th

Cultured different bacteria in M9 medium with sodium terephthalate at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂: The growth of each bacterium was not as expected, so we considered culturing bacteria in improved W medium.

Co-cultured different pairs in improved W medium in the same condition (using two tubes in each group):

Co-cultured different pairs in LB medium in the same condition(using two tubes in each group):

Aug.6th

Co-cultured different pairs in improved W medium in the same condition (using two tubes in each group):

Microscopic examination of each bacterium and each pair by means of Fuchsin or Gram dye. To our surprise, there were some pairs (P.p + R.j) living well in the same tube and also there were some pairs we were not sure. It seemed that P.putida KT2440 was the dominant bacterium when it was co-cultured with others. We were thinking about limiting its growth by control ingredients in different media.

Construction of PBBR

Aug.5th

Extraction of plasmid pBBR1MCS-2

Aug.6th

Cut pBBR1MCS-2 with restricted enzymes Xba1 and Sac1 and checked by agarose gel electrophoresis:
Failed. After gel recovery, the concentration of digested pBBR1MCS-2 was 2.4 ng/μl, which was too low to be used in the next step.

Gene Knockout of Escherichia coli

We tried to copy tet gene(tetracycline resistance gene) in two overlap area of the fragment(about 500bp and 1000bp,called ‘tet-1’ and ‘tet-2’) and the homologous arms of the knockout gene-atpF and atpH(about 450bp each,called ‘left’ and ‘right’)with the help of PCR.

Modification of Cyanobacteria

Jul.31th

1.Cultivation: 30µL mother liquid of Synechococcus sp PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.
2.The genome DNA of 7942 was extracted using a Genomic DNA Isolation Kit.

Week3(8/7/2016-8/13/2016)

Optimization of Culture Conditions

Aug.7th

For each pair, we checked the bacterial concentration at OD_600 and detected the concentration of TPA by UV at Abs_242.According to results of the UV tests, if R.j existed, whatever the strategy was, the concentration of TPA would significantly decrease. Especially for pairs R.j+P.p and R.j + B.s, results showed greater reductions in the concentration of TPA compared with control group.

Pre-culture and acclimatize P.putida KT2440 in a condition with 5% EG To our surprise, this wild type of P.putida could grow well in this condition, we considered increasing the concentration of EG in the condition.

Aug.9th

1.Extract 5ml YPD medium to 8 test tubes, respectively.
2.Added bacteria solution as following table (use two tubes each group)

3.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂

Aug.10th

1.Prepared 300ml W0 medium and add 1.5g Yeast Extract, then regarded it W1 medium.
2.Extracted 8ml W1 medium to 16 test tubes, respectively.
3.Added bacteria solution as following table (use two tubes each group).

4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Aug.12th

1.Prepared 200ml W0 medium and add 0.4g Urea, then regarded it W2 medium.
2.Extracted 8ml W2 medium to 16 test tubes, respectively.
3.Added bacteria solution as following table (use two tubes each group)

4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

1.Prepared 200ml W0 medium and add 0.4g KNO3, then regarded it W3 medium.
2.Extracted 8ml W3 medium to 16 test tubes, respectively.
3.Added bacteria solution as following table (use two tubes each group)

4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Aug.13th

1.Extract 5ml W medium to 6 test tubes and add 20μL, 50μL, 80μL EG to them, respectively;
2.Add 10 μL bacteria solution of Pseudomonas putida KT2440 to the six test tubes, respectively;
3. Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀.

Added bacteria solution to W2, W3 medium as following table (use two tubes each group)

Construction of PBBR

Aug.7th

Repeated the digestion and agarose gel electrophoresis:
Also Failed. After gel recovery, the concentration of digested pBBR1MCS-2 was only 2.4 ng/μl. We needed to increase the concentration of pBBR1MCS-2 in EB Solution.

Aug.8th

Colony PCR of P.putida KT2440 to get genes AcoA & AceA:

Result: Failed. Only primer dimers existed.

Aug.9th

Repeated the colony PCR above:

Result: Also Failed. Only primer dimers existed. We considered using boiled bacteria as the template in the next PCR.

Aug.11th

Extraction of plasmid pBBR1MCS-2.

The nucleic acid concentration of new EB solutions increased a lot. Increasing the amount of E.coli and decreasing the amount of EB did work.

Aug.12th

PCR of P.putida KT2440 to get genes AcoA & AceA:

Result: Successful PCR. Obvious bright bands located at 900+ and 1300+ bp (AcoA 978bp and AceA 1326 bp). Using boiled P.putida as the template in the PCR worked well.

Aug.13th

Cut pBBR1MCS-2 with restricted enzymes Xba1 and Sac1 and checked by agarose gel electrophoresis.
Result: Obvious bright bands located over 5000 bp.
After gel recovery, we got the EB solution with cut plasmid pBBR1MCS-2, but the concentration is only 6.6 ng/μl, which is still quite low.

Experiments about Bacillus subtilis

Aug.9th

Inoculated B.subtilis 168 for transformation.

Aug.10th

Plasmids of PETase and plasmid of pHP13-p43 in E.coli were isolated. Enzyme digestion using EcoR I and BamH I, then do gel extraction and then link them.

Aug.11th

The plasmids from DNA ligation were transferred into B.subtilis 168, then cultivated on chloramphenicol containing LB plates to observe whether successful.

Aug.12th

B.subtilis Transformation was observed failure，3 times.

Gene Knockout of Escherichia coli

Instead of enzyme-cut and link up, we used the overlap of each fragment to increase our aim gene(‘tet’, ‘left-right’, ‘left-tet-right’).

Week4(8/14/2016-8/20/2016)

Optimization of Culture Conditions

Aug.15th

1.Prepared 200ml W0 medium and add 0.6g sucrose, then regarded it W4 medium.
2.Extracted 8ml W4 medium to 16 test tubes, respectively.
3.Added bacteria solution as following table (use two tubes each group)

4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Aug.16th

Prepared 200ml W0 medium.
2.Extracted 8ml W0 medium to 16 test tubes, respectively.
3.Added bacteria solution as following table (use two tubes each group)

4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Construction of PBBR

Aug.14th

Ligation of cut pBBR1MCS-2 and CFP and Chemical transformation of pBBR into E.coli:
Ligation sites: Sac1 and Xba1

Result:
The ligation was successful, because we found the special band which represented CFP during the verification by PCR. We also successfully transformed the plasmid into E.coli which was cultured on the medium with kanamycin. However, the expression of CFP was not detected, which needed to be figure out.

Aug.15th

Cut pBBR1MCS-2 with restricted enzymes EcoR1 and Sac1 and checked by agarose gel electrophoresis:
Result: Obvious bright bands located over 5000 bp.
After gel recovery, we got the EB solution with cut plasmid pBBR1MCS-2, and the concentration is 14.7 ng/μl.

Aug.16th

Extraction of plasmid pBBR1MCS-2.

Aug.17th

Ligation of cut pBBR1MCS-2 with AcoA and AceA and Chemical transformation of pBBRAA into E.coli:
Ligation sites: Sac1 and EcoR1

Result:
The ligation was successful, because we found the special band which represented AcoA and AceA during the verification by PCR. We also successfully transformed the plasmid into E.coli which was cultured on the medium with kanamycin.

Experiments about Bacillus subtilis

Aug.14th

B.subtilis Transformation was observed failure.
Reconstruct the plasmids.

Aug.15th

E.coli transformation was observed failure.
Enzyme digestion again.
E.coli transformation again.

Aug.16th

E.coli transformation was observed failure.
Plasmid was enzyme digested and not observed correct band.

Aug.17th

Plasmids of PETase was enzyme digestion again.
Inoculate B.subtilis DB104.

Aug.19th

Inoculate E.coli of pHP13-p43.

Gene Knockout of Escherichia coli

We aimed to put the left-tet-right gene and plasmid which have λ-red gene into the the genome of E.coli. After two screening, we could gain the target strains which knocked out the atpF and atpH gene.

Modification of Cyanobacteria

Synechococcus sp PCC 7942 had no signals of life.

Week5(8/21/2016-8/27/2016)

Optimization of Culture Conditions

Aug.22th

        1.Prepared 200ml W0 medium and add 0.3g NaOAc, then regarded it W7 medium.
        2.Extracted 8ml W7 medium to 16 test tubes, respectively.
        3.Added bacteria solution as following table (use two tubes each group)

4.cultured them at 30℃ and check growing situation and the situation of TPA degradation.

Experiments about Bacillus subtilis

Aug.24th

Cultivated transformed E.coli on chloramphenicol containing LB plates to observe whether successful.

Gene Knockout of Escherichia coli

We cultivateD Saccharomyces cerevisiae and E.coli which has knocked out atpF and atpH gene and introduced GFP gene in special culture medium which the only carbon source was xylose.

Week6(8/28/2016-9/3/2016)

Experiments about Bacillus subtilis

Aug.29th

B.subtilis DB104 transformation.

Aug.30th

B.subtilis DB104 transformation is successful.

Sep.2th

Construct plamid of MHETase without success.
Transform into E.coli, in chloramphenicol containing LB culture.

Modification of Cyanobacteria

Aug.29th

1.pMV-G19 and pMV-G15 containing our target genes were received.
2.Colonies were used to inoculate overnight cultures.

Aug.30th

1.Plasmids were isolated using a miniprep kit.
2.Amplification of 19 and 15 with Q5 High-Fidelity DNA Polymerase out of E.coli.
  19 amplification at 65.0°C with 19.rev/fwd primes.
        PCR worked, positive control worked, no amplification of 19.
  15 amplification at 65.0°C with 15.rev/fwd primes.
        PCR worked, positive control worked, no amplification of 15.
3.A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.
  This result was confirmed by sequencing.
4.The fragments of 19 and 15 were purified with PCR Purification Kit.

Aug.31th

1.Ligation of 15 with 19.
2.We add a single 3`-adenine overhang to each end of the fragment of 19 and then purified with DNA Purification Kit.
3.19 was ligated into T vectors via TA clone and transformed into E.coli via heat shock.

Sep.1th

1.A colony PCR was performed with five colonies.
2.Gel electrophoresis showed that 5 colonies were positive for insertion of 19.
3.Two of these colonies containing 19 were used to inoculate overnight cultures.
4.15 gene fragment was phosphorylated.

Sep.2th

1.Plasmids containing T vector with 19(pT-19)were isolated using a miniprep kit.
2.Mono-restriction digest of pT-19 with stu I.
3.The enzyme-digested product was dephosphorylation.
4.Dephosphorylated plasmid and phosphorylated gene 15 were connected.
5.Ligation product was transformed into E.coli via heat shock.

Sep.3th

1.A colony PCR was performed with twelve colonies.
2.Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.
3.These two colonies were used to inoculate overnight cultures.

Week7(9/4/2016-9/10/2016)

Experiments about Bacillus subtilis

Sep.8th

Inoculate PETase transformed B.stubtilis 168 in 3 flask culture.
1.LB, chloramphenicol, PETase transformed B.stubtilis 168, PET film.
2.LB, chloramphenicol, PETase transformed B.stubtilis 168, no PET film.
3.LB, chloramphenicol, B.stubtilis 168, PET film.

Sep.9th

Construct plamid of MHETase without success.

Sep.10th

Construct plamid of MHETase without success.

Modification of Cyanobacteria

Sep.4th

1.Plasmids with correct sequence of 19-15 were isolated using a miniprep kit.
2.Mono-restriction digest of pT-19-15 with Nru I.
3.The enzyme-digested product was dephosphorylation.

Sep.6th

Colonies containing gene 13 were used to inoculate overnight cultures.

Sep.7th

1.Plasmids were isolated using a miniprep kit.
2.Amplification of 13 with Q5 High-Fidelity DNA Polymerase out of E.coli
3.13 amplification at 65.0°C with 13.rev/fwd primes
PCR worked, positive control worked, no amplification of 13
4.The fragments of 13 were purified with PCR Purification Kit.

Sep.8th

13 gene fragment was phosphorylated.

Sep.9th

1.Insertion of Ni promoter and ligation of 13-19-15
2.Ni inducible promoter was ligated into pCPC-3301 vector.
3.Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.
4.Single colonies were obtained by plating.

Sep.10th

1.A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.
Two of the successful ones were used to inoculate overnight cultures.
2.A colony PCR of pT-13-19-15 was performed with 7 colonies.
Two of the successful ones were used to inoculate overnight cultures.

Week8(9/11/2016-9/17/2016)

Optimization of Culture Conditions

Sep.12th

Optimize the growing environment of Bacillus stubtilis by change medium components.Firstly, we prepared 400ml W medium, then, we devided the medium into four pieces averagely and number them No.1, No.2, No.3, No.4. Next, we added 0.1g NaCl, 0.1g NH4Cl, 0.3g sucrose to No.2, No.3, No.4, respectively. After that, we Extracted 5 ml No.1, No.2, No.3, No.4 and added them to four test tubes, then, we added 10 μL bacteria solution of Bacillus stubtilis 168 to four test tubes, respectively. Eventually, we cultured them at 37℃ and check growing situation.

Sep.16th

        1.Prepared 200ml W0 medium and add 0.2g NH4Cl, then regarded it W8 medium.
        2.Extracted 10ml W8 medium to 16 test tubes, respectively.
        3.Added bacteria solution as following table (use two tubes each group)

4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Experiments about Bacillus subtilis

Sep.11th

Construct plamid of MHETase without success.

Sep.12th

Construct plamid of MHETase without success.

Modification of Cyanobacteria

Sep.11th

Two kinds of plasmids were isolated using a miniprep kit.

Sep.12th

Two kinds of plasmids were isolated using a miniprep kit.

Sep.14th

The sequencing results for both of them were error.

Sep.15th

1.Colonies containing Ni inducible promoter were used to inoculate overnight cultures.
2.PCR was performed to check if the gene fragments were ligated correctly.
13_ fwd and 15_rev on pT-13-19-15
3.Gel electrophoresis showed that it failed.

Sep.16th

Plasmids pCPC-3031-Ni were isolated using a miniprep kit.

Sep.17th

1.Several PCRs were performed to check if the gene fragments were ligated correctly.
  13_fwd and 13_rev on pT-13-19-15
  19_fwd and 19_rev on pT-13-19-15
  15_fwd and 15_rev on pT-13-19-15
  13_fwd and 19_rev on pT-13-19-15
  19_fwd and 15_rev on pT-13-19-15
  13_ wd and 15_rev on pT-13-19-15
The fourth and sixth ones were not successful.
2.Repetition: Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.

Week9(9/18/2016-9/24/2016)

Optimization of Culture Conditions

Sep.19th

1. Prepared 1L W medium and add 2.5g TPA and 1.1875g NaOH.
2. devided the medium into five pieces averagely and add chemicals as following table

3. Extracted 5ml W9, W10, W11, W12, W13 medium to 40 test tubes, respectively.
4. Added bacteria solution as following table (use two tubes each group)

5. cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Sep.21th

Repeat experiments of W9, W10 the day before yesterday.

Sep.22th

        1. Use five mediums of Sep.19th and W0, W8 medium of Sep.16th.
        2. Extracted 5ml W0, W8, W9, W10, W11, W12, W13 medium to 56 test tubes, respectively.
        3. Added bacteria solution as following table (use two tubes each group)

4. cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Sep.23th

1. Prepared 400L W medium and add 1.2g KNO3 and 1.2g glucose.
2. devided the medium into four pieces averagely and add chemicals as following table

3. Extracted 5ml W0, W14, W15, W16 medium to 34 test tubes, respectively.
4. Added bacteria solution as following table (use two tubes each group)

5. cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD₆₀₀ and detected the concentration of TPA by UV at OD₂₄₂.

Experiments about Bacillus subtilis

Sep.19th

Construct plasmid of MHETase without success.
Cultivated MHETase transformed B.stubtilis 168 on Erythromycin containing LB plates to observe, without success.

16SrDNA

Sep.19th

we firstly tried to cultivate each of them(Rhodococcus RHA1, Pseudomonas putida KT2440, bacillus subtilis 168) in LB culture medium for amplification.

Sep.21th

We use orthogonal test to prove each bacteria had each special DNA stripe from the method of bacteria colony-PCR.

Sep.22th

Then we cultivate each of them and the mixture of three in W0 culture medium for amplification.

Sep.23th

We did the same 4*3(bacteria liquid and six primer sequences) orthogonal test.

Sep.24th

We cultivate each of them and the mixture of three in in modified W0 culture medium which changed the carbon source from glucose to sugar for amplification.

Modification of Cyanobacteria

Sep.18th

Repetition: Several PCRs were performed to check if the gene fragments were ligated correctly.
13_fwd and 13_rev on pT-13-19-15
13_fwd and 19_rev on pT-13-19-15
The second one was failed.

Sep.21th

1.Medium preparation :BG-11.
2.19_ fwd and 15_rev were used to amplify 19-15.

Sep.22th

1.Gel electrophoresis showed that amplification of fragments was successfull.
2.Ligated 13 and 19-15 via overlap PCR.
3.This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.
4.Restriction digest on pCPC-3031-Ni with Sac I.
5.The fragment 13-19-15 was ligated onto T vector and transformed into E.coli via heat shock.

Sep.23th

1.A colony PCR of pT-13-19-15 was performed with 12 colonies.
2.Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.
3.13-19-15 gene fragment was phosphorylated.
4.Phosphorylated 13-19-15 was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.

Sep.24th

Plasmids pT-13-19-15 were isolated using a miniprep kit.

Week10(9/25/2016-10/1/2016)

16SrDNA

Sep.25th

We still did the same 4*3(bacteria liquid and six primer sequences) orthogonal test.

Modification of Cyanobacteria

Sep.25th

1.A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.
2.Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.

Sep.26th

Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.
The sequencing results for them were correct.

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+	<header class="entry-header">
+		<div class="entry-title" align="center" >Notes for Bacterial Consortium</div>
+	</header><!-- .entry-header -->
+        <h1 class="entry-title">Week1(7/24/2016-7/30/2016)</h1>
+		<div class="entry-content">
+         <div class="note-content">
-<head>
+<li><h1 style="font-size:135%">Optimization of Culture Conditions</h1><br/>
-  <meta charset="utf-8">
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Jul.26th</b></h1>
-  <title>Achievement</title>
+<div style="padding-left:32px;">Prepare M9 medium with TPA and culture <i>Pseudomonas putida KT2440</i> at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub>.</div><br/>
-  <link href="images/img_1.jpg" rel="icon">
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Jul.27th</b></h1>
-  <style>
+<div style="padding-left:32px;">1.Extract 10 ml LB medium to 5 test tubes and add 0μl, 50μl,, 100μl,, 250μl,, 500μl EG to them, respectively.<br/>
-    .entry-content li{list-style:none;
+.Add 5μl bacteria solution of <i>Pseudomonas putida KT2440</i> to five test tubes above.<br/>
-	   height:75px;
+.Culture them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub>. <br/>
-	   line-height:inherit;
+<br/></div>
-	   padding-left:100px;
-	   background:url(images/Bielefeld-CeBiTec-Checkbox_red.png) no-repeat left center;
-	   font-size:20px;
-	}
-	</style>
-</head>
-<body>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Jul.28th</b></h1>
+<div style="padding-left:32px;">Add 6g TPA, 2.9gNaOH and 1.2g glucose to M9 medium, and culture them in the improved M9 medium and M9 medium of Jul.26 at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.<br/>
+<br/></div>
-  <nav class="navbar navbar-TeamTianjin" role="navigation" id="TeamTianjinNavbar">
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Jul.30th</b></h1>
-	<div class="container-fluid">
+<div style="padding-left:32px;">Cultured different bacteria in M9 medium with sodium terephthalate and different concentration of glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.<br/>
-    	<div class="navbar-header">
+<br/></div>
-                <div class="nameTeamTianjin">TEAM TIANJIN</div>
-    		<button type="button" class="navbar-toggle" data-toggle="collapse" data-target="#bs-example-navbar-collapse-1">
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-    		<img class="img-responsive logoBG" src="images/img_1.jpg" alt="Logo on left" href="http://127.0.0.1">
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+</li>
-   	  <div class="collapse navbar-collapse" id="bs-example-navbar-collapse-1">
-    		<ul class="nav navbar-nav">
-				<li><a href="#" id="Overview">OVERVIEW</a></li>
-        		<li><a href="#" id="Achievements">ACHIEVEMENTS</a></li>
+</div>
-        		<li class="dropdown">
+<a class="expand-btn">Show More</a>
-        			<a href="#" class="dropdown-toggle" data-toggle="dropdown" id="Projects">PROJECTS</a>
-        			<ul class="dropdown-menu">
-					  <li><a href="#" id="Vision">VISION</a></li>
-					  <li><a href="#" id="Experiment">EXPERIMENT</a></li>
+		</div><!-- .entry-content -->
-					  <li><a href="#" id="Modeling">MODELING</a></li>
-					  <li><a href="#" id="Results">RESULTS</a></li>
-                      <li class="divider"></li>
-				      <li><a href="#" id="Parts">PARTS</a></li>
+	     </article><!-- #post-4252 -->
-                      <li><a href="#" id="LaboratoryNotes">LABORATORY NOTES</a></li>
-        			</ul>
+<!------------------------------------week1 end------------------------------------------------>
-        		</li>
-        		<li class="dropdown">
-        			<a href="#" class="dropdown-toggle" data-toggle="dropdown" id="People">PEOPLE</a>
-        			<ul class="dropdown-menu">
+<!------------------------------------week2 start------------------------------------------------>
-					  <li><a href="#" id="Team">TEAM</a></li>
+<div id="Week2"></div>
-					  <li><a href="#" id="Collaboration">COLLABORATION</a></li>
+<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
-					  <li><a href="#" id="SocialMedia">SOCIAL MEDIA</a></li>
+	<header class="entry-header">
-        			</ul>
+		 <h1 class="entry-title">Week2(7/31/2016-8/6/2016)</h1>
-        		</li>
+	</header><!-- .entry-header -->
-        		<li class="dropdown">
-        			<a href="#" class="dropdown-toggle" data-toggle="dropdown" id="Practice">PRACTICE</a>
+		<div class="entry-content">
-        			<ul class="dropdown-menu">
+	<div class="note-content2">
-					  <li><a href="#" id="Community">COMMUNITY</a></li>
-					  <li><a href="#" id="BusinessPlan">BUSINESS PLAN</a></li>
+<li>
-        			</ul>
+<h1 style="font-size:135%">Optimization of Culture Conditions</h1><br/>
-        		</li>
-    		</ul>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Jul.31th</b></h1>
-    	</div>
+<div style="padding-left:32px;">Cultured <i>Rhodococcus jostii RHA1</i> in M9 medium with sodium terephthalate and glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.</div><br/>
-	</div>
-</nav>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.1th</b></h1>
-  <header role="banner">
+<div style="padding-left:32px;">Cultured <i>Rhodococcus jostii RHA1</i> in W medium with sodium terephthalate and glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.</div><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.2th</b></h1>
+<div style="padding-left:32px;">Cultured <i>Pseudomonas putida KT2440</i> in W medium with sodium terephthalate and glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.</div><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.3th</b></h1>
+<div style="padding-left:32px;">Cultured <i>Pseudomonas putida KT2440</i> , <i>Rhodococcus jostii RHA1</i> , <i>Pseudomonas putida KT2440</i> and <i>Rhodococcus jostii RHA1</i>in W medium with sodium terephthalate and glucose at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.</div><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.4th</b></h1>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Observed the growth of 1-day pre-cultured bacteria in LB at 30℃:
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/3/3e/T--Tianjin--table_8.4.png"></div>
+<div style="padding-left:32px;">All above were kept culturing for one more day and were checked on the next day. The next step was to explore the optimal condition for each bacterium and to co-culture each teo of them to see whether the consortium could work well.<br/>
+<br/>
+The most probable pairs:<br/>
+. P.putida KT2440 + R.jostii RHA1;<br/>
+. B.subtilis 168 + R.jostii RHA1;<br/>
+. B.subtills 168 + P.putida KT2440;<br/>
+. R.jostii RHA1 + Y.lipolytia;<br/>
+. P.putida KT2440 + Y.lipolytia;<br/>
+. E.coli(RFP) + Y.lipolytia.<br/></div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.5th</b></h1>
+<div style="padding-left:32px;">Cultured different bacteria in M9 medium with sodium terephthalate at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>:
+The growth of each bacterium was not as expected, so we considered culturing bacteria in improved W medium.</div>
+<br/>
+<div style="padding-left:32px;">Co-cultured different pairs in improved W medium in the same condition (using two tubes in each group):</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/9/92/T--Tianjin--table_8.5-1.png"></div>
+<br/>
+<div style="padding-left:32px;">Co-cultured different pairs in LB medium in the same condition(using two tubes in each group):</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tianjin--table_8.5-2.png"></div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.6th</b></h1>
+<div style="padding-left:32px;">Co-cultured different pairs in improved W medium in the same condition (using two tubes in each group):</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/9/92/T--Tianjin--table_8.5-1.png"></div>
+<br/>
+<div style="padding-left:32px;">Microscopic examination of each bacterium and each pair by means of Fuchsin or Gram dye.
+To our surprise, there were some pairs (P.p + R.j) living well in the same tube and also there were some pairs we were not sure. It seemed that P.putida KT2440 was the dominant bacterium when it was co-cultured with others. We were thinking about limiting its growth by control ingredients in different media.
+</div>
+<br/>
+</li>
+<li><h1 style="font-size:135%">Construction of PBBR</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.5th</b></h1>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Extraction of plasmid pBBR1MCS-2<br/>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/d/d8/T--Tianjin--table_8.5.png"></div>
+<br/><b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.6th</b></h1>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Cut pBBR1MCS-2 with restricted enzymes Xba1 and Sac1 and checked by agarose gel electrophoresis: <br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Failed. After gel recovery, the concentration of digested pBBR1MCS-2 was 2.4 ng/μl, which was too low to be used in the next step.<br/>
+</li>
+<br/>
+<li>
+<h1 style="font-size:135%">Gene Knockout of <i>Escherichia coli</i></h1><br/>
+<div style="padding-left:32px;">We tried to copy tet gene(tetracycline resistance gene) in two overlap area of the fragment(about 500bp and 1000bp,called ‘tet-1’ and ‘tet-2’) and the homologous arms of the knockout gene-atpF and atpH(about 450bp each,called ‘left’ and ‘right’)with the help of PCR.</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Jul.31th</b></h1>
+<div style="padding-left:32px;">1.Cultivation: 30µL mother liquid of Synechococcus sp PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.<br/>
+.The genome DNA of 7942 was extracted using a Genomic DNA Isolation Kit.</div><br/>
+</div>
+<a class="expand-btn2">Show More</a>
+		</div><!-- .entry-content -->
+	     </article>
+<!------------------------------------week2 end------------------------------------------------>
-  <h1><a href="/">Achievement</a></h1>
-    <h2>Reasons Why We Can Get A Gold Metal</h2>
-  </header>  <!--
+<!------------------------------------week3 start------------------------------------------------>
-  <nav role="navigation">
+<div id="Week3"></div>
-      <ul class="subscription" data-subscription="rss">
+<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
-        <li><a href="/atom.xml" rel="subscribe-rss" title="subscribe via RSS">RSS</a></li>
+	<h1 class="entry-title">Week3(8/7/2016-8/13/2016)</h1>
-      </ul>
+		<div class="entry-content">
-      <form action="http://google.com/search" method="get">
-         <fieldset role="search">
-           <input type="hidden" name="q" value="IGEM" />
-           <input class="search" type="text" name="q" results="0" placeholder="Search"/>
-         </fieldset>
-      </form>
-      <ul class="main-navigation">
-        <li><a href="/">Blog</a></li>
-        <li><a href="/archives/">Archives</a></li>
-      </ul>
-   </nav>    -->
-  <div id="main">
-    <div id="content">
-      <div>
-         <article class="entry" role="article">
-           <header>
-             <h1 class="entry-title">
-               IGEM Medal Requirements
-             </h1>
- <!--        <p class="meta">
-                <time datetime="2011-02-17T11:52:00-08:00" pubdate data-updated="true">Feb 17<span>th</span>, 2011</time>
-         &bull; <a rel="bookmark" href="/blog/2011/02/17/using-sass-with-the-1kb-grid-system/">Permalink</a>
-             </p>  -->
-           </header>
-           <div class="entry-content">
-             <h2>Bronze Medal Requirements</h2>
-             <ul>
-                <li>The 1KB CSS Grid is a simplified grid system and is one of many dozens of different grids available.</li>
-                <li>The 1KB CSS Grid is a simplified grid system and is one of many dozens of different grids available.</li>
-                <li>The 1KB CSS Grid is a simplified grid system and is one of many dozens of different grids available.</li>
-                <li>The 1KB CSS Grid is a simplified grid system and is one of many dozens of different grids available.</li>
-              </ul>
-             <p>The 1KB CSS Grid is a simplified grid system and is one of many dozens of different grids available. I started using a personal variant of this grid system recently because it seemed like a very simple code-base to understand. A common complaint about CSS grids is that they require classes in the markup that aren't much different that simply hard-coding widths in a style attribute. Of course SASS can help remove that complaint and makes grids even more flexible. Here's a tour of the 1KB CSS Grid, modified to suit my tastes and rewritten in SASS.
-             </p>
-<!--more-->
-                <h2 id="basic-css">Silver Medal Requirements</h2>
-                   <p>The above CSS is very similar to the <a href="http://www.usabilitypost.com/2009/05/29/the-1kb-css-grid-part-1/">original source</a> with a few stylistic differences (dashes instead of underscores) and the removal of the <code>overflow: hidden</code>
-	clearfix hack and <a href="http://haslayout.net/css/Double-Margin-Bug">IE6 hacks</a>. My intention is to use the grid with <a href="http://html5boilerplate.com/">HTML5 Boilerplate</a> which has a better clearfix built-in (<a href="http://www.yuiblog.com/blog/2010/09/27/clearfix-reloaded-overflowhidden-demystified/"><code>overflow: hidden</code> isn't appealing</a> as a clearfix hack). I've also chosen to <a href="http://windowsteamblog.com/ie/b/ie/archive/2010/09/01/internet-explorer-usage-share-in-august.aspx">remove IE6</a> from my list of supported browsers for any project that doesn't require it.
-                   </p>
-                   <table>
-               <tr>
-                  <td><img src="images/Bielefeld-CeBiTec-Checkbox_red.png" width="76px"></td>
-                  <td>let's hit dinghaoran </td>
-               </tr>
-               <tr>
-                  <td><img src="images/Bielefeld-CeBiTec-Checkbox_red.png" width="76px"></td>
-                  <td>d</td>
-               </tr>
-               <tr>
-                  <td><img src="images/Bielefeld-CeBiTec-Checkbox_red.png" width="76px"></td>
-                  <td>f</td>
-               </tr>
-               <tr>
-                  <td><img src="images/Bielefeld-CeBiTec-Checkbox_red.png" width="76px"></td>
-                  <td>h</td>
-               </tr>
-              </table>
-<h2 id="basic-example-using-only-css">Gold Medal Requirements</h2>
-<p>
-	It's not immediately obvious how to piece this all together but the concepts are pretty simple.
-</p>
- <table>
-               <tr>
-                  <td><img src="images/Bielefeld-CeBiTec-Checkbox_red.png" width="76px"></td>
-                  <td>let's hit dinghaoran </td>
-               </tr>
-               <tr>
-                  <td><img src="images/Bielefeld-CeBiTec-Checkbox_red.png" width="76px"></td>
-                  <td>d</td>
-               </tr>
-               <tr>
-                  <td><img src="images/Bielefeld-CeBiTec-Checkbox_red.png" width="76px"></td>
-                  <td>f</td>
-               </tr>
-               <tr>
-                  <td><img src="images/Bielefeld-CeBiTec-Checkbox_red.png" width="76px"></td>
-                  <td>h</td>
-               </tr>
-              </table>
-<p>
-	Above is a typical page using the new HTML5 container tags and based on the <a href="https://github.com/paulirish/html5-boilerplate/blob/master/index.html">example HTML5 Boilerplate markup</a>. This example is shown with hard-coded class names in the HTML. As expected, there's a page wrapper (<code>#container</code>
-	), <code>header</code>
-	, <code>#main</code>
-	and <code>footer</code>
-	containers, and some columns in the main area. This page would create a typical 2 column layout (<code>#left-column</code>
-	and <code>#main-column</code>
-	). The <code>#main-column</code>
-	, as shown, has a <code>.hero</code>
-	area that spans the full width and a content area is further sub-divided into 2 columns (<code>#content</code>
-	and <code>#right-column</code>
-	).
-</p>
-<p>
-	On a typical site, the <code>header</code>
-	and <code>footer</code>
-	would probably contain their own columns and rows. The <code>.hero</code>
-	is used for promotional space at the top of content. This is a typical design choice and might contain a banner or a slideshow or some introductory copy.
-</p>
-<p>
-	Personally I find the inclusion of the class names in the markup to be a little ugly. The most offensive part is the <code>grid-3</code>
-	and <code>grid-6</code>
-	, etc. This is barely different than hard-coding the width with an inline style. I prefer to use SASS mixins to remove those class names from the markup altogether and control it directly from the CSS.
-</p>
-<h2 id="rewritten-in-sass">Rewritten in SASS</h2>
+<div class="note-content3">
-<p>
+<li>
-	Writing the same thing in SASS opens up a world of possibilities. Using <a href="http://sass-lang.com/docs/yardoc/file.SASS_REFERENCE.html#defining_a_mixin">mixins</a> allows the column measurements to be easily changed and opens up the possibility of removing the extra class names from the markup. This SASS file was used to generate the CSS at the top of the article.
+<h1 style="font-size:135%">Optimization of Culture Conditions</h1><br/>
-</p>
-<h3 id="default-variables">Default Variables</h3>
-<ul>
-	<li>
-		<code>$column-width: 60px;</code>
-		is the width of a sinlge column
-	</li>
-	<li>
-		<code>$gutter-width: 20px;</code>
-		is the space between two columns
-	</li>
-	<li>
-		<code>$columns: 12;</code>
-		is the total number of columns
-	</li>
-	<li>
-		(60px + 20px) * 12 = 960px
-	</li>
-</ul>
-<h3 id="grid-mixins">Grid Mixins</h3>
-<p>
-	<code>grid</code>
-	and <code>grid-plus</code>
-	are simply used for setting a width on a column. <code>grid-plus</code>
-	can be used to account for padding or otherwise arbitrarily altering the standard column width. This is especially useful when dealing with designers that like grids but like violating them even more.
-</p>
-<p>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.7th</b></h1>
-	The example above shows 3 equivalent ways to specify a 6-column-wide column. The shortcut <code>grid-column</code>
+<div style="padding-left:32px;">For each pair, we checked the bacterial concentration at OD_600 and detected the concentration of TPA by UV at Abs_242.According to results of the UV tests, if R.j existed, whatever the strategy was, the concentration of TPA would significantly decrease. Especially for pairs R.j+P.p and R.j + B.s, results showed greater reductions in the concentration of TPA compared with control group.</div>
-	mixin makes it easy to create a standard column. But in the case where a column might require <code>border</code>
+<br/>
-	or <code>padding</code>
-	, the <code>grid-plus</code>
-	mixin can be used to account for the difference.
-</p>
-<ul>
-	<li>
-		<code>@include grid(&lt;number&gt;);</code>
-		is for column widths
-		<ul>
-			<li>
-				<code>@include grid-plus(&lt;number&gt;, &lt;length&gt;);</code>
-				is for columns that need a non-standard width
-			</li>
-		</ul>
-	</li>
-	<li>
-		<code>@include grid-column;</code>
-		is for floating columns
-		<ul>
-			<li>
-				<code>@include grid-column(&lt;number&gt;);</code>
-				is a shortcut, the same as calling <code>grid(&lt;number&gt;)</code>
-				and <code>grid-column</code>
-				consecutively
-			</li>
-			<li>
-				<code>@include grid-column-empty(&lt;number&gt;, &lt;position&gt;);</code>
-				adds margin to create empty space either before or after the column
-			</li>
-		</ul>
-	</li>
-	<li>
-		<code>@include grid-row;</code>
-		is a column container
-	</li>
-	<li>
-		<code>@include grid-page;</code>
-		is the main page wrapper with a full width and is horizontally centered
-		<ul>
-			<li>
-				<code>@include grid-page(&lt;number&gt;);</code>
-				creates a page container that is less than the default page width; useful for modal pop-ups
-			</li>
-		</ul>
-	</li>
-</ul>
-<h2 id="basic-example-using-sass">Basic Example Using SASS</h2>
-<p>
-	Using the ID's and class names already in the markup, it's possible to utilize the mixins that were created earlier to achieve the exact same effect. I typically do this in a layout file that contains all of the column layouts for the various templates in the site.
-</p>
+<div style="padding-left:32px;">Pre-culture and acclimatize P.putida KT2440 in a condition with 5% EG
+To our surprise, this wild type of P.putida could grow well in this condition, we considered increasing the concentration of EG in the condition.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.9th</b></h1>
+<div style="padding-left:32px;">1.Extract 5ml YPD medium to 8 test tubes, respectively.<br/>
+.Added bacteria solution as following table (use two tubes each group)</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/4/4b/T--Tianjin--table_8.9-1.png"></div>
+<div style="padding-left:32px;">3.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub></div>
+<br/>
-<h4>To Be Clear</h4>
-<ul>
-	<li>
-		<code>#container</code>
-		is specified as a <code>grid-page</code>
-	</li>
-	<li>
-		<code>header</code>
-		, <code>#main</code>
-		and <code>footer</code>
-		are set up as <code>grid-row(true)</code>
-		<ul>
-			<li>
-				This essentially applies <code>.clearfix</code>
-				to those container
-			</li>
-			<li>
-				<code>grid-row(true)</code>
-				is used for rows contained directly within a <code>grid-page</code>
-				which ommits the negative margins that would otherwise be applied.
-			</li>
-		</ul>
-	</li>
-	<li>
-		<code>#left-column</code>
-		and <code>#right column</code>
-		are specified as 3-columns wide
-	</li>
-	<li>
-		The <code>#main-column</code>
-		is 9 columns wide. More importantly, all <code>section</code>
-		tags are specified as <code>grid-row</code>
-		, allowing them to contain columns.
-	</li>
-	<li>
-		The <code>#content</code>
-		column is 6 columns wide.
-	</li>
-</ul>
-<p>
-	From the above example it should be clear how to use mixins to apply the grid styles directly through CSS without needing to hard-code the grid related class names into the markup.
-</p>
-<h3 id="compiled-to-css">Compiled to CSS</h3>
-<p>
-	Because SASS must be compiled to CSS for it to work in a browser, it's useful to see what CSS is being generated. The file below is what the layout.scss example file generated.
-</p>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.10th</b></h1>
+<div style="padding-left:32px;">1.Prepared 300ml W0 medium and add 1.5g Yeast Extract, then regarded it W1 medium.<br/>
+.Extracted 8ml W1 medium to 16 test tubes, respectively.<br/>
+.Added bacteria solution as following table (use two tubes each group).</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tianjin--table_8.22.png"></div>
+<div style="padding-left:32px;">4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.</div>
+<br/>
-<h3 id="html-without-the-hard-coded-class-names">HTML Without the Hard-coded Class Names</h3>
-<p>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.12th</b></h1>
-	SASS makes it possible to clean up the HTML and remove the extra classes.
+<div style="padding-left:32px;">1.Prepared 200ml W0 medium and add 0.4g Urea, then regarded it W2 medium.<br/>
-</p></div>
+.Extracted 8ml W2 medium to 16 test tubes, respectively.<br/>
+.Added bacteria solution as following table (use two tubes each group)<br/>
+</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tianjin--table_8.22.png"></div>
+<div style="padding-left:32px;">4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.</div>
+<br/>
-  <footer>
+<div style="padding-left:32px;">1.Prepared 200ml W0 medium and add 0.4g KNO3, then regarded it W3 medium.<br/>
-    <p class="meta">
+.Extracted 8ml W3 medium to 16 test tubes, respectively.<br/>
+.Added bacteria solution as following table (use two tubes each group)<br/>
-<span class="byline author vcard">Posted by <span class="fn">Grady Kuhnline</span></span>
+</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tianjin--table_8.22.png"></div>
+<div style="padding-left:32px;">4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.</div>
+<br/>
-<time datetime="2011-02-17T11:52:00-08:00" pubdate data-updated="true">Feb 17<span>th</span>, 2011</time>
-<span class="categories">
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.13th</b></h1>
+<div style="padding-left:32px;">1.Extract 5ml W medium to 6 test tubes and add 20μL, 50μL, 80μL EG to them, respectively;<br/>
-    <a class='category' href='/categories/compass/'>compass</a>, <a class='category' href='/categories/css/'>css</a>, <a class='category' href='/categories/grid/'>grid</a>, <a class='category' href='/categories/sass/'>sass</a>
+.Add 10 μL bacteria solution of <i>Pseudomonas putida KT2440</i> to the six test tubes, respectively;<br/>
+. Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub>.<br/>
-</span>
+</div>
+<br/>
+<div style="padding-left:32px;">Added bacteria solution to W2, W3 medium as following table (use two tubes each group)</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/d/d5/T--Tianjin--table_8.13.png"></div>
+<br/>
+</li>
-    </p>
+<li><h1 style="font-size:135%">Construction of PBBR</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.7th</b></h1>
-      <div class="sharing">
+<div style="padding-left:32px;">Repeated the digestion and agarose gel electrophoresis:<br/>
+Also Failed. After gel recovery, the concentration of digested pBBR1MCS-2 was only 2.4 ng/μl. We needed to increase the concentration of pBBR1MCS-2 in EB Solution.</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.8th</b></h1>
+<div style="padding-left:32px;">Colony PCR of P.putida KT2440 to get genes AcoA & AceA:</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/8/82/T--Tianjin--table_8.7.png"></div>
+<div style="padding-left:32px;">Result:
+Failed. Only primer dimers existed.</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.9th</b></h1>
+<div style="padding-left:32px;">Repeated the colony PCR above:</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/2/2c/T--Tianjin--table_8.9.png"></div>
+<div style="padding-left:32px;">Result:
+Also Failed. Only primer dimers existed. We considered using boiled bacteria as the template in the next PCR.
 </div>
+<br/>
-     <p class="meta">
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.11th</b></h1>
+<div style="padding-left:32px;">Extraction of plasmid pBBR1MCS-2.</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/a/ad/T--Tianjin--table_8.11.png"></div>
-        <a class="basic-alignment right" href="/blog/2011/02/24/cross-browser-rounded-corners-overview/" title="Next Post: Cross-Browser Rounded Corners Overview">Cross-Browser Rounded Corners Overview &raquo;</a>
+<div style="padding-left:32px;">The nucleic acid concentration of new EB solutions increased a lot. Increasing the amount of E.coli and decreasing the amount of EB did work.</div>
+<br/>
-    </p>
-  </footer>
-</article>
-  <section>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.12th</b></h1>
-    <h1>Comments</h1>
+<div style="padding-left:32px;">PCR of P.putida KT2440 to get genes AcoA & AceA:</div>
-<!--    <div id="disqus_thread" aria-live="polite"><noscript>Please enable JavaScript to view the <a href="http://disqus.com/?ref_noscript">comments powered by Disqus.</a></noscript>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/4/49/T--Tianjin--table_8.12.png"></div>
-    </div>  -->
+<div style="padding-left:32px;">Result:
-    <h2>shit</h2>
+Successful PCR. Obvious bright bands located at 900+ and 1300+ bp (AcoA 978bp and AceA 1326 bp). Using boiled P.putida as the template in the PCR worked well.
-  </section>
 </div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.13th</b></h1>
+<div style="padding-left:32px;">Cut pBBR1MCS-2 with restricted enzymes Xba1 and Sac1 and checked by agarose gel electrophoresis.<br/>
+Result: Obvious bright bands located over 5000 bp.<br/>
+After gel recovery, we got the EB solution with cut plasmid pBBR1MCS-2, but the concentration is only 6.6 ng/μl, which is still quite low.
-<aside class="sidebar">
+</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Experiments about <i>Bacillus subtilis</i></h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.9th</b></h1>
+<div style="padding-left:32px;">Inoculated <i>B.subtilis 168</i> for transformation.</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.10th</b></h1>
+<div style="padding-left:32px;">Plasmids of PETase and plasmid of pHP13-p43 in <i>E.coli</i> were isolated. Enzyme digestion using <i>EcoR I</i> and <i>BamH I</i>, then do gel extraction and then link them.</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.11th</b></h1>
+<div style="padding-left:32px;">The plasmids from DNA ligation were transferred into <i>B.subtilis 168</i>, then cultivated on chloramphenicol containing LB plates to observe whether successful.</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.12th</b></h1>
+<div style="padding-left:32px;"><i>B.subtilis</i> Transformation was observed failure，3 times.
+</div>
+<br/>
+</li>
+<br/>
+<li>
+<h1 style="font-size:135%">Gene Knockout of <i>Escherichia coli</i></h1><br/>
+<div style="padding-left:32px;">Instead of enzyme-cut and link up, we used the overlap of each fragment to increase our aim gene(‘tet’, ‘left-right’, ‘left-tet-right’).</div>
+<br/>
+</li>
+</div>
+<a class="expand-btn3">Show More</a>
+		</div><!-- .entry-content -->
+	     </article>
+<!------------------------------------week3 end------------------------------------------------>
-    <section>
-  <h1>Game Skills</h1>
-  <ul id="recent_posts">
+<!------------------------------------week4 start------------------------------------------------>
+<div id="Week4"></div>
-       <li class="post">
+<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
-         <a href="/blog/2012/07/03/state-of-browsers-july-2012/">State of Browsers, July 2012</a>
+	<h1 class="entry-title">Week4(8/14/2016-8/20/2016)</h1>
-      </li>
+		<div class="entry-content">
+<div class="note-content4">
+<li>
+<h1 style="font-size:135%">Optimization of Culture Conditions</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.15th</b></h1>
+<div style="padding-left:32px;">1.Prepared 200ml W0 medium and add 0.6g sucrose, then regarded it W4 medium.<br/>
+.Extracted 8ml W4 medium to 16 test tubes, respectively.<br/>
+.Added bacteria solution as following table (use two tubes each group)<br/>
+</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tianjin--table_8.22.png"></div>
+<div style="padding-left:32px;">4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.16th</b></h1>
+<div style="padding-left:32px;">Prepared 200ml W0 medium.<br/>
+.Extracted 8ml W0 medium to 16 test tubes, respectively.<br/>
+.Added bacteria solution as following table (use two tubes each group)<br/>
+</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tianjin--table_8.22.png"></div>
+<div style="padding-left:32px;">4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Construction of PBBR</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.14th</b></h1>
+<div style="padding-left:32px;">Ligation of cut pBBR1MCS-2 and CFP and Chemical transformation of pBBR into E.coli:</br>
+Ligation sites: Sac1 and Xba1</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/5/54/T--Tianjin--table_8.14.png"></div>
+<div style="padding-left:32px;">Result:<br/>
+The ligation was successful, because we found the special band which represented CFP during the verification by PCR. We also successfully transformed the plasmid into E.coli which was cultured on the medium with kanamycin. However, the expression of CFP was not detected, which needed to be figure out.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.15th</b></h1>
+<div style="padding-left:32px;">Cut pBBR1MCS-2 with restricted enzymes EcoR1 and Sac1 and checked by agarose gel electrophoresis: 	<br/>
+Result: Obvious bright bands located over 5000 bp.<br/>
+After gel recovery, we got the EB solution with cut plasmid pBBR1MCS-2, and the concentration is 14.7 ng/μl.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.16th</b></h1>
+<div style="padding-left:32px;">Extraction of plasmid pBBR1MCS-2.</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/3/30/T--Tianjin--table_8.16.png"></div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.17th</b></h1>
+<div style="padding-left:32px;">Ligation of cut pBBR1MCS-2 with AcoA and AceA and Chemical transformation of pBBRAA into E.coli:<br/>
+Ligation sites: Sac1 and EcoR1</div>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/6/69/T--Tianjin--table_8.18.png"></div>
+<div style="padding-left:32px;">Result:<br/>
+The ligation was successful, because we found the special band which represented AcoA and AceA during the verification by PCR. We also successfully transformed the plasmid into E.coli which was cultured on the medium with kanamycin.
+</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Experiments about <i>Bacillus subtilis</i></h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.14th</b></h1>
+<div style="padding-left:32px;"><i>B.subtilis</i> Transformation was observed failure.<br/>
+Reconstruct the plasmids.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.15th</b></h1>
+<div style="padding-left:32px;"><i>E.coli</i> transformation was observed failure.<br/>
+Enzyme digestion again.<br/>
+<i>E.coli</i> transformation again.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.16th</b></h1>
+<div style="padding-left:32px;"><i>E.coli</i> transformation was observed failure.<br/>
+Plasmid was enzyme digested and not observed correct band.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.17th</b></h1>
+<div style="padding-left:32px;">Plasmids of PETase was enzyme digestion again.<br/>
+Inoculate <i>B.subtilis DB104</i>.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.19th</b></h1>
+<div style="padding-left:32px;">Inoculate <i>E.coli</i> of pHP13-p43.
+</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Gene Knockout of <i>Escherichia coli</i></h1><br/>
+<div style="padding-left:32px;">We aimed to put the left-tet-right gene and plasmid which have λ-red gene into the the genome of E.coli. After two screening, we could gain the target strains which knocked out the atpF and atpH gene.</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
+<div style="padding-left:32px;">Synechococcus sp PCC 7942 had no signals of life.</div><br/>
+ </div>
+<a class="expand-btn4">Show More</a>
+		</div><!-- .entry-content -->
+	     </article>
+<!------------------------------------week4 end------------------------------------------------>
+<!------------------------------------week5 start------------------------------------------------>
+<div id="Week5"></div>
+<article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
+	<h1 class="entry-title">Week5(8/21/2016-8/27/2016)</h1>
+		<div class="entry-content">
+<div class="note-content4">
+			<li><h1 style="font-size:135%">Optimization of Culture Conditions</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.22th</b></h1>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.Prepared 200ml W0 medium and add 0.3g NaOAc, then regarded it W7 medium.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2.Extracted 8ml W7 medium to 16 test tubes, respectively.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3.Added bacteria solution as following table (use two tubes each group)<br/>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tianjin--table_8.22.png"></div>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4.cultured them at 30℃ and check growing situation and the situation of TPA degradation.
+</li>
+<li>
+<h1 style="font-size:135%">Experiments about <i>Bacillus subtilis</i></h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.24th</b></h1>
+<div style="padding-left:32px;">Cultivated transformed <i>E.coli</i> on chloramphenicol containing LB plates to observe whether successful.
+</div>
+<br/>
+</li>
+<br/>
+<li>
+<h1 style="font-size:135%">Gene Knockout of <i>Escherichia coli</i></h1><br/>
+<div style="padding-left:32px;">We cultivateD Saccharomyces cerevisiae and E.coli which has knocked out atpF and atpH gene and introduced GFP gene in special culture medium which the only carbon source was xylose.</div>
+<br/>
+</li>
+ </div>
+<a class="expand-btn4">Show More</a>
+		</div><!-- .entry-content -->
+	     </article>
+<!------------------------------------week5 end------------------------------------------------>
+<!------------------------------------week6 start------------------------------------------------>
+ <div id="Week6"></div>
+        <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
+	<header class="entry-header">
+		<h1 class="entry-title">Week6(8/28/2016-9/3/2016)</h1>
+	</header><!-- .entry-header -->
+		<div class="entry-content">
+	<div class="note-content4">
+<li>
+<h1 style="font-size:135%">Experiments about <i>Bacillus subtilis</i></h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.29th</b></h1>
+<div style="padding-left:32px;"><i>B.subtilis DB104</i> transformation.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.30th</b></h1>
+<div style="padding-left:32px;"><i>B.subtilis DB104</i> transformation is successful.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.2th</b></h1>
+<div style="padding-left:32px;">Construct plamid of MHETase without success.<br/>
+Transform into <i>E.coli</i>, in chloramphenicol containing LB culture.
+</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.29th</b></h1>
+<div style="padding-left:32px;">1.pMV-G19 and pMV-G15  containing our target genes were received.<br/>
+.Colonies were used to inoculate overnight cultures.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.30th</b></h1>
+<div style="padding-left:32px;">1.Plasmids were isolated using a miniprep kit.<br/>
+.Amplification of <i>19</i> and <i>15</i> with Q5 High-Fidelity DNA Polymerase out of E.coli.<br/>
+&nbsp;&nbsp;<i>19</i> amplification at 65.0°C with 19.rev/fwd primes.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR worked, positive control worked, no amplification of <i>19</i>.<br/>
+&nbsp;&nbsp;<i>15</i> amplification at 65.0°C with 15.rev/fwd primes.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;PCR worked, positive control worked, no amplification of <i>15</i>.<br/>
+.A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.<br/>
+&nbsp;&nbsp;This result was confirmed by sequencing.<br/>
+.The fragments of <i>19</i> and <i>15</i> were purified with PCR Purification Kit.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Aug.31th</b></h1>
+<div style="padding-left:32px;">1.Ligation of <i>15</i> with <i>19</i>.<br/>
+.We add a single 3`-adenine overhang to each end of the fragment of <i>19</i> and then purified with DNA Purification Kit.<br/>
+.<i>19</i> was ligated into T vectors via TA clone and transformed into E.coli via heat shock.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.1th</b></h1>
+<div style="padding-left:32px;">1.A colony PCR was performed with five colonies.<br/>
+.Gel electrophoresis showed that 5 colonies were positive for insertion of <i>19</i>.<br/>
+.Two of these colonies containing <i>19</i> were used to inoculate overnight cultures.<br/>
+.<i>15</i> gene fragment was phosphorylated.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.2th</b></h1>
+<div style="padding-left:32px;">1.Plasmids containing T vector with <i>19</i>(pT-19)were isolated using a miniprep kit.<br/>
+.Mono-restriction digest of pT-19 with stu I. <br/>
+.The enzyme-digested product was dephosphorylation.<br/>
+.Dephosphorylated plasmid and phosphorylated gene <i>15</i> were connected.<br/>
+.Ligation product was transformed into E.coli via heat shock.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.3th</b></h1>
+<div style="padding-left:32px;">1.A colony PCR was performed with twelve colonies.<br/>
+.Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.<br/>
+.These two colonies were used to inoculate overnight cultures.<br/>
+</div>
+<br/>
+ </div>
+<a class="expand-btn4">Show More</a>
+		</div><!-- .entry-content -->
+	     </article>
+<!------------------------------------week6 end------------------------------------------------>
+<!------------------------------------week7 start------------------------------------------------>
+ <div id="Week7"></div>
+         <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
+	<header class="entry-header">
+		<h1 class="entry-title">Week7(9/4/2016-9/10/2016)</h1>
+	</header><!-- .entry-header -->
+		<div class="entry-content">
+	<div class="note-content4">
+<li>
+<h1 style="font-size:135%">Experiments about <i>Bacillus subtilis</i></h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.8th</b></h1>
+<div style="padding-left:32px;">Inoculate PETase transformed <i>B.stubtilis 168</i> in 3 flask culture.<br/>
+.LB, chloramphenicol, PETase transformed <i>B.stubtilis 168</i>, PET film.<br/>
+.LB, chloramphenicol, PETase transformed <i>B.stubtilis 168</i>, no PET film.<br/>
+.LB, chloramphenicol, <i>B.stubtilis 168</i>, PET film.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.9th</b></h1>
+<div style="padding-left:32px;">Construct plamid of MHETase without success.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.10th</b></h1>
+<div style="padding-left:32px;">Construct plamid of MHETase without success.
+</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.4th</b></h1>
+<div style="padding-left:32px;">1.Plasmids with correct sequence of  <i>19-15</i>  were isolated using a miniprep kit.<br/>
+.Mono-restriction digest of pT-19-15 with Nru I.<br/>
+.The enzyme-digested product was dephosphorylation.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.6th</b></h1>
+<div style="padding-left:32px;">Colonies containing gene <i>13</i> were used to inoculate overnight cultures.
+<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.7th</b></h1>
+<div style="padding-left:32px;">1.Plasmids were isolated using a miniprep kit.<br/>
+.Amplification of <i>13</i> with Q5 High-Fidelity DNA Polymerase out of E.coli <br/>
+.<i>13</i> amplification at 65.0°C with 13.rev/fwd primes<br/>
+&nbsp;&nbsp;PCR worked, positive control worked, no amplification of <i>13</i><br/>
+.The fragments of <i>13</i> were purified with PCR Purification Kit.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.8th</b></h1>
+<div style="padding-left:32px;"><i>13</i> gene fragment was phosphorylated.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.9th</b></h1>
+<div style="padding-left:32px;">1.Insertion of Ni promoter and ligation of <i>13-19-15</i><br/>
+.Ni inducible promoter was ligated into pCPC-3301 vector.<br/>
+.Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.<br/>
+.Single colonies were obtained by plating.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.10th</b></h1>
+<div style="padding-left:32px;">
+.A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.<br/>
+&nbsp;&nbsp;Two of the successful ones were used to inoculate overnight cultures.<br/>
+.A colony PCR of pT-13-19-15 was performed with 7 colonies.<br/>
+&nbsp;&nbsp;Two of the successful ones were used to inoculate overnight cultures.<br/>
+</div>
+<br/>
+ </div>
+<a class="expand-btn4">Show More</a>
+		</div><!-- .entry-content -->
+	     </article>
+<!------------------------------------week7 end------------------------------------------------>
+<!------------------------------------week8 start------------------------------------------------>
+ <div id="Week8"></div>
+        <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
+	<header class="entry-header">
+		<h1 class="entry-title">Week8(9/11/2016-9/17/2016)</h1>
+	</header><!-- .entry-header -->
+		<div class="entry-content">
+	<div class="note-content4">
+<li><h1 style="font-size:135%">Optimization of Culture Conditions</h1><br/>
+    <b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.12th</b></h1>
+<div style="padding-left:32px;">Optimize the growing environment of Bacillus stubtilis by change medium components.Firstly, we prepared 400ml W medium, then, we devided the medium into four pieces averagely and number them No.1, No.2, No.3, No.4. Next, we added 0.1g NaCl, 0.1g NH4Cl, 0.3g sucrose to No.2, No.3, No.4, respectively. After that, we Extracted 5 ml No.1, No.2, No.3, No.4 and added them to four test tubes, then, we added 10 μL bacteria solution of <i>Bacillus stubtilis 168</i> to four test tubes, respectively. Eventually, we cultured them at 37℃ and check growing situation.</div>
+</li>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.16th</b></h1>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1.Prepared 200ml W0 medium and add 0.2g NH4Cl, then regarded it W8 medium.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2.Extracted 10ml W8 medium to 16 test tubes, respectively.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3.Added bacteria solution as following table (use two tubes each group)<br/>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tianjin--table_8.22.png"></div>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4.Cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.
+<br/><br/>
+<li>
+<h1 style="font-size:135%">Experiments about <i>Bacillus subtilis</i></h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.11th</b></h1>
+<div style="padding-left:32px;">Construct plamid of MHETase without success.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.12th</b></h1>
+<div style="padding-left:32px;">Construct plamid of MHETase without success.
+</div>
+<br/>
+<li>
+<h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.11th</b></h1>
+<div style="padding-left:32px;">Two kinds of plasmids were isolated using a miniprep kit.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.12th</b></h1>
+<div style="padding-left:32px;">Two kinds of plasmids were isolated using a miniprep kit.
+<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.14th</b></h1>
+<div style="padding-left:32px;">The sequencing results for both of them were error.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.15th</b></h1>
+<div style="padding-left:32px;">1.Colonies containing Ni inducible promoter were used to inoculate overnight cultures.<br/>
+.PCR was performed to check if the gene fragments were ligated correctly.<br/>
+&nbsp;&nbsp;13_ fwd and 15_rev on pT-13-19-15<br/>
+.Gel electrophoresis showed that it failed.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.16th</b></h1>
+<div style="padding-left:32px;">Plasmids pCPC-3031-Ni were isolated using a miniprep kit.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.17th</b></h1>
+<div style="padding-left:32px;">
+.Several PCRs were performed to check if the gene fragments were ligated correctly.<br/>
+&nbsp;&nbsp;13_fwd and 13_rev on pT-13-19-15<br/>
+&nbsp;&nbsp;19_fwd and 19_rev on pT-13-19-15<br/>
+&nbsp;&nbsp;15_fwd and 15_rev on pT-13-19-15<br/>
+&nbsp;&nbsp;13_fwd and 19_rev on pT-13-19-15<br/>
+&nbsp;&nbsp;19_fwd and 15_rev on pT-13-19-15<br/>
+&nbsp;&nbsp;13_ wd and 15_rev on pT-13-19-15<br/>
+<b>The fourth and sixth ones were not successful.</b><br/>
+.<b>Repetition:</b>  Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.<br/>
+</div>
+<br/>
+</li>
+</div>
+<a class="expand-btn4">Show More</a>
+		</div><!-- .entry-content -->
+	     </article>
+<!------------------------------------week8 end------------------------------------------------>
+<!------------------------------------week9 start------------------------------------------------>
+ <div id="Week9"></div>
+        <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
+	<header class="entry-header">
+		<h1 class="entry-title">Week9(9/18/2016-9/24/2016)</h1>
+	</header><!-- .entry-header -->
+		<div class="entry-content">
+	<div class="note-content4">
+<li><h1 style="font-size:135%">Optimization of Culture Conditions</h1><br/>
+    <b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.19th</b></h1>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1. Prepared 1L W medium and add 2.5g TPA and 1.1875g NaOH.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2. devided the medium into five pieces averagely and add chemicals as following table<br/>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/f/f4/T--Tianjin--table_9.19-1.png"></div>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3. Extracted 5ml W9, W10, W11, W12, W13 medium to 40 test tubes, respectively.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4. Added bacteria solution as following table (use two tubes each group)<br/>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/d/d1/T--Tianjin--table_9.19-2.png"></div>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5. cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.<br/>
+<b><h1 style="font-size:108%"><br/>&nbsp;&nbsp;Sep.21th</b></h1>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Repeat experiments of W9, W10 the day before yesterday.
+<b><h1 style="font-size:108%"><br/>&nbsp;&nbsp;Sep.22th</b></h1>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1. Use five mediums of Sep.19th and W0, W8 medium of Sep.16th.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2. Extracted 5ml W0, W8, W9, W10, W11, W12, W13 medium to 56 test tubes, respectively.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3. Added bacteria solution as following table (use two tubes each group)<br/>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/0/04/T--Tianjin--table_9.22.png"></div>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4. cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.
+<b><h1 style="font-size:108%"><br/>&nbsp;&nbsp;Sep.23th</b></h1>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1. Prepared 400L W medium and add 1.2g KNO3 and 1.2g glucose.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2. devided the medium into four pieces averagely and add chemicals as following table<br/>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/c/c3/T--Tianjin--table_9.23.png"></div>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3. Extracted 5ml W0, W14, W15, W16 medium to 34 test tubes, respectively.<br/>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4. Added bacteria solution as following table (use two tubes each group)<br/>
+<div style="text-align:center"><img src="https://static.igem.org/mediawiki/2016/b/bc/T--Tianjin--table_9.23-2.png"></div>
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5. cultured them at 30℃ & 200 rpm, checked the bacterial concentration at OD<sub>600</sub> and detected the concentration of TPA by UV at OD<sub>242</sub>.<br/>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Experiments about <i>Bacillus subtilis</i></h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.19th</b></h1>
+<div style="padding-left:32px;">Construct plasmid of MHETase without success.<br/>
+Cultivated MHETase transformed <i>B.stubtilis 168</i> on Erythromycin containing LB plates to observe, without success.
+</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">16SrDNA</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.19th</b></h1>
+<div style="padding-left:32px;">we firstly tried to cultivate each of them(<i>Rhodococcus RHA1, Pseudomonas putida KT2440, bacillus subtilis 168</i>) in LB culture medium for amplification.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.21th</b></h1>
+<div style="padding-left:32px;">We use orthogonal test to prove each bacteria had each special DNA stripe from the method of bacteria colony-PCR.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.22th</b></h1>
+<div style="padding-left:32px;">Then we cultivate each of them and the mixture of three in W0 culture medium for amplification.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.23th</b></h1>
+<div style="padding-left:32px;">We did the same 4*3(bacteria liquid and six primer sequences) orthogonal test.
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.24th</b></h1>
+<div style="padding-left:32px;">We cultivate each of them and the mixture of three in in modified W0 culture medium which changed the carbon source from glucose to sugar for amplification.
+</div>
+<br/>
+</li>
+<li>
+<h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.18th</b></h1>
+<div style="padding-left:32px;"><b>Repetition:</b> Several PCRs were performed to check if the gene fragments were ligated correctly.<br/>
+&nbsp;&nbsp;13_fwd and 13_rev on pT-13-19-15<br/>
+&nbsp;&nbsp;13_fwd and 19_rev on pT-13-19-15<br/>
+<b>The second one was failed.</b><br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.21th</b></h1>
+<div style="padding-left:32px;">1.Medium preparation :BG-11.<br/>
+.19_ fwd and 15_rev were used to amplify <i>19-15</i>.
+<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.22th</b></h1>
+<div style="padding-left:32px;">1.Gel electrophoresis showed that amplification of fragments was successfull.<br/>
+.Ligated <i>13</i> and <i>19-15</i> via overlap PCR.<br/>
+.This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.<br/>
+.Restriction digest on pCPC-3031-Ni with Sac I.<br/>
+.The fragment <i>13-19-15</i> was ligated onto T vector and transformed into E.coli via heat shock.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.23th</b></h1>
+<div style="padding-left:32px;">1.A colony PCR of pT-13-19-15 was performed with 12 colonies.<br/>
+.Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.<br/>
+.<i>13-19-15</i> gene fragment was phosphorylated.<br/>
+.Phosphorylated <i>13-19-15</i> was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.24th</b></h1>
+<div style="padding-left:32px;">Plasmids pT-13-19-15 were isolated using a miniprep kit.<br/>
+</div>
+<br/>
+</div>
+<a class="expand-btn4">Show More</a>
+		</div><!-- .entry-content -->
+	     </article>
+<!------------------------------------week9 end------------------------------------------------>
+<!------------------------------------week10 start------------------------------------------------>
+ <div id="Week10"></div>
+        <article id="post-4252" class="post-4252 post type-post status-publish format-standard has-post-thumbnail hentry category-150 tag-174 tag-xinjiang tag-173">
+	<header class="entry-header">
+		<h1 class="entry-title">Week10(9/25/2016-10/1/2016)</h1>
+	</header><!-- .entry-header -->
+		<div class="entry-content">
+	<div class="note-content4">
+<li>
+<h1 style="font-size:135%">16SrDNA</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.25th</b></h1>
+<div style="padding-left:32px;">We still did the same 4*3(bacteria liquid and six primer sequences) orthogonal test.
+</div>
+<br/>
+<li>
+<h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.25th</b></h1>
+<div style="padding-left:32px;">1.A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.<br/>
+.Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.<br/>
+</div>
+<br/>
+<b><h1 style="font-size:108%">&nbsp;&nbsp;Sep.26th</b></h1>
+<div style="padding-left:32px;">Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.<br/>
+<b>The sequencing results for them were correct.</b>
+<br/>
+</div>
+<br/>
+</div>
+<a class="expand-btn4">Show More</a>
+		</div><!-- .entry-content -->
+	     </article>
+<!------------------------------------week10 end------------------------------------------------>
+        </div><!-- #content -->
+	</div><!-- #main -->
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+	  $( '.expand-btn3' ).click(function() {
+		var $text = $(this).html();
+		$(this).siblings( '.note-content3' ).toggle();
+		if( $text === 'Show More' ) {
+			$(this).html( 'Show Less' );
+		} else {
+			$(this).html( 'Show More' );
+		}
+	});
+});
+        </script>
+<script>   /* show more */
+      $(document).ready(function() {
+	  $( '.expand-btn4' ).click(function() {
+		var $text = $(this).html();
+		$(this).siblings( '.note-content4' ).toggle();
+		if( $text === 'Show More' ) {
+			$(this).html( 'Show Less' );
+		} else {
+			$(this).html( 'Show More' );
+		}
+	});
+});
+        </script>
+<script>   /* show more */
+      $(document).ready(function() {
+	  $( '.expand-btn5' ).click(function() {
+		var $text = $(this).html();
+		$(this).siblings( '.note-content5' ).toggle();
+		if( $text === 'Show More' ) {
+			$(this).html( 'Show Less' );
+		} else {
+			$(this).html( 'Show More' );
+		}
+	});
+});
+        </script>
+<script>   /* show more */
+      $(document).ready(function() {
+	  $( '.expand-btn6' ).click(function() {
+		var $text = $(this).html();
+		$(this).siblings( '.note-content6' ).toggle();
+		if( $text === 'Show More' ) {
+			$(this).html( 'Show Less' );
+		} else {
+			$(this).html( 'Show More' );
+		}
+	});
+});
+        </script>
+         <script type="text/javascript">
+/* chrome ie8下均正常 ,实现滚动效果*/
+$(document).ready(function() {
+    $("a.topLink").click(function() {
+        $("html, body").animate({
+            scrollTop: $($(this).attr("href")).offset().top + "px"
+        }, {
+            duration: 1000,
+            easing: "swing"
+        });
+        return false;
+    });
+});
+</script>
 </html>
-{{:Team:Tianjin/Templates/AddJS|:Team:Tianjin/Achievements/js/hey.js}}
-{{:Team:Tianjin/Templates/AddJS|:Team:Tianjin/Achievements/js/bootstrap.js}}
+{{:Team:Tianjin/Templates/Sponsor|}}
-{{:Team:Tianjin/Templates/AddJS|:Team:Tianjin/Achievements/js/material.js}}
-{{:Team:Tianjin/Templates/AddJS|:Team:Tianjin/Achievements/js/material-kit.js}}

Difference between revisions of "Team:Tianjin/Achievements"

Revision as of 10:40, 13 October 2016

Week1(7/24/2016-7/30/2016)

Optimization of Culture Conditions

Jul.26th

Jul.27th

Jul.28th

Jul.30th

Optimization of Culture Conditions

Jul.31th

Aug.1th

Aug.2th

Aug.3th

Aug.4th

Aug.5th

Aug.6th

Construction of PBBR

Aug.5th

Aug.6th

Gene Knockout of Escherichia coli

Modification of Cyanobacteria

Jul.31th

Week3(8/7/2016-8/13/2016)

Optimization of Culture Conditions

Aug.7th

Aug.9th

Aug.10th

Aug.12th

Aug.13th

Construction of PBBR

Aug.7th

Aug.8th

Aug.9th

Aug.11th

Aug.12th

Aug.13th

Experiments about Bacillus subtilis

Aug.9th

Aug.10th

Aug.11th

Aug.12th

Gene Knockout of Escherichia coli

Week4(8/14/2016-8/20/2016)

Optimization of Culture Conditions

Aug.15th

Aug.16th

Construction of PBBR

Aug.14th

Aug.15th

Aug.16th

Aug.17th

Experiments about Bacillus subtilis

Aug.14th

Aug.15th

Aug.16th

Aug.17th

Aug.19th

Gene Knockout of Escherichia coli

Modification of Cyanobacteria

Week5(8/21/2016-8/27/2016)

Optimization of Culture Conditions

Aug.22th

Experiments about Bacillus subtilis

Aug.24th

Gene Knockout of Escherichia coli

Experiments about Bacillus subtilis

Aug.29th

Aug.30th

Sep.2th

Modification of Cyanobacteria

Aug.29th

Aug.30th

Aug.31th

Sep.1th

Sep.2th

Sep.3th

Experiments about Bacillus subtilis

Sep.8th

Sep.9th

Sep.10th