Team:ZJU-China/Notebook

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<div class="main">
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<div class="bgpart3"></div>
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      <table style="background-color:transparent;" width="750" border="0" cellspacing="0" cellpadding="1">
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  <tr>
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    <td ><h3>Modeling|biobrick</h3></td>
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    <td > <div class="roundcorner" >
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    <p><a name="jumptop" id="p_intro">&nbsp;Introduction&nbsp;</a>|<a id="p_model">&nbsp;Model&nbsp;</a>|<a id="p_simul">&nbsp;Simulation&nbsp;</a></p>
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<p style="margin-left:10px;">This model is used for simulating the behavior of three genetic circuits we designed</p>
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</div>
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 +
<h4 class="current">Lab Notes</h4>
 +
<div class="pane" style="display: block;"><a
 +
href="https://2011.igem.org/Team:ZJU-China/Notebook.html">July</a> <a
 +
href="https://2011.igem.org/Team:ZJU-China/August.html">August<br />
 +
</a> <a href="https://2011.igem.org/Team:ZJU-China/September.html">September<br />
 +
</a>
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<a href="https://2011.igem.org/Team:ZJU-China/October.html">October</a></div>
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<h4>Protocol</h4>
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    <div id="leftul"><div class="leftul">
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<h4>Overview</h4>
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<div class="pane" >
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                <a  href="https://2011.igem.org/Team:ZJU-China/Project">Summary</a>
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<a  href="https://2011.igem.org/Team:ZJU-China/Project/Stratifiedsystem">Background<br/></a>
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-
<a  href="https://2011.igem.org/Team:ZJU-China/Project/Result">Results<br/></a>
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        <a  href="https://2011.igem.org/Team:ZJU-China/Project/Future">Future Application</a>
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</div>
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<h4 class="current">Modeling</h4>
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-
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<div class="pane" style="display:block;">
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<a  href="https://2011.igem.org/Team:ZJU-China/Modeling">|Biofilm formation<br/></a>
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<a  href="https://2011.igem.org/Team:ZJU-China/Modeling/Biobrick">|Biobrick<br/></a>
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-
        <a  href="#">|Results<br/></a>
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</div>
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<h4>Results</h4>
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<div class="pane" >
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<a  href="https://2011.igem.org/Team:ZJU-China/Datapage">Data page</a>
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                <a  href="https://2011.igem.org/Team:ZJU-China/Parts">Parts</a>
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<p></p>
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<h1>Lab Notes - July</h1>
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<div id="framecontent">
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<h1>Biobrick Group</h1>
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<h3>Week1</h3><hr/>  
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<div id="page1">
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<h1>Week1</h1>
 +
<table id="notesheet" style="width:690px;"  border="1" cellspacing="0"
 +
cellpadding="1" style="vertical-align: middle">
 +
<tr>
 +
<td width="76"><strong>Day</strong></td>
 +
<td width="349"><strong>Note</strong></td>
 +
</tr>
 +
 
 +
<tr>
 +
<td id="sheetleft">Jul.4th Monday</td>
 +
<td>
 +
<table id="intable" width="328" border="0" cellspacing="0"
 +
cellpadding="1">
   
   
-
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">
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<td width="128">▪ Aerobic cultivation of DH5α</td>
-
  <tr><td width="76">Day</td><td width="349">Note</td></tr>
+
-
 
+
-
  <tr>
+
-
    <td id="sheetleft">Jul.4th Monday</td>
+
-
    <td><table id="intable" width="328" border="0" cellspacing="0" cellpadding="1">
+
-
 
+
-
    <td width="128">• Aerobic cultivation of DH5α</td>
+
-
 
+
-
 
+
-
    <td width="196">• Preparation of apparatus for the formation of biofilm</td>
+
-
 
+
-
</table></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td id="sheetleft">Jul 5th Tuesday
+
-
</td>
+
-
    <td>&nbsp;</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td id="sheetleft">Jul.6th
+
-
Wednesday
+
-
</td>
+
-
    <td><table width="217" border="0" cellspacing="0" cellpadding="1">
+
-
  <tr>
+
-
    <td width="215">•Receiving  primers ordered previously</td>
+
-
   
+
-
  </tr>
+
-
</table>
+
-
</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td id="sheetleft">Jul.7th
+
-
Thursday
+
-
</td>
+
-
    <td><table width="238" border="0" cellspacing="0" cellpadding="1">
+
-
  <tr>
+
-
    <td width="200">• Preparation of the aliquot of the primers</td>
+
-
    <td width="200">• Something wrong with a shaking incubator</td>
+
-
  </tr>
+
-
</table>
+
-
</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td id="sheetleft">Jul.8th
+
-
Friday
+
-
</td>
+
-
    <td><table width="222" border="0" cellspacing="0" cellpadding="1">
+
-
      <tr>
+
-
        <td width="155">• Preparation of culture plates for the transformations</td>
+
-
      </tr>
+
-
    </table></td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td id="sheetleft">Jul.9th
+
-
Saturday
+
-
</td>
+
-
    <td><table width="313" border="0" cellspacing="0" cellpadding="1">
+
-
  <tr>
+
-
    <td width="127">• Preparation of culture plates for the transformations</td>
+
-
    <td width="182">• protocols of transformation</td>
+
-
  </tr>
+
-
</table>
+
-
</td>
+
-
  </tr>
+
-
  <tr>
+
-
    <td id="sheetleft">Jul.10th
+
-
Sunday
+
-
</td>
+
-
    <td><table width="246" border="0" cellspacing="0" cellpadding="1">
+
-
      <tr>
+
-
        <td>• Several colonies were picked up and cultivated in  5mL LB medium.
+
-
•Cryosectioning of biofilm
+
-
</td>
+
-
      </tr>
+
-
    </table></td>
+
-
  </tr>
+
-
+
-
</table>
+
-
<p>&nbsp;</p>
+
-
</div>
+
-
<script type="text/javascript">
+
 +
 +
<td width="196">▪ Preparation of apparatus for the formation of
 +
biofilm</td>
 +
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul 5th Tuesday</td>
 +
<td>&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.6th Wednesday</td>
 +
<td>
 +
<table width="217" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="215">▪ Receiving primers ordered previously</td>
 +
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.7th Thursday</td>
 +
<td>
 +
<table width="238" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="200">▪ Preparation of the aliquot of the primers</td>
 +
<td width="200">▪ Something wrong with a shaking incubator</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.8th Friday</td>
 +
<td>
 +
<table width="222" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="155">▪ Preparation of culture plates for the
 +
transformations</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.9th Saturday</td>
 +
<td>
 +
<table width="313" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="127">▪ Preparation of culture plates for the
 +
transformations</td>
 +
<td width="182">▪ protocols of transformation</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.10th Sunday</td>
 +
<td>
 +
<table width="246" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td>▪ Several colonies were picked up and cultivated in 5mL LB
 +
medium. ▪ryosectioning of biofilm</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
 +
</table>
 +
</p>
 +
<h1>Week2</h1>
 +
<table id="notesheet" width="650" border="1" cellspacing="0"
 +
cellpadding="1">
 +
<tr>
 +
<td width="76"><strong>Day</strong></td>
 +
<td width="349"><strong>Note</strong></td>
 +
</tr>
 +
 +
<tr>
 +
<td id="sheetleft">Jul.11th Monday</td>
 +
<td>
 +
<table id="intable" width="541" border="0" cellspacing="0"
 +
cellpadding="1">
 +
 +
<td width="128">▪ Set up new LB culture plates with ampicillin
 +
and kanamycin
 +
</p>
 +
</td>
 +
 +
 +
<td width="196">▪ Sterilization of Glycerol and <br />
 +
Preparation of 25mg/mL kanamycin</td>
 +
<td>▪Transformation of the parts mentioned on Jul.9th for the
 +
second time</td>
 +
<td>▪Observation the sections</td>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul 12th Tuesday</td>
 +
<td>
 +
<table id="intable" width="560" border="0" cellspacing="0"
 +
cellpadding="1">
 +
 +
<td width="128">▪ Pick two colonies of each parts and cultivate
 +
them in LB medium</td>
 +
 +
 +
<td width="203">▪ Transformation of three parts(20J,20H.22B from
 +
Kit plates of 2011 Distribution ) which are related to the
 +
degradation of Cellulose</td>
 +
<td width="91">▪Min prep to isolate 10I,12I and 22M</td>
 +
<td width="130">▪Conservation of 10I,12I,22M and 11P</td>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.13th Wednesday</td>
 +
<td>
 +
<table width="618" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="104">▪ Place the culture plate of 20J,20H and 22B in
 +
the fridge.</td>
 +
<td width="102">▪min prep to isolate 13K ▪onservation of 13K</td>
 +
<td width="163">▪estriction digest of the
 +
parts(12I,10I,22M,13K) with EcoRI and PstI</td>
 +
<td width="111">▪Gel electrophoresis to analyse restriction
 +
fragments</td>
 +
<td width="128">▪Test the Tm of Primers CP1&amp;CS,NP&amp;NS
 +
with 13K. The result of Gel electrophoresis shows that 60.2℃ is the
 +
Tm of NS and NP, and 57.4℃ is the Tm of CS and CP1.</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.14th Thursday</td>
 +
<td>
 +
<table width="618" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td>▪ Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers.
 +
The result of Gel electrophoresis shows that the Tm for the primers
 +
of Vgb is 54℃ and the Tm for the primers of nirB is 55℃ The RCR of
 +
YFP,RFP and tetR failed.</td>
 +
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.15th Friday</td>
 +
<td>
 +
<table width="600" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="155">▪ PCR(Phusion)<br />
 +
▪ Digest 10I, 22M, 13K ▪ Used wrong cutter, digestion again.</td>
 +
<td>▪ Run the results of PCR and the first digestion. The
 +
annealing temperature of YFP needed change. The digestion results
 +
confirmed</td>
 +
<td>▪ Run the digestion results of second time. The bands are
 +
confirmed.</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.16th Saturday</td>
 +
<td>
 +
<table width="620" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="127">▪ Cut the linearized pSB1k3 with E+P</td>
 +
<td width="123">▪ Purify the digestion results of 22M, 13K, 10I</td>
 +
<td width="133">▪ Confirm digestion of pSB1k3 by
 +
electrophoresis, then purification</td>
 +
<td width="113">▪ Test Tm of YFP<br />
 +
▪ ligation: 22M+10I, 13K+10I</td>
 +
<td width="114">▪ Tm of YFP is 54 degree ▪ transform the
 +
ligation results.</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.17th Sunday</td>
 +
<td>
 +
<table width="620" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td>▪ PCR 22M<br />
 +
▪ purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR.</td>
 +
<td>▪ ligation the purified the fragments in yesterday.</td>
 +
<td>▪ 22M PCR<br />
 +
▪ Transform the ligation results.</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
 +
</table>
 +
 +
<p>&nbsp;</p>
 +
<h1>Week3</h1>
 +
<table id="notesheet" width="713" border="1" cellspacing="0"
 +
cellpadding="1">
 +
<tr>
 +
<td width="87"><strong>Day</strong></td>
 +
<td width="616"><strong>Note</strong></td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.18th Monday</td>
 +
<td>
 +
<table id="intable" width="541" border="0" cellspacing="0"
 +
cellpadding="1">
 +
 +
<td width="128">▪ Genome extraction of E.coli</td>
 +
 +
 +
<td width="196">▪ PCR of NirB<br />
 +
▪ Cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P</td>
 +
<td>▪ Ligate: 10I+13K, 10I+22M <br />
 +
▪ Repeat NirB PCR</td>
 +
<td>
 +
<p align="left">▪ Culture 11P<br />
 +
▪ Miniprep 10I, 22M, 13K</p>
 +
</td>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul 19th Tuesday</td>
 +
<td>
 +
<table id="intable" width="615" border="0" cellspacing="0"
 +
cellpadding="1">
 +
 +
<td width="140">▪ mini-prep: 10I+13K, 10I+22M</td>
 +
 +
 +
<td width="213">▪Insert 10I+13K, 10I+22M into pSB1k3</td>
 +
<td width="110">▪Transform: 5E, 3C, 7C, 1K, 1I from the
 +
distribution plate</td>
 +
<td width="144">▪Transform 10I+22M, 10I+13K</td>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.20th Wednesday</td>
 +
<td>
 +
<table width="610" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="170">▪Colonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K,
 +
1I confirmed</td>
 +
<td width="102">▪ Gibson PCR</td>
 +
<td width="250">▪Colony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K,
 +
1I</td>
 +
<td width="80">▪PCR NirB</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.21th Thursday</td>
 +
<td>
 +
<table width="618" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td>▪PCR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I
 +
</td>
 +
<td>▪ Gibson PCR ▪ Run the results of PCR verification. Bands
 +
confirmed.</td>
 +
<td>▪ Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I ▪
 +
Purification of Gibson PCR results</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.22th Friday</td>
 +
<td>
 +
<table width="618" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="117">▪ PCR amplification of Gibson assembly results
 +
</td>
 +
<td width="144">▪ Mini prep: 22M+10I, 22.Presever in -20<br />
 +
▪ Mini prep: 1K,1I,3C,5E,7C<br />
 +
▪ Gibson Assembly fail.</td>
 +
<td width="164">▪ PCR NirB by Phusion<br />
 +
▪Repeat PCR by changing Pnibr to Gnirbr<br />
 +
▪Cut the mini and medi prep results with E</td>
 +
<td width="185">▪ Run the results of PCR and digestion. Fail in
 +
PCR of NirB, succeed in 10I+3K and 10I+22M ligation.</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.23th Saturday</td>
 +
<td>
 +
<table width="613" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="237">▪Double digestion of 13K+10I, 22M+10I, pSB1c3
 +
 +
</td>
 +
<td width="169">▪ Fail in purification of Medi prep</td>
 +
<td width="201">▪ PCR NirB<br />
 +
▪ Ligate NirB+13K+10I, Vgb+22M+10I</td>
 +
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.24th Sunday</td>
 +
<td>
 +
<table width="608" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="244">▪ Gibson assembly: NirB+RFP+tetR<br />
 +
▪ Cut results of Gibson assembly and pSB1c3.</td>
 +
<td width="164">▪ Purify the ligation results in yesterday</td>
 +
<td width="194">▪ ligate with backbone ▪ Culture 1I, 1K, 3C,
 +
5E, 7C, 11P</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
 +
</table>
 +
<p>&nbsp;</p>
 +
<h1>Week4</h1>
 +
<table id="notesheet" width="691" border="1" cellspacing="0"
 +
cellpadding="1">
 +
<tr>
 +
<td width="63"><strong>Day</strong></td>
 +
<td width="630"><strong>Note</strong></td>
 +
</tr>
 +
 +
<tr>
 +
<td id="sheetleft">Jul.25th Monday</td>
 +
<td>
 +
<table id="intable" width="541" border="0" cellspacing="0"
 +
cellpadding="1">
 +
 +
<td width="128">▪ Transform Pnirb+13k+10I+pSBK3-2</td>
 +
 +
 +
<td>▪ Place the culture in 4 degree</td>
 +
<td>▪ Medi-and mini-prep of five cultre</td>
 +
<td>▪Gibson Assembly confirmation by gel</td>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul. 26th Tuesday</td>
 +
<td>
 +
<table id="intable" width="610" border="0" cellspacing="0"
 +
cellpadding="1">
 +
 +
<td width="134">▪ Cut Pnirb, Pvgb,(E+S)<br />
 +
▪ Cut 10I+13K, 10I+22M.(X+P)</td>
 +
 +
 +
<td width="167">▪ Ligation: vgb+10I+22M, nirB+10I+13K,</td>
 +
<td width="172">▪ Ligation: pSBK13+vgb+10I+22M,
 +
pSBK13+nirB+10I+13K</td>
 +
<td width="129">▪ Transform the ligation results.</td>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.27th Wednesday</td>
 +
<td>
 +
<table width="608" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="170">▪ No colony on the plate<br />
 +
▪ Gibson assembly</td>
 +
<td width="184">▪ Run the results of Gibson assembly</td>
 +
<td width="248">▪ Excise the bands, the purify the DNA<br />
 +
▪ Gibson Assenmly</td>
 +
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.28th Thursday</td>
 +
<td>
 +
<table width="609" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="607">▪ 2011 China Meet-up @ Hefei</td>
 +
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.29th Friday</td>
 +
<td>
 +
<table width="607" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td>▪ 2011 China Meet-up @ Hefei</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.30th Saturday</td>
 +
<td>
 +
<table width="620" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
 +
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td id="sheetleft">Jul.31th Sunday</td>
 +
<td>
 +
<table width="600" border="0" cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td width="368">▪ Culture 22M+10I, 13K+10I</td>
 +
<td width="228">▪ Cut a0H, 20J, 22B</td>
 +
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
 +
</table>
 +
<br />
 +
<br />
 +
</div>
 +
<!--page1-->
 +
<div id="importantcontainer">
 +
<div class="frame" id="important">
 +
<div class="bgcolors" id="round">
 +
<h1>Data/Protocol</h1>
 +
</div>
 +
</div>
 +
</div>
 +
<div id="framecontent">
 +
<h1>Jul.13th Wednesday</h1>
 +
 +
<p><strong>Systems of restriction digestion with EcoRI and
 +
PstI</strong></p>
 +
<p>
 +
<table style="margin: 15px;" width="110" height="200" border="1"
 +
cellspacing="0" cellpadding="1">
 +
<tr>
 +
<td>Plasmid</td>
 +
<td>1μL (>100ng)</td>
 +
</tr>
 +
<tr>
 +
<td>EcoRI</td>
 +
<td>1μL</td>
 +
</tr>
 +
<tr>
 +
<td>PstI</td>
 +
<td>1μL</td>
 +
</tr>
 +
<tr>
 +
<td>10×Buffer Tango</td>
 +
<td>2μL</td>
 +
</tr>
 +
<tr>
 +
<td>ddH2O</td>
 +
<td>15μL</td>
 +
</tr>
 +
</table>
 +
<img src="https://static.igem.org/mediawiki/2011/c/c3/0713program.png"
 +
width="200" height="200" /></p>
 +
<p><strong>Temperature grad</strong><br />
 +
56℃ 57.4℃ 60.2℃ 62.9℃ 64.3℃ 65.8℃ /p>
 +
<div id="framecontent">
 +
<p>&nbsp;</p>
 +
 +
<p><strong>PCR system (test the Tm of the primers
 +
CP1&amp;CS, NP&amp;NS)</strong></p>
 +
 +
<table style="margin: 15px;" width="110" border="1" cellspacing="0"
 +
cellpadding="1">
 +
<tr>
 +
<td>10×Buffer</td>
 +
<td>2μL</td>
 +
</tr>
 +
<tr>
 +
<td>dNTPs</td>
 +
<td>0.5μL</td>
 +
</tr>
 +
<tr>
 +
<td>primers CP1 (NP)</td>
 +
<td>1μL</td>
 +
</tr>
 +
<tr>
 +
<td>primers CS (NS)</td>
 +
<td>1μL</td>
 +
</tr>
 +
<tr>
 +
<td>Template</td>
 +
<td>1μL</td>
 +
</tr>
 +
<tr>
 +
<td>rTaq</td>
 +
<td>0.2μL</td>
 +
</tr>
 +
<tr>
 +
<td>H2O</td>
 +
<td>14.5μL</td>
 +
</tr>
 +
<tr>
 +
<td>Total</td>
 +
<td>20μL</td>
 +
</tr>
 +
</table>
 +
</p>
 +
<p>Within each compartment are<strong> components</strong>: include
 +
different types of biomass ,substrates , products. biomass is often
 +
divided into active microbial species, inert cells, and extracellular
 +
polymeric substances(EPS).</p>
 +
<p>The components can undergo transformation, transport, and
 +
transfer <strong>processes</strong>. For example, substrate is consumed,
 +
and this leads to the synthesis of new active biomass.</p>
 +
<p>All process affecting each component in each compartment are
 +
mathematically linked together into a<strong> mass balance
 +
equation</strong> that contains rate terms and parameters for each process.</p>
 +
<p><strong>Model Selection:</strong>Many kinds of Mathematics models
 +
have been founded to describe a system of biofilm. Models of different
 +
dimensions (1d, 2d, 3d) focus on different properties of a biofilm.
 +
Since we care most about the oxygen concentration gradients
 +
perpendicular to the substratum, <strong>numerical
 +
1-dimensional dynamic model(N1)</strong> would be a proper choice for us.</p>
 +
</div>
 +
<!--bgcolors--></div>
 +
<!--important--></div>
 +
<!--importantcontainer-->
 +
<p style="clear: both;"></p>
 +
<div id="page2">
 +
<div class="block" id="nsheet"><img
 +
src="http://ung.igem.org/wiki/images/4/42/Biofilmcomp.png" width="640"
 +
height="464" style="margin: 5px;" />
 +
<p>&nbsp;</p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h1>Early July</h1>
 +
<p>Pre-experiments with biofilm formation with circular and non
 +
circular silicone tube, 24 well plate on different support including
 +
glass, paper, plastic film, rubber. The final material are used based on
 +
the easiness biofilm form on them and on the easiness to observe under
 +
microscope.</p>
 +
<img
 +
src="https://static.igem.org/mediawiki/2011/b/be/20110711-3nP4100X0877.jpg"
 +
width="400" style="margin-left: 20px;">
 +
<p>&nbsp;</p></div>
 +
<div class="block" id="nsheet">
 +
<h1>28th July</h1>
 +
<p>13.30: DH5α 11p 5mlX4<br />
 +
23.00: silicone tube set at 37℃ /p>
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h1>29th July</h1>
 +
<p>13.00: LB culture 50ml with circular culture medium. LB culture
 +
50ml with noncircular culture medium.</p>
 +
 +
</div>
 +
<div class="block" id="nsheet">
 +
<h1>31st July</h1>
 +
<p>13.00: -80℃ storing silicone tube. A thick white and loosely bond
 +
substance is seen on the inner wall of the 5mm silicone tube, and on the
 +
inner wall of the 1mm silicone tube a flatter and smoother white
 +
substance. Especially obvious where the tube turns. Possibly because the
 +
speed of culture flow is slower.</p>
 +
 +
</div>
 +
</div>
 +
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Latest revision as of 19:59, 28 October 2011

Notebook - July

Lab Notes

July August
 September
 October

Protocol

Protocol

Brainstorm

Brainstorm

Lab Notes - July

Biobrick Group

Week1

Day Note
Jul.4th Monday
▪ Aerobic cultivation of DH5α ▪ Preparation of apparatus for the formation of biofilm
Jul 5th Tuesday  
Jul.6th Wednesday
▪ Receiving primers ordered previously
Jul.7th Thursday
▪ Preparation of the aliquot of the primers ▪ Something wrong with a shaking incubator
Jul.8th Friday
▪ Preparation of culture plates for the transformations
Jul.9th Saturday
▪ Preparation of culture plates for the transformations ▪ protocols of transformation
Jul.10th Sunday
▪ Several colonies were picked up and cultivated in 5mL LB medium. ▪ryosectioning of biofilm

Week2

Day Note
Jul.11th Monday
▪ Set up new LB culture plates with ampicillin and kanamycin

▪ Sterilization of Glycerol and
Preparation of 25mg/mL kanamycin		 ▪Transformation of the parts mentioned on Jul.9th for the second time ▪Observation the sections
Jul 12th Tuesday
▪ Pick two colonies of each parts and cultivate them in LB medium ▪ Transformation of three parts(20J,20H.22B from Kit plates of 2011 Distribution ) which are related to the degradation of Cellulose ▪Min prep to isolate 10I,12I and 22M ▪Conservation of 10I,12I,22M and 11P
Jul.13th Wednesday
▪ Place the culture plate of 20J,20H and 22B in the fridge. ▪min prep to isolate 13K ▪onservation of 13K ▪estriction digest of the parts(12I,10I,22M,13K) with EcoRI and PstI ▪Gel electrophoresis to analyse restriction fragments ▪Test the Tm of Primers CP1&CS,NP&NS with 13K. The result of Gel electrophoresis shows that 60.2℃ is the Tm of NS and NP, and 57.4℃ is the Tm of CS and CP1.
Jul.14th Thursday
▪ Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers. The result of Gel electrophoresis shows that the Tm for the primers of Vgb is 54℃ and the Tm for the primers of nirB is 55℃ The RCR of YFP,RFP and tetR failed.
Jul.15th Friday
▪ PCR(Phusion)
▪ Digest 10I, 22M, 13K ▪ Used wrong cutter, digestion again.		 ▪ Run the results of PCR and the first digestion. The annealing temperature of YFP needed change. The digestion results confirmed ▪ Run the digestion results of second time. The bands are confirmed.
Jul.16th Saturday
▪ Cut the linearized pSB1k3 with E+P ▪ Purify the digestion results of 22M, 13K, 10I ▪ Confirm digestion of pSB1k3 by electrophoresis, then purification ▪ Test Tm of YFP
▪ ligation: 22M+10I, 13K+10I		 ▪ Tm of YFP is 54 degree ▪ transform the ligation results.
Jul.17th Sunday
▪ PCR 22M
▪ purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR.		 ▪ ligation the purified the fragments in yesterday. ▪ 22M PCR
▪ Transform the ligation results.

 

Week3

Day Note
Jul.18th Monday
▪ Genome extraction of E.coli ▪ PCR of NirB
▪ Cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P		 ▪ Ligate: 10I+13K, 10I+22M
▪ Repeat NirB PCR

▪ Culture 11P
▪ Miniprep 10I, 22M, 13K
Jul 19th Tuesday
▪ mini-prep: 10I+13K, 10I+22M ▪Insert 10I+13K, 10I+22M into pSB1k3 ▪Transform: 5E, 3C, 7C, 1K, 1I from the distribution plate ▪Transform 10I+22M, 10I+13K
Jul.20th Wednesday
▪Colonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I confirmed ▪ Gibson PCR ▪Colony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I ▪PCR NirB
Jul.21th Thursday
▪PCR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I ▪ Gibson PCR ▪ Run the results of PCR verification. Bands confirmed. ▪ Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I ▪ Purification of Gibson PCR results
Jul.22th Friday
▪ PCR amplification of Gibson assembly results ▪ Mini prep: 22M+10I, 22.Presever in -20
▪ Mini prep: 1K,1I,3C,5E,7C
▪ Gibson Assembly fail.		 ▪ PCR NirB by Phusion
▪Repeat PCR by changing Pnibr to Gnirbr
▪Cut the mini and medi prep results with E		 ▪ Run the results of PCR and digestion. Fail in PCR of NirB, succeed in 10I+3K and 10I+22M ligation.
Jul.23th Saturday
▪Double digestion of 13K+10I, 22M+10I, pSB1c3 ▪ Fail in purification of Medi prep ▪ PCR NirB
▪ Ligate NirB+13K+10I, Vgb+22M+10I
Jul.24th Sunday
▪ Gibson assembly: NirB+RFP+tetR
▪ Cut results of Gibson assembly and pSB1c3.		 ▪ Purify the ligation results in yesterday ▪ ligate with backbone ▪ Culture 1I, 1K, 3C, 5E, 7C, 11P

 

Week4

Day Note
Jul.25th Monday
▪ Transform Pnirb+13k+10I+pSBK3-2 ▪ Place the culture in 4 degree ▪ Medi-and mini-prep of five cultre ▪Gibson Assembly confirmation by gel
Jul. 26th Tuesday
▪ Cut Pnirb, Pvgb,(E+S)
▪ Cut 10I+13K, 10I+22M.(X+P)		 ▪ Ligation: vgb+10I+22M, nirB+10I+13K, ▪ Ligation: pSBK13+vgb+10I+22M, pSBK13+nirB+10I+13K ▪ Transform the ligation results.
Jul.27th Wednesday
▪ No colony on the plate
▪ Gibson assembly		 ▪ Run the results of Gibson assembly ▪ Excise the bands, the purify the DNA
▪ Gibson Assenmly
Jul.28th Thursday
▪ 2011 China Meet-up @ Hefei
Jul.29th Friday
▪ 2011 China Meet-up @ Hefei
Jul.30th Saturday
Jul.31th Sunday
▪ Culture 22M+10I, 13K+10I ▪ Cut a0H, 20J, 22B


Data/Protocol

Jul.13th Wednesday

Systems of restriction digestion with EcoRI and PstI

Plasmid 1μL (>100ng)
EcoRI 1μL
PstI 1μL
10×Buffer Tango 2μL
ddH2O 15μL

Temperature grad
56℃ 57.4℃ 60.2℃ 62.9℃ 64.3℃ 65.8℃ /p>

 

PCR system (test the Tm of the primers CP1&CS, NP&NS)

10×Buffer 2μL
dNTPs 0.5μL
primers CP1 (NP) 1μL
primers CS (NS) 1μL
Template 1μL
rTaq 0.2μL
H2O 14.5μL
Total 20μL

Within each compartment are components: include different types of biomass ,substrates , products. biomass is often divided into active microbial species, inert cells, and extracellular polymeric substances(EPS).

The components can undergo transformation, transport, and transfer processes. For example, substrate is consumed, and this leads to the synthesis of new active biomass.

All process affecting each component in each compartment are mathematically linked together into a mass balance equation that contains rate terms and parameters for each process.

Model Selection:Many kinds of Mathematics models have been founded to describe a system of biofilm. Models of different dimensions (1d, 2d, 3d) focus on different properties of a biofilm. Since we care most about the oxygen concentration gradients perpendicular to the substratum, numerical 1-dimensional dynamic model(N1) would be a proper choice for us.

 

Early July

Pre-experiments with biofilm formation with circular and non circular silicone tube, 24 well plate on different support including glass, paper, plastic film, rubber. The final material are used based on the easiness biofilm form on them and on the easiness to observe under microscope.

 

28th July

13.30: DH5α 11p 5mlX4
23.00: silicone tube set at 37℃ /p>

29th July

13.00: LB culture 50ml with circular culture medium. LB culture 50ml with noncircular culture medium.

31st July

13.00: -80℃ storing silicone tube. A thick white and loosely bond substance is seen on the inner wall of the 5mm silicone tube, and on the inner wall of the 1mm silicone tube a flatter and smoother white substance. Especially obvious where the tube turns. Possibly because the speed of culture flow is slower.

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