Revision as of 10:03, 7 October 2016

TEAM TIANJIN

Team Tianjin-Attribution

Notes For CFPS system

7.27 - 8.2

PCR (Q5 DNA Polymerase Protocol) was applied to amplify single stranded DNA of site mutation for overlap in next step, 48 wells on plate was used. Gel electrophoresis implied that N205L_Reverse, S92A_Reverse, Q119A_Forward, Q199A_Reverse, S207T_Forward and M154A_Reverse were not properly amplified. After several trials and errors, annealing temperature was slightly reduced. Forward and reverse single stranded DNA of all 24 mutation sites were properly prepared.

All PCR product was purified (DNA Purification Protocol).

Overlapping forward and reverse single stranded DNA of all 24 mutation sites (Q5 DNA Polymerase Protocol). Considering several sites had bad gel electrophoresis results, touchdown PCR was introduced. We set the annealing temperature varying from 62 degrees centigrade to 55 degrees centigrade, which meant temperature would touch down 0.2 degrees centigrade every cycle in 35 PCR cycles.

After several trials and errors, optimized condition was found. All mutation sites PCR product were inspected through gel electrophoresis. Sites W159H, W159A, R90T, R90I, R90A, S92A, S207A were discarded for their bad stripe images implied the original primer might had been degraded due to exposed to relatively high temperature (our laboratory room temperature could occasionally rise to above 30 degrees centigrade). These sites were prepared again later.

Genome was extracted from Saccharomyces cerevisiae, and was used as template to prepare alpha-factor signal peptide. PCR was firstly applied with Q5 DNA Polymerase Protocol as annealing temperature was 65 degrees centigrade. Gel electrophoresis implied that alpha-factor signal peptide was not properly prepared. The second time we used Pfu DNA Polymerase Protocol and annealing temperature was set to 50 degrees centigrade. Gel electrophoresis implied alpha-factor signal peptide was properly prepared. Product was purified (DNA Purification Protocol).

8.3 - 8.10

Help Cell-Free team prepare site mutation DNA segments. Q5 DNA Polymerase Protocol and Pfu Polymerase Protocol were both applied. To attain the best quality PCR product, more than 8 times PCR was performed on 96-wells plates, each time more than 20 wells were used. Touchdown PCR was also introduced.

Help Consortia team perform PCR on pBBR plasmid.

PCR (Pfu DNA Polymerase Protocol) was applied to prepare CYC1 terminator and was extracted from gel (Gel DNA Purification Protocol).

PCR (Pfu DNA Polymerase Protocol) was applied to prepare PGK promoter and was extracted from gel (Gel DNA Purification Protocol). After several trials and errors we found the rational condition to prepare PGK promoter gene.

Colony PCR was applied to help Consortia team verify genes on E.Coli genome.

Plasmid pRS416 was performed PCR with primers of alpha-factor signal peptide to check whether pRS416 was correctly linked with alpha-factor signal peptide. Result was negative, one hypothetical explanation was our plasmid donated by other laboratory fellows might have been damaged after a long period (at least 2 years) storage in fridge. We decide to substitute pRS415 for pRS416.

Help Consortia team perform PCR to prepare several genes.

8.11 - 8.18

PCR (Q5 DNA Polymerase Protocol) was applied to amplify PETase wild type gene, for future use.

We tried to combine all components including PGK promoter, CYC1 terminator, PETase mutant gene as well as alpha-factor signal peptide on plasmid pUC19. We tried to apply DNA Ligase Protocol to all 4 components simultaneously but found the efficiency was rather low. After several trials and errors we successfully prepared recombinant plasmid though it took several days.

We tried to link PETase mutant I208V gene to plasmid pRS415, and check whether it was successful use the same method described before. Gel electrophoresis confirmed that pRS415 was correctly linked with PETase mutant I208V gene. We also noticed that there exist 2 EcoRI cutting sites on pRS416 (one on resistance gene), which led to the failure in last week.

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Week1
Week2
Week3
Week4
Week5

Notice: This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release. — 2016 iGEM Team Tianjin

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 <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">
-		<div class="entry-title" align="center" >Notes For R-R system</div>
+		<div class="entry-title" align="center" >Notes For CFPS system</div>
 	</header><!-- .entry-header -->
-         <h1 class="entry-title">Week1(8/24/2016-8/30/2016)</h1>
+         <h1 class="entry-title">7.27 - 8.2</h1>
 		<div class="entry-content">
                    <div class="note-content">
-    <li>We used PCR to amplify the <b><i>CpxR</i></b> promoter and <b><i>RFP</i></b> gene from plasmid <b><i>pUC57</i></b>, and then we recycled the amplified fragment from the agarose gel. Then we used<b><i> Xba1</i></b> and <b><i>Pst1</i></b> enzymes to cut the plasmid <b><i>pUC19</i></b> and <b><i>CpxR-RFP</i></b> fragment. </li>
+<p>
-   <li>We linked the cut plasmid and<b><i> CpxR-RFP</i></b> fragment together and transformed the recombinant plasmid to <b><i>E.coli</i></b>.
+ <li>PCR (Q5 DNA Polymerase Protocol) was applied to amplify single stranded DNA of site mutation for overlap in next step, 48 wells on plate was used. Gel electrophoresis implied that N205L_Reverse, S92A_Reverse, Q119A_Forward, Q199A_Reverse, S207T_Forward and M154A_Reverse were not properly amplified. After several trials and errors, annealing temperature was slightly reduced. Forward and reverse single stranded DNA of all 24 mutation sites were properly prepared.</li><br/>
-We used PCR to amplify the <b><i>PETase</i></b> gene and then recycled them from the agarose gel.</li>
+ <li>All PCR product was purified (DNA Purification Protocol).</li><br/>
-<li>We cultured the grown-up<b><i> E.coli</i></b> which had been transformed into recombinant <b><i>pUC19</i></b> into liquid LB+Amp culture medium overnight.</li>
+ <li>Overlapping forward and reverse single stranded DNA of all 24 mutation sites (Q5 DNA Polymerase Protocol). Considering several sites had bad gel electrophoresis results, touchdown PCR was introduced. We set the annealing temperature varying from 62 degrees centigrade to 55 degrees centigrade, which meant temperature would touch down 0.2 degrees centigrade every cycle in 35 PCR cycles.</li><br/>
-<li>We isolated the recombinant plasmid from the<b><i> E.coli</i></b> cultured last night. Then we use<b><i> Xba1</i></b> and <b><i>Pst1</i></b> enzyme to cut the plasmid to verify the plasmid was successfully constructed. The result was we succeeded.</li>
+ <li>After several trials and errors, optimized condition was found. All mutation sites PCR product were inspected through gel electrophoresis. Sites W159H, W159A, R90T, R90I, R90A, S92A, S207A were discarded for their bad stripe images implied the original primer might had been degraded due to exposed to relatively high temperature (our laboratory room temperature could occasionally rise to above 30 degrees centigrade). These sites were prepared again later.</li><br/>
-<li>We cut the recombinant plasmid <b><i>pUC19</i></b> with enzyme<b><i> EcoR1</i></b> and <b><i>Sac1</i></b> and then we recycled it from the agarose gel. We stored the recycled product in -30℃ in order to wait for the <b><i>PETase</i></b> gene transformed into it.</li>
+<li>Genome was extracted from Saccharomyces cerevisiae, and was used as template to prepare alpha-factor signal peptide. PCR was firstly applied with Q5 DNA Polymerase Protocol as annealing temperature was 65 degrees centigrade. Gel electrophoresis implied that alpha-factor signal peptide was not properly prepared. The second time we used Pfu DNA Polymerase Protocol and annealing temperature was set to 50 degrees centigrade. Gel electrophoresis implied alpha-factor signal peptide was properly prepared. Product was purified (DNA Purification Protocol).
-<li>In order to verify the inclusion body sensing effects of <b><i>CpxR</i></b> promoter, we selected a colony of <b><i>E.coli</i></b> with recombinant plasmid<b><i> pUC19</i></b> and cultured them in 37℃ for 6 hours and then rose the temperature to 42℃ and culture it overnight.</li>
+</li><br/>
-<li>The result of the verification experiment last night was unsuccessful for there was only ultralow red fluorescence was detected, which was considered the basic expression of <b><i>RFP</i></b>.</li>
+                         </p>
-<li>We redesigned the experiment and set 3 groups:<br/>
-. E.coli with standard <b><i>RFP</i></b> gene from our own laboratory.<br/>
-. E.coli with our recombinant plasmid<b><i> pUC19</i></b> and we cultured them in 37℃ all through.<br/>
-. E.coli with our recombinant plasmid <b><i>pUC19</i></b> and we first cultured them in 37℃ and after 6 hours transferred them to 42℃.
-</li>
-<li>The result was still unsuccessful for the 2nd and 3rd group showed ultralow red fluorescence and only 1st group showed high enough red fluorescence.</li>
-<li>We redesigned the experiment again. We decided to transformed the recombinant plasmid <b><i>pUC19</i></b> and<b><i> pET21a</i></b> which was from the protein modification group and had <b><i>PETase</i></b> gene in it into<b><i> E.coli</i></b> at the same time.</li>
-<li>We cut the<b><i> pUC57</i></b> with enzyme <b><i>Xba1</i></b> and <b><i>Pst1</i></b> and recycled the skeleton part from the agarose gel.</li>
 </div>
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-	     </article> #post-4252 -->
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 	<header class="entry-header">
-		 <h1 class="entry-title">Week2(8/31/2016-9/6/2016)</h1>
+		 <h1 class="entry-title">8.3 - 8.10</h1>
 	</header><!-- .entry-header -->
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 <div class="note-content2">
-<li>We linked the remained cut <b><i>CpxR-RFP</i></b> fragment into the skeleton and then transformed the recombinant <b><i>pUC57</i></b> and the <b><i>pET21a</i></b> into<b><i> E.coli</i></b> at the same time.</li>
+<p><li>Help Cell-Free team prepare site mutation DNA segments. Q5 DNA Polymerase Protocol and Pfu Polymerase Protocol were both applied. To attain the best quality PCR product, more than 8 times PCR was performed on 96-wells plates, each time more than 20 wells were used. Touchdown PCR was also introduced.</li><br />
+<li>Help Consortia team perform PCR on pBBR plasmid.</li><br />
-<li>The transformation last night turned to be a failure. We tried it again.</li>
+<li>PCR (Pfu DNA Polymerase Protocol) was applied to prepare CYC1 terminator and was extracted from gel (Gel DNA Purification Protocol).</li><br />
-<li>The transformation last day seemed to be successful for the colonies were visible in LB+Amp plate. However, we use PCR to verify and it turned out that the fragment had not been linked into the plasmid.</li>
+<li>PCR (Pfu DNA Polymerase Protocol) was applied to prepare PGK promoter and was extracted from gel (Gel DNA Purification Protocol). After several trials and errors we found the rational condition to prepare PGK promoter gene.</li><br/>
-<li>We finally gave up the former design and decided to link the<b><i> PETase</i></b> gene into the plasmid<b><i> pUC19</i></b>. However, we did not have the key enzyme <b><i>Sal1</i></b> so we started to construct the TPA positive feedback system.</li>
+  <li>Colony PCR was applied to help Consortia team verify genes on E.Coli genome.</li><br/>
-<li>We first prepared the TPA standard solution (5g/L) for further use. Then we use PCR to amplify the <b><i>TPA-sensing leader sequence</i></b>,<b><i> PGK1 promoter</i></b>, <b><i>CYC1 terminator</i></b>, <b><i>RFP gene</i></b>, TPA regulation protein gene (<b><i>tpaR</i></b>), TPA transporting protein gene (<b><i>tpaK</i></b>). Then we cut the fragments above and plasmid <b><i>pRS413</i></b>, <b><i>pRS415</i></b>, and <b><i>pYES2</i></b> with corresponding enzymes and recycled the fragments from agarose gel.</li>
+  <li>Plasmid pRS416 was performed PCR with primers of alpha-factor signal peptide to check whether pRS416 was correctly linked with alpha-factor signal peptide. Result was negative, one hypothetical explanation was our plasmid donated by other laboratory fellows might have been damaged after a long period (at least 2 years) storage in fridge. We decide to substitute pRS415 for pRS416.</li><br/>
-<li>We linked the fragments together by this way:<br/>
+  <li>Help Consortia team perform PCR to prepare several genes.</li><br/>
-<b><i>1. pYES2-leader-PGK1-RFP.<br/>
+</p>
-. pRS413-PGK1-tpaK-CYC1.<br/>
-. pRS415-PGK1-tpaR-CYC1</i></b>
-</li>
-<li>Then we used PCR to verify the success and all of the plasmids were correctly constructed. Then we transformed the there plasmids into <b><i>Saccharomyces cerevisiae</i></b>.
-</li>
-<li>The key enzyme <b><i>Sal1</i></b> arrived and we isolate the plasmid <b><i>pET21a</i></b>. Then we use <b><i>BamH1</i></b> and <b><i>Sal1</i></b> to cut both plasmid and <b><i>PETase</i></b> gene, then linked them together and transformed the recombinant plasmid into <b><i>E.coli</i></b>.</li>
 </div>
 <a class="expand-btn2">Show More</a>
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 <!------------------------------------week2 end------------------------------------------------>
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 <div id="Week3"></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">Week3(9/7/2016-9/13/2016)</h1>
+	<h1 class="entry-title">8.11 - 8.18</h1>
 		<div class="entry-content">
 <div class="note-content3">
-<li>We cultured the transformed<b><i> E.coli</i></b> and isolated the plasmid. Then we use PCR to amplify the whole fragment in <b><i>pET21a</i></b> from <b><i>T7 promoter</i></b> to <b><i>T7 terminator</i></b>. Then we recycled this fragment from agarose gel.</li>
+<p><li>PCR (Q5 DNA Polymerase Protocol) was applied to amplify PETase wild type gene, for future use.</li><br/>
-<li>The transformed <b><i>Saccharomyces cerevisiae</i></b> had grown to visible colony in Sc-Ura-Leu-His plate. Then we use colony PCR to verify the plasmids had been transformed into the cells. The result is successful so that we streaked more plates.</li>
+<li>We tried to combine all components including PGK promoter, CYC1 terminator, PETase mutant gene as well as alpha-factor signal peptide on plasmid pUC19. We tried to apply DNA Ligase Protocol to all 4 components simultaneously but found the efficiency was rather low. After several trials and errors we successfully prepared recombinant plasmid though it took several days. </li><br/>
-<li>We cut the <b><i>T7 promoter-PETase gene-T7 terminator</i></b> fragment with enzymes <b><i>EcoR1</i></b> and <b><i>Sac1</i></b>. Then we linked it to the already cut plasmid <b><i>pUC19</i></b> (cut in August 28th). Then we transformed the recombinant plasmid into<b><i> E.coli</i></b>.</li>
+<li>We tried to link PETase mutant I208V gene to plasmid pRS415, and check whether it was successful use the same method described before. Gel electrophoresis confirmed that pRS415 was correctly linked with PETase mutant I208V gene. We also noticed that there exist 2 EcoRI cutting sites on pRS416 (one on resistance gene), which led to the failure in last week.</li>
-<li>We cultured the transformed <b><i>Saccharomyces cerevisiae</i></b> into Sc-Ura-Leu-His culture medium in 30℃. We added TPA standard solution in this way:<br/>
+</p>
-. Group 1: did not add TPA.<br/>
-. Group 2: added 1000μL TPA standard solution.<br/>
-. Group 3: added 100μL TPA standard solution.<br/>
-. Group 4: added 10μL TPA standard solution.<br/>
-.Group 5: added 1μL TPA standard solution.
-</li>
-<li>We cultured the transformed <b><i>E.coli</i></b> into LB+Amp culture medium. Then add 1.5μL IPTG to induce the expression of <b><i>PETase</i></b> gene.</li>
-<li>We first detected the red fluorescence of <b><i>E.coli</i></b>, however, the experiment group had almost no increase of red fluorescence relative to control group. We changed the induction wavelength and scan the whole wavelength of emission, but we did not receive any result we expected.</li>
-<li>The TPA positive feedback system seemed to have minor effection for there were a little increment of red fluorescence of the 5th group relative to the 1st one.</li>
-<li>We doubted that it might be the<b><i> RFP</i></b> in the kit was useless. We isolated the <b><i>pET21a</i></b> and used PCR to amplify the <b><i>RFP </i></b>gene.</li>
-<li>We cut the <b><i>RFP</i></b> gene and <b><i>pET21a</i></b> with enzymes <b><i>Xba1</i></b> and <b><i>Sac1</i></b>, then we linked them and transformed it into <b><i>E.coli</i></b>.</li>
-<li>We cultured the transformed <b><i>E.coli</i></b> and added IPTG to induce the expression of<b><i> RFP</i></b>, and this time the red fluorescence was clear enough that could be seen directly.</li>
 </div>
 <a class="expand-btn3">Show More</a>
 		</div><!-- .entry-content -->
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@@ Line 192: / Line 150: @@
 <!------------------------------------week4 start------------------------------------------------>
-<div id="Week4"></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">Week4(9/14/2016-9/20/2016)</h1>
-		<div class="entry-content">
-<div class="note-content4">
-<li>We started to construct another regulation way, the <b><i>E.coli</i></b> lysis regulation pathway. We first used colony PCR to obtain the <b><i>ddpX</i></b> gene from the <b><i>E.coli</i></b> genome and recycled the <b><i>ddpX</i></b> from the agarose gel.</li>
-<li>We found that there was no enzyme cleavage site between the<b><i> CpxR</i></b> promoter and <b><i>RFP</i></b> gene in the part we use. We had to design the primers and amplified the <b><i>CpxR</i></b> promoter by PCR.</li>
-<li>We used PCR to amplify the<b><i> CpxR</i></b> promoter. Then we recycled it from agarose gel.</li>
-<li>We cut the<b><i> CpxR</i></b> promoter with enzymes <b><i>Xba1</i></b> and<b><i> BamH1</i></b>, <b><i>ddpX</i></b> gene with enzymes <b><i>BamH1</i></b> and<b><i> EcoR1</i></b>, first batch of <b><i>pET21a</i></b> with<b><i> Xba1</i></b> and<b><i> EcoR1</i></b>, second batch of <b><i>pET21a</i></b> with<b><i> BamH1</i></b> and <b><i>EcoR1</i></b>.</li>
-<li>Then we linked these fragment in the following two ways:<br/>
-<b><i>1. pET21a-CpxR-ddpX.<br/>
-. pET21a-ddpX.</i></b>
-</li>
- </div>
-<a class="expand-btn4">Show More</a>
-		</div><!-- .entry-content -->
 <!------------------------------------week4 end------------------------------------------------>
@@ Line 230: / Line 159: @@
 <!------------------------------------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(9/21/2016-9/27/2016)</h1>
-		<div class="entry-content">
-<div class="note-content5">
-<li>We used PCR to amplify the whole fragments in<b><i> pET21a</i></b> (from <b><i>CpxR </i></b>to <b><i>T7 terminator</i></b>). However, the band in the agarose gel was disperse so that we were unable to recycle it.</li>
-<li>We used colony PCR to verify if the <b><i>pET21a</i></b> had been correctly constructed, the result is yes.</li>
-<li>We changed the DNA polymerase and annealing temperature several times and redid the PCR, however, the disperse band were always existed.</li>
-<li>We cultured the <b><i>E.coli</i></b> transformed into the <b><i>pET21a-ddpX</i></b> fragment and detect the OD600 in order to verify the lysis effection of <b><i>ddpX</i></b>. </li>
-<li>Considering the <b><i>pYES2</i></b> is multicopy plasmid so that the copy number would affect the <b><i>RFP</i></b> expression level, we decided to change the <b><i>pYES2</i></b> to single-copy plasmid <b><i>pRS416</i></b>. Since the <b><i>pRS416</i></b> does not have terminator in its backbone, we used PCR to amplify the <b><i>CYC1</i></b> terminator from plasmid <b><i>pYES2</i></b>.</li>
-<li>We cut the <b><i>pYES2</i></b> with enzyme <b><i>Hind3</i></b> and <b><i>EcoR1</i></b>, <b><i>CYC1</i></b> with<b><i> EcoR1</i></b> and <b><i>Sal1</i></b>,<b><i> pRS416</i></b> with <b><i>Hind3</i></b> and<b><i> Sal1</i></b>. Then we linked the three part together.</li>
-<li>We transformed the three plasmids into <b><i>Saccharomyces cerevisiae</i></b> together. </li>
-<li>The new primers using to amplify the<b><i> CpxR-ddpX-T7</i></b> terminator fragment arrived and we redid the PCR. However, the disperse band was still existed. </li>
-<li>The transformation of Saccharomyces cerevisiae turned out to be a failure because no colony was found on the Sc-Ura-Leu-His plate. </li>
- </div>
-<a class="expand-btn5">Show More</a>
-		</div><!-- .entry-content -->
@@ Line 269: / Line 166: @@
-<!------------------------------------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(9/28/2016-10/2/2016)</h1>
-	</header><!-- .entry-header -->
-		<div class="entry-content">
-<div class="note-content6">
-<li>We redid the inclusion body reporting experiment, and this time we directly observed the color of bacterial after centrifugation (12000rpm, 1min). The group with <b><i>PETase</i></b> gene and <b><i>CpxR-RFP</i></b> fragment showed the deepest red.</li>
- </div>
-<a class="expand-btn6">Show More</a>
-		</div><!-- .entry-content -->
-<!------------------------------------week6 end------------------------------------------------>
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 							<li><a class="topLink" href="#Topp" style="color:#5555FF">Top</a></li>
-                                                        <li><a class="topLink" href="#Week1" style="color:#5555FF">Week1</a></li>
+                            <li><a class="topLink" href="#Week1" style="color:#5555FF">Week1</a></li>
 							<li><a class="topLink" href="#Week2" style="color:#5555FF">Week2</a></li>
 							<li><a class="topLink" href="#Week3" style="color:#5555FF">Week3</a></li>
 							<li><a class="topLink" href="#Week4" style="color:#5555FF">Week4</a></li>
 							<li><a class="topLink" href="#Week5" style="color:#5555FF">Week5</a></li>
-							<li><a class="topLink" href="#Week6" style="color:#5555FF">Week6</a></li>
-							<li><a class="topLink" href="#Week7" style="color:#5555FF">Week7</a></li>
 						</ul>
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Difference between revisions of "Team:Tianjin/Note/Protein Engineering"

Revision as of 10:03, 7 October 2016

7.27 - 8.2

8.11 - 8.18