Team:Osaka/Notebook

From 2010.igem.org

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-
__NOTOC__
+
<html>
-
==Calendar==
+
<style type="text/css">
-
{|{| class="toccolours" style="float:{{{float|left}}}; width:{{{width|200px}}}; margin-left:1em; text-align:center;"
+
<!--
 +
h2 {
 +
font-weight:bold;
 +
}
 +
 
 +
#contentSub {
 +
display:none;
 +
}
 +
 
 +
#siteSub {
 +
display:none;
 +
}
 +
 
 +
#search-controls {
 +
display:none;
 +
}
 +
 
 +
.firstHeading {
 +
display:none;
 +
}
 +
 
 +
#search-controls {
 +
display:none;
 +
}
 +
 
 +
#footer-box {
 +
 
 +
}
 +
 
 +
#top-section {
 +
height:25px;
 +
width:1000px;
 +
color: blue;
 +
background-color: transparent;
 +
background-image: url(https://static.igem.org/mediawiki/2010/f/f1/Osaka_top_section.jpg)
 +
}
 +
 
 +
#p-logo {
 +
display:none;
 +
}
 +
 
 +
#menubar {
 +
top:0px;
 +
}
 +
 
 +
#globalWrapper {
 +
background-color: transparent;
 +
background-image: url(https://static.igem.org/mediawiki/2010/a/af/Osaka_sand2.jpg);
 +
padding-bottom: 20px;
 +
padding-left:0px;
 +
padding-right:0px;
 +
}
 +
}
 +
 
 +
#content {
 +
position: relative;
 +
width:1000px;
 +
padding:0px;
 +
}
 +
.wrap{
 +
 +
}
 +
.top{
 +
width:1000px;
 +
background-color:black;
 +
}
 +
.logo{  width:160px;
 +
float:left;
 +
padding:0;
 +
margin:0;
 +
background-color:#0342ff;
 +
 +
}
 +
.banner{
 +
width:840px;
 +
float:left;
 +
padding:0;
 +
margin:0;
 +
background-color:black;
 +
 +
}
 +
 
 +
.bottom{
 +
width:1000px;}
 +
.right{
 +
width:840px;
 +
float:right;
 +
}
 +
.contents2{
 +
        width:820px;
 +
        float:right;
 +
        padding:0 10px;
 +
        margin:0;
 +
        background-color:white;
 +
}
 +
.twitter{
 +
width:250px;
 +
float:right;
 +
padding:0;
 +
margin:0;
 +
background-color:white;
 +
}
 +
.contents{
 +
width:750px;
 +
float:left;
 +
padding:0 10px;
 +
margin:0;
 +
background-color:white;
 +
}
 +
 
 +
.menu{
 +
width:160px;
 +
float:left;
 +
background-color:white;
 +
        padding:0;
 +
}
 +
.clear{
 +
clear:both;}
 +
.clear hr{
 +
display:none;}
 +
.bold {
 +
      font-weight:bold;
 +
}
 +
-->
 +
</style>
 +
</head>
 +
 
 +
 
 +
<body>
 +
 
 +
<div class="wrap">
 +
<div class="top">
 +
    <div class="banner">
 +
  <img src="https://static.igem.org/mediawiki/2010/d/d6/Osaka_banner3.jpg" width="1000" height="250" alt="iGEM Osaka banner" usemap="#banner_map">
 +
 
 +
<map name="banner_map">
 +
  <area shape="rect" coords="540,180,750,220" href="https://2010.igem.org" alt="iGEM 2010 home"/>
 +
  <area shape="rect" coords="430,230,750,270" href="http://www.osaka-u.ac.jp/en" alt="Osaka University"/>
 +
  <area shape="circle" coords="100,200,85" href="https://2010.igem.org/Team:Osaka" alt="iGEM Osaka home"/>
 +
</map>
 +
 
 +
  </div><!-- banner -->
 +
  <div class="clear"><hr></div>
 +
</div><!-- top -->
 +
 
 +
<div class="bottom">
 +
  <div class="menu">
 +
</html>
 +
{{Osaka_menu}}
 +
<html>
 +
  </div>
 +
 
 +
 
 +
 
 +
 
 +
<html>
 +
<div class="right">
 +
<div class="contents">
 +
<div style="margin:40px 0">
 +
</html>
 +
==Notebook==
 +
{| class="toccolours" style="float:{{{float|left}}}; width:{{{width|240px}}}; text-align:center;"
|- style="background:{{{color1|#ccccff}}};"
|- style="background:{{{color1|#ccccff}}};"
-
|colspan="8"|{{{header|July}}}
+
|colspan="8"|{{{header|July / August}}}
|-
|-
|- style="background:{{{color2|#eeeeff}}};"
|- style="background:{{{color2|#eeeeff}}};"
-
|wodth=100px|Week
+
|width=100px|Week
|width="14%"|<span style="color:red;">S</span>
|width="14%"|<span style="color:red;">S</span>
|width="14%"|M
|width="14%"|M
Line 14: Line 175:
|width="14%"|F
|width="14%"|F
|width="14%"|<span style="color:deepskyblue;">S</span>
|width="14%"|<span style="color:deepskyblue;">S</span>
-
|-
 
-
|colspan ="5"|
 
-
|<span style={{{s1|"color:{{{c1|inherit}}};"}}}>1</span>
 
-
|<span style={{{s2|"color:{{{c2|inherit}}};"}}}>2</span>
 
-
|<span style={{{s3|"color:{{{c3|deepskyblue}}};"}}}>3</span>
 
-
|-
 
-
|<span style></span>
 
-
|<span style={{{s4|"color:{{{c4|red}}};"}}}>4</span>
 
-
|<span style={{{s5|"color:{{{c5|inherit}}};"}}}>5</span>
 
-
|<span style={{{s6|"color:{{{c6|inherit}}};"}}}>6</span>
 
-
|<span style={{{s7|"color:{{{c7|inherit}}};"}}}>7</span>
 
-
|<span style={{{s8|"color:{{{c8|inherit}}};"}}}>8</span>
 
-
|<span style={{{s9|"color:{{{c9|inherit}}};"}}}>9</span>
 
-
|<span style={{{s10|"color:{{{c10|deepskyblue}}};"}}}>10</span>
 
-
|-
 
-
|<span style></span>
 
-
|<span style={{{s11|"color:{{{c11|red}}};"}}}>11</span>
 
-
|<span style={{{s12|"color:{{{c12|inherit}}};"}}}>12</span>
 
-
|<span style={{{s13|"color:{{{c13|inherit}}};"}}}>13</span>
 
-
|<span style={{{s14|"color:{{{c14|inherit}}};"}}}>14</span>
 
-
|<span style={{{s15|"color:{{{c15|inherit}}};"}}}>15</span>
 
-
|<span style={{{s16|"color:{{{c16|inherit}}};"}}}>16</span>
 
-
|<span style={{{s17|"color:{{{c17|deepskyblue}}};"}}}>17</span>
 
-
|-
 
-
|<span style></span>
 
-
|<span style={{{s18|"color:{{{c18|red}}};"}}}>18</span>
 
-
|<span style={{{s19|"color:{{{c19|inherit}}};"}}}>19</span>
 
-
|<span style={{{s20|"color:{{{c20|inherit}}};"}}}>20</span>
 
-
|<span style={{{s21|"color:{{{c21|inherit}}};"}}}>21</span>
 
-
|<span style={{{s22|"color:{{{c22|inherit}}};"}}}>22</span>
 
-
|<span style={{{s23|"color:{{{c23|inherit}}};"}}}>23</span>
 
-
|<span style={{{s24|"color:{{{c24|deepskyblue}}};"}}}>24</span>
 
|-
|-
|<span style>[[Team:Osaka/week1|week1]]</span>
|<span style>[[Team:Osaka/week1|week1]]</span>
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|<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span>
|<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span>
|[[Team:Osaka/week1#July 31 (Sat)|31]]<span style={{{s31|"color:{{{c31|deepskyblue}}};"}}}></span>
|[[Team:Osaka/week1#July 31 (Sat)|31]]<span style={{{s31|"color:{{{c31|deepskyblue}}};"}}}></span>
-
|-
 
-
|- style="background:{{{color3|#eeeeff}}};"
 
-
|colspan="7"|{{{footer|}}}
 
-
|}<noinclude>
 
-
{| class="toccolours" style="float:{{{float|left}}}; width:{{{width|200px}}}; margin-left:1em; text-align:center;"
 
-
|- style="background:{{{color1|#ccccff}}};"
 
-
|colspan="8"|{{{header|August}}}
 
-
|-
 
-
|- style="background:{{{color2|#eeeeff}}};"
 
-
|width=100px|Week
 
-
|width="14%"|<span style="color:red;">S</span>
 
-
|width="14%"|M
 
-
|width="14%"|T
 
-
|width="14%"|W
 
-
|width="14%"|T
 
-
|width="14%"|F
 
-
|width="14%"|<span style="color:deepskyblue;">S</span>
 
|-
|-
|<span style>[[Team:Osaka/week2|week2]]</span>
|<span style>[[Team:Osaka/week2|week2]]</span>
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|-
|-
|<span style>[[Team:Osaka/week6|week6]]</span>
|<span style>[[Team:Osaka/week6|week6]]</span>
-
|<span style={{{s29|"color:{{{c29|red}}};"}}}>29</span>
+
|[[Team:Osaka/week6#August 29 (Sun)|29]]<span style={{{s29|"color:{{{c29|red}}};"}}}></span>
-
|<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span>
+
|[[Team:Osaka/week6#August 30 (Mon)|30]]<span style={{{s30|"color:{{{c30|inherit}}};"}}}></span>
-
|<span style={{{s31|"color:{{{c31|inherit}}};"}}}>31</span>
+
|[[Team:Osaka/week6#August 31 (Tue)|31]]<span style={{{s31|"color:{{{c31|inherit}}};"}}}></span>
|colspan="4"|
|colspan="4"|
|- style="background:{{{color3|#eeeeff}}};"
|- style="background:{{{color3|#eeeeff}}};"
|colspan="7"|{{{footer|}}}
|colspan="7"|{{{footer|}}}
|}<noinclude>
|}<noinclude>
-
{| class="toccolours" style="float:{{{float|left}}}; width:{{{width|150px}}}; margin-left:1em; text-align:center;"
+
{| class="toccolours" style="float:{{{float|left}}}; width:{{{width|240px}}}; text-align:center;"
|- style="background:{{{color1|#ccccff}}};"
|- style="background:{{{color1|#ccccff}}};"
-
|colspan="7"|{{{header|September}}}
+
|colspan="8"|{{{header|September}}}
|-
|-
|- style="background:{{{color2|#eeeeff}}};"
|- style="background:{{{color2|#eeeeff}}};"
 +
|width=100px|Week
|width="14%"|<span style="color:red;">S</span>
|width="14%"|<span style="color:red;">S</span>
|width="14%"|M
|width="14%"|M
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|width="14%"|<span style="color:deepskyblue;">S</span>
|width="14%"|<span style="color:deepskyblue;">S</span>
|-
|-
-
|colspan ="3"|
+
|<span style>[[Team:Osaka/week6|week6]]</span>
-
|<span style={{{s1|"color:{{{c1|inherit}}};"}}}>1</span>
+
|<span style></span>
-
|<span style={{{s2|"color:{{{c2|inherit}}};"}}}>2</span>
+
|<span style></span>
-
|<span style={{{s3|"color:{{{c3|inherit}}};"}}}>3</span>
+
|<span style></span>
-
|<span style={{{s4|"color:{{{c4|deepskyblue}}};"}}}>4</span>
+
|[[Team:Osaka/week6#September 1 (Wed)|1]]<span style={{{s1|"color:{{{c1|inherit}}};"}}}></span>
 +
|[[Team:Osaka/week6#September 2 (Thu)|2]]<span style={{{s2|"color:{{{c2|inherit}}};"}}}></span>
 +
|[[Team:Osaka/week6#September 3 (Fri)|3]]<span style={{{s3|"color:{{{c3|inherit}}};"}}}></span>
 +
|[[Team:Osaka/week6#September 4 (Sat)|4]]<span style={{{s4|"color:{{{c4|deepskyblue}}};"}}}></span>
|-
|-
-
|<span style={{{s5|"color:{{{c5|red}}};"}}}>5</span>
+
|<span style>[[Team:Osaka/week7|week7]]</span>
-
|<span style={{{s6|"color:{{{c6|inherit}}};"}}}>6</span>
+
|[[Team:Osaka/week7#September 5 (Sun)|5]]<span style={{{s5|"color:{{{c5|red}}};"}}}></span>
-
|<span style={{{s7|"color:{{{c7|inherit}}};"}}}>7</span>
+
|[[Team:Osaka/week7#September 6 (Mon)|6]]<span style={{{s6|"color:{{{c6|inherit}}};"}}}></span>
-
|<span style={{{s8|"color:{{{c8|inherit}}};"}}}>8</span>
+
|[[Team:Osaka/week7#September 7 (Tue)|7]]<span style={{{s7|"color:{{{c7|inherit}}};"}}}></span>
-
|<span style={{{s9|"color:{{{c9|inherit}}};"}}}>9</span>
+
|[[Team:Osaka/week7#September 8 (Wed)|8]]<span style={{{s8|"color:{{{c8|inherit}}};"}}}></span>
-
|<span style={{{s10|"color:{{{c10|inherit}}};"}}}>10</span>
+
|[[Team:Osaka/week7#September 9 (Thu)|9]]<span style={{{s9|"color:{{{c9|inherit}}};"}}}></span>
-
|<span style={{{s11|"color:{{{c11|deepskyblue}}};"}}}>11</span>
+
|[[Team:Osaka/week7#September 10 (Fri)|10]]<span style={{{s10|"color:{{{c10|inherit}}};"}}}></span>
 +
|[[Team:Osaka/week7#September 11 (Sat)|11]]<span style={{{s11|"color:{{{c11|deepskyblue}}};"}}}></span>
|-
|-
-
|<span style={{{s12|"color:{{{c12|red}}};"}}}>12</span>
+
|<span style>[[Team:Osaka/week8|week8]]</span>
-
|<span style={{{s13|"color:{{{c13|inherit}}};"}}}>13</span>
+
|[[Team:Osaka/week8#September 12 (Sun)|12]]<span style={{{s12|"color:{{{c12|red}}};"}}}></span>
-
|<span style={{{s14|"color:{{{c14|inherit}}};"}}}>14</span>
+
|[[Team:Osaka/week8#September 13 (Mon)|13]]<span style={{{s13|"color:{{{c13|inherit}}};"}}}></span>
-
|<span style={{{s15|"color:{{{c15|inherit}}};"}}}>15</span>
+
|[[Team:Osaka/week8#September 14 (Tue)|14]]<span style={{{s14|"color:{{{c14|inherit}}};"}}}></span>
-
|<span style={{{s16|"color:{{{c16|inherit}}};"}}}>16</span>
+
|[[Team:Osaka/week8#September 15 (Wed)|15]]<span style={{{s15|"color:{{{c15|inherit}}};"}}}></span>
-
|<span style={{{s17|"color:{{{c17|inherit}}};"}}}>17</span>
+
|[[Team:Osaka/week8#September 16 (Thu)|16]]<span style={{{s16|"color:{{{c16|inherit}}};"}}}></span>
-
|<span style={{{s18|"color:{{{c18|deepskyblue}}};"}}}>18</span>
+
|[[Team:Osaka/week8#September 17 (Fri)|17]]<span style={{{s17|"color:{{{c17|inherit}}};"}}}></span>
 +
|[[Team:Osaka/week8#September 18 (Sat)|18]]<span style={{{s18|"color:{{{c18|deepskyblue}}};"}}}></span>
|-
|-
-
|<span style={{{s19|"color:{{{c19|red}}};"}}}>19</span>
+
|<span style>[[Team:Osaka/week9|week9]]</span>
-
|<span style={{{s20|"color:{{{c20|inherit}}};"}}}>20</span>
+
|[[Team:Osaka/week9#September 19 (Sun)|19]]<span style={{{s19|"color:{{{c19|red}}};"}}}></span>
-
|<span style={{{s21|"color:{{{c21|inherit}}};"}}}>21</span>
+
|[[Team:Osaka/week9#September 20 (Mon)|20]]<span style={{{s20|"color:{{{c20|inherit}}};"}}}></span>
-
|<span style={{{s22|"color:{{{c22|inherit}}};"}}}>22</span>
+
|[[Team:Osaka/week9#September 21 (Tue)|21]]<span style={{{s21|"color:{{{c21|inherit}}};"}}}></span>
-
|<span style={{{s23|"color:{{{c23|inherit}}};"}}}>23</span>
+
|[[Team:Osaka/week9#September 22 (Wed)|22]]<span style={{{s22|"color:{{{c22|inherit}}};"}}}></span>
-
|<span style={{{s24|"color:{{{c24|inherit}}};"}}}>24</span>
+
|[[Team:Osaka/week9#September 23 (Thu)|23]]<span style={{{s23|"color:{{{c23|inherit}}};"}}}></span>
-
|<span style={{{s25|"color:{{{c25|deepskyblue}}};"}}}>25</span>
+
|[[Team:Osaka/week9#September 24 (Fri)|24]]<span style={{{s24|"color:{{{c24|inherit}}};"}}}></span>
 +
|[[Team:Osaka/week9#September 25 (Sat)|25]]<span style={{{s25|"color:{{{c25|deepskyblue}}};"}}}></span>
|-
|-
-
|<span style={{{s26|"color:{{{c26|red}}};"}}}>26</span>
+
|<span style>[[Team:Osaka/week10|week10]]</span>
-
|<span style={{{s27|"color:{{{c27|inherit}}};"}}}>27</span>
+
|[[Team:Osaka/week10#September 26 (Sun)|26]]<span style={{{s26|"color:{{{c26|red}}};"}}}></span>
-
|<span style={{{s28|"color:{{{c28|inherit}}};"}}}>28</span>
+
|[[Team:Osaka/week10#September 27 (Mon)|27]]<span style={{{s27|"color:{{{c27|inherit}}};"}}}></span>
-
|<span style={{{s29|"color:{{{c29|inherit}}};"}}}>29</span>
+
|[[Team:Osaka/week10#September 28 (Tue)|28]]<span style={{{s28|"color:{{{c28|inherit}}};"}}}></span>
-
|<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span>
+
|[[Team:Osaka/week10#September 29 (Wed)|29]]<span style={{{s29|"color:{{{c29|inherit}}};"}}}></span>
 +
|[[Team:Osaka/week10#September 30 (Thu)|30]]<span style={{{s30|"color:{{{c30|inherit}}};"}}}></span>
|colspan="2"|
|colspan="2"|
|- style="background:{{{color3|#eeeeff}}};"
|- style="background:{{{color3|#eeeeff}}};"
|colspan="7"|{{{footer|}}}
|colspan="7"|{{{footer|}}}
|}<noinclude>
|}<noinclude>
-
{| class="toccolours" style="float:{{{float|left}}}; width:{{{width|150px}}}; margin-left:1em; text-align:center;"
+
{| class="toccolours" style="float:{{{float|left}}}; width:{{{width|240px}}}; text-align:center;"
|- style="background:{{{color1|#ccccff}}};"
|- style="background:{{{color1|#ccccff}}};"
-
|colspan="7"|{{{header|October}}}
+
|colspan="8"|{{{header|October}}}
|-
|-
|- style="background:{{{color2|#eeeeff}}};"
|- style="background:{{{color2|#eeeeff}}};"
 +
|width=100px|Week
|width="14%"|<span style="color:red;">S</span>
|width="14%"|<span style="color:red;">S</span>
|width="14%"|M
|width="14%"|M
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|width="14%"|<span style="color:deepskyblue;">S</span>
|width="14%"|<span style="color:deepskyblue;">S</span>
|-
|-
-
|colspan ="5"|
+
|<span style>[[Team:Osaka/week10|week10]]</span>
-
|<span style={{{s1|"color:{{{c1|inherit}}};"}}}>1</span>
+
|<span style></span>
-
|<span style={{{s2|"color:{{{c2|deepskyblue}}};"}}}>2</span>
+
|<span style></span>
 +
|<span style></span>
 +
|<span style></span>
 +
|<span style></span>
 +
|[[Team:Osaka/week10#October 1 (Fri)|1]]<span style={{{s1|"color:{{{c1|inherit}}};"}}}></span>
 +
|[[Team:Osaka/week10#October 2 (Sat)|2]]<span style={{{s2|"color:{{{c2|deepskyblue}}};"}}}></span>
|-
|-
-
|<span style={{{s3|"color:{{{c3|red}}};"}}}>3</span>
+
|<span style>[[Team:Osaka/week11|week11]]</span>
-
|<span style={{{s4|"color:{{{c4|inherit}}};"}}}>4</span>
+
|[[Team:Osaka/week11#October 3 (Sun)|3]]<span style={{{s3|"color:{{{c3|red}}};"}}}></span>
-
|<span style={{{s5|"color:{{{c5|inherit}}};"}}}>5</span>
+
|[[Team:Osaka/week11#October 4 (Mon)|4]]<span style={{{s4|"color:{{{c4|inherit}}};"}}}></span>
-
|<span style={{{s6|"color:{{{c6|inherit}}};"}}}>6</span>
+
|[[Team:Osaka/week11#October 5 (Tue)|5]]<span style={{{s5|"color:{{{c5|inherit}}};"}}}></span>
-
|<span style={{{s7|"color:{{{c7|inherit}}};"}}}>7</span>
+
|[[Team:Osaka/week11#October 6 (Wed)|6]]<span style={{{s6|"color:{{{c6|inherit}}};"}}}></span>
-
|<span style={{{s8|"color:{{{c8|inherit}}};"}}}>8</span>
+
|[[Team:Osaka/week11#October 7 (Thu)|7]]<span style={{{s7|"color:{{{c7|inherit}}};"}}}></span>
-
|<span style={{{s9|"color:{{{c9|deepskyblue}}};"}}}>9</span>
+
|[[Team:Osaka/week11#October 8 (Fri)|8]]<span style={{{s8|"color:{{{c8|inherit}}};"}}}></span>
 +
|[[Team:Osaka/week11#October 9 (Sat)|9]]<span style={{{s9|"color:{{{c9|deepskyblue}}};"}}}></span>
|-
|-
-
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----
 
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==Notebook==
 
-
 
-
 
-
===August 29 (Sun)===
 
-
# Miniprep: 1-1K, 1-1I, 3-3G, 2-4A, 3-11I
 
-
# Restriction digests
 
-
#* for checking: 1-1K, 1-1I, 3-3G with EcoRI, PstI
 
-
#* for assembly: 2-4A, 3-11I with EcoRI, SpeI (''upstream parts'')
 
-
# Gel electrophoresis for confirmation
 
-
#* Inserts seem to be present in all samples
 
-
# 3A assembly ligations:
 
-
## 2-4A as upstream, 2-2O as downstream, 1-5A as vector; product designated as 002
 
-
## 3-11I as upstream, 2-2O as downstream, 1-5A as vector; product designated as 003
 
-
#* 2-2O using XbaI, PstI digest from yesterday
 
-
#* 1-5A has Kan resistance
 
-
#* ligation reaction for 10 mins at room temperature followed by 20min inactivation at 80°C
 
-
# Transformation of ligation products 002 and 003
 
-
 
-
===August 30 (Mon)===
 
-
# Restriction digests for 3A assembly
 
-
#* 2-22P (OmpA) & 1-2J (PelB) with EcoRI, SpeI
 
-
#* 2-20J (CenA), 2-20H (Cex), F1 with XbaI, PstI
 
-
# Gel electrophoresis of the digests to confirm inserts
 
-
#* all OK
 
-
# Transfer of 002 and 003 to solution culture (3 colonies each)
 
-
 
-
===August 31 (Tue)===
 
-
# Miniprep of 002, 003
 
-
# Cut check of 002, 003 with EcoRI, SpeI
 
-
#* 003 was properly cut, but the insert length was inconsistent; looking back at 8/28 gel result, length of 2-2O (downstream part in 003) also seemed to be longer than expected
 
-
# Repeat colony pick-up and solution culture of 2-2O (5 colonies this time)
 
-
 
-
===September 1 (Wed)===
 
-
# Transformation (See Table 7)
 
-
# Miniprep of 5 separate cultures of 2-2O inoculated yesterday
 
-
# Cut check of 2-2O with XbaI, PstI
 
-
#* 0.7kbp bands in all 5 samples even though insert is supposed to be only 18bp - problem with the part (inconsistency confirmed from registry info page)
 
-
#* obtain Kozak sequence by PCR instead?
 
-
{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
 
-
|+Table 7
 
-
!ID!!Part Name!!Resistance!!Description
 
-
|-
 
-
|1-12D||<bbpart>BBa_E2030</bbpart>||K||yeast-optimized  EYFP
 
-
|-
 
-
|1-12B||<bbpart>BBa_E2020</bbpart>||K||yeast-optimized  ECFP
 
-
|-
 
-
|3-2K||<bbpart>BBa_K165001</bbpart>||A||yeast GAL1 promoter
 
-
|-
 
-
|1-7D||<bbpart>BBa_J63006</bbpart>||A||yeast GAL1 promoter + Kozak sequence
 
-
|}
 
-
 
-
===September 2 (Thu)===
 
-
# Colony check
 
-
#* 1-12D, 3-2K, 1-7D produced colonies -> inoculated into solution culture
 
-
#* 1-12B did not transform successfully
 
-
# 3A assembly ligations:
 
-
## 001 as upstream, 1-2J as downstream, 1-3A as vector; product designated as 004
 
-
## 001 as upstream, 2-22P as downstream, 1-3A as vector; product designated as 005
 
-
# Transformation of ligation products
 
-
 
-
===September 3 (Fri)===
 
-
# Colony check: yesterday's transformations seem to have failed; repeat of transformations of 004 and 005 with 50μl competent cells, 2μl ligation product (''note: colonies appeared later; these repeats were then discarded'')
 
-
# Miniprep of 1-12D, 3-2K, 1-7D followed by cut check with XbaI, PstI
 
-
#* all lengths ok
 
-
# Transfer of yesterday's transformations (''colonies appeared later in the evening'') to culture solution (2 colonies picked up from each plate)
 
-
 
-
===September 4 (Sat)===
 
-
# Miniprep of 004, 005
 
-
# Restriction digest of 004, 005 and 1-7D (as control) with EcoRI, SpeI
 
-
# Gel electrophoresis
 
-
#* 1-7D -> OK
 
-
#* 004 -> insert length same as 001; ''since both upstream part 001 and vector 1-3A were C resistance, 3A assembly must have failed to yield ligation product; try Standard Assembly!''
 
-
#* 005 -> ??
 
-
# Gel electrophoresis followed by purification of 001 to isolate insert -> Standard Assembly
 
-
#* gel purification performed according to protocol in QIAquick Spin Handbook
 
-
# Ligation of gel-purified 001 to 1-2J or 2-22P, with vector 1-3A, to make 004 or 005 respectively (''same 004 and 005 as designed before'')
 
-
 
-
===September 5 (Sun)===
 
-
# Heat inactivation of yesterday's ligation mixes at 80°C for 20min followed by transformation.
 
-
 
-
===September 6 (Mon)===
 
-
# Transfer of yesterday's transformations to solution culture
 
-
# Transformation of the following registry parts (See Table 8)
 
-
{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
 
-
|+Table 8
 
-
!ID!!Part Name!!Resistance!!Description
 
-
|-
 
-
|2-10F||<bbpart>BBa_K081005</bbpart>||A||constitutive promoter from combinatorial library + RBS
 
-
|-
 
-
|2-10H||<bbpart>BBa_K081006</bbpart>||A||lambda phage promoter + RBS
 
-
|}
 
-
 
-
===September 7 (Tue)===
 
-
# Miniprep of 004, 005
 
-
# Cut check with EcoRI, SpeI
 
-
#* both insert lengths ok!
 
-
# Transformation of DNA for PGA synthesis-related genes (See Table 9)
 
-
# Transfer to solution culture
 
-
#* 004, 005 transformed on 9/5 (pick up from fresh colonies) -> ''needed more plasmid''
 
-
#* 2-10F, 2-10H transformed yesterday
 
-
{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
 
-
|+Table 9
 
-
!ID!!Part Name!!Resistance!!Description
 
-
|-
 
-
|A01||pTPG01-1||A||plasmid pTrc99A with pgs genes inserted
 
-
|-
 
-
|A02||pTPG01-2||A||<nowiki>''</nowiki>
 
-
|-
 
-
|A03||pBSGR3||K||glutamine racemase
 
-
|}
 
-
 
-
===September 8 (Wed)===
 
-
# Miniprep 2-10F, 2-10H, 004, 005
 
-
# Restriction digest of above parts with EcoRI, SpeI; also with only EcoRI as negative control
 
-
# Gel electrophoresis
 
-
#* ''new batch of EtBr for staining''
 
-
#* (RESULTS?)
 
-
# Transfer of A01~A03 to solution culture
 
-
# PCR to make Silver standard-compatible parts from 2-20J (CenA) and 2-20J (Cex) based on protocol in Takara Ex Taq polymerase kit
 
-
#* it took several tries to get a successful reaction
 
-
#** 1st attempt: template DNA was used directly; concentration too high (failure)?
 
-
#** 2nd attempt: 100X, 1000X dilutions attempted without success; this time, over-dilution or stringent annealing temp (68°C) may have been culprit?
 
-
#** 3rd attempt: 10X dilutions, annealing temp lowered to 65°C -> success!
 
-
#* note: reactions were evaluated by gel electrophoresis of crude PCR product - if band appears at approximately correct length then reaction judged as successful
 
-
#* '''special note of thanks to Nakamura who stayed in lab overnight to run the PCRs'''
 
-
 
-
===September 9 (Thu)===
 
-
# Miniprep of A01, A02, A03 followed by restriction digests with EcoRI
 
-
# Purification of 9/8 PCR products from gel using QIA Quick Spin gel extraction kit (''why not use PCR purification kit??'')
 
-
# Restriction digest of A01~03 with EcoRI, PCR products with XbaI, PstI
 
-
# Gel electrophoresis of digested parts together with 1-5A ''1-5A supposed to be receiving vector, but digested at wrong sites''
 
-
#* CenA PCR product -> OK (Silver-compatible part designated FcenA)
 
-
#* Cex PCR product -> ?
 
-
# Another round of PCR to amplify Cex as Silver standard part (''why?'')
 
-
#* 10X dilution of template
 
-
#* 68°C annealing temp
 
-
# Ligation
 
-
#* FcenA: PCR product with 1-5A as vector (cut/ligated at X, P)
 
-
#* 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A (vector)
 
-
#* 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector)
 
-
#* 008: 3A assembly of 001 (upstream), Cex PCR product (downstream), 1-5A (vector) ''bad insert?''
 
-
#* 009: 3A assembly of 001 (upstream), FcenA (downstream), 1-5A (vector)
 
-
# Transformation of newly assembled parts 006~009
 
-
# Transfer of 006~009 to solution culture.
 
-
 
-
===September 10 (Fri)===
 
-
# Gel electrophoresis of PCR product from Cex and 2-20H (original Cex part) for comparison
 
-
#* PCR product seems ok -> purification from gel; Silver-compatible part designated Fcex
 
-
# Restriction digests of 004 & 005 (9/4 ligations) with EcoRI, SpeI; followed by gel electrophoresis
 
-
#* (RESULTS?)
 
-
# Restriction digests of Fcex (purified today), FcenA (amplified on 9/9) with　XbaI, PstI followed by gel electrophoresis
 
-
#* (RESULTS?)
 
-
# Ligations
 
-
#* Fcex: PCR product from Cex with 1-5A as vector (cut/ligated at X, P)
 
-
#* 006: 3A assembly of 004 (upstream), FcenA (downstream), 1-5A　(vector); ''repeat''
 
-
#* 007: 3A assembly of 005 (upstream), FcenA (downstream), 1-5A (vector); ''repeat''
 
-
#* 010: 3A assembly of 004 (upstream), Fcex (downstream), 1-5A (vector)
 
-
#* 011: 3A assembly of 005 (upstream), Fcex (downstream), 1-5A (vector)
 
-
# Transformation of newly assembled parts Fcex, 006, 007, 010, 011.
 
-
# Colony check of 9/9 transformations
 
-
#* 006: no colonies
 
-
#* 007: no colonies
 
-
#* 008: >100 colonies ''bad insert?''
 
-
#* 009: >100 colonies
 
-
#* FcenA: >100 colonies
 
-
# Transfer of 008, 009, FcenA, 001, 2-10F to solution culture. (''more 001, 2-10F needed'')
 
-
 
-
===September 11 (Sat)===
 
-
# Miniprep of 008, 009, FcenA, 001, 2-10F.
 
-
# Ristriction digest of 008, 009, 001, 2-10F with EcoRI,　SpeI and FcenA with XbaI, PstI.
 
-
# Gel electrophoresis of digested  008, 009, FcenA, 001, 2-10F.
 
-
#* 008 -> ???
 
-
#* 009 -> O.K.
 
-
#* ''add 1-13D as terminator to 008 and 009'
 
-
#* ''FcenA was not digested by XbaI''
 
-
# Restriction digest of FcenA with EcoRI.
 
-
# Gel electrophoresis of FcenA
 
-
#* FcenA was digested by EcoRI -> O.K.
 
-
# Ligations for 3A assembly
 
-
#* 012: 009 (upstream), 1-13D(terminator,　downstream), 1-3A (vector)
 
-
#* 013: 008 (upstream), 1-13D(terminator,　downstream), 1-5A (vector) ''bad insert?''
 
-
# Transfomation of newly assembled parts 012, 013
 
-
# Transfer of Fcex, 006, 007, 010, 011 (transformed yesterday) to solution culture.
 
-
 
-
===September 12 (Sun)===
 
-
# Miniprep of Fcex, 006, 007, 010, 011
 
-
# Restriction digests of 006, 007, 010, 011 with EcoRI, SpeI; Fcex with XbaI, PstI
 
-
# Gel electrophoresis of digests
 
-
#* (RESULTS)
 
-
# Ligation for 3A assembly
 
-
#* 014: 011 as upstream, 1-13D as downstream, 1-3A as vector
 
-
# Transformation of 014 using 2μl ligation product with 50μl competent cells
 
-
# Moved yesterday's transformation plates (012, 013) to 4°C refrigerator ''no RFP expression from vector plasmids at all?''
 
-
# Transfer of A01, A02, A03 (previously transformed) to solution culture
 
-
 
-
===September 13 (Mon)===
 
-
# Miniprep of A01, A03; restriction digest with EcoRI followed by electrophoresis
 
-
#* (RESULTS?)
 
-
# PCR test
 
-
#* re-cloning of beta-glucosidase from F1 (received from Edinburgh team) into Silver standard-compatible format
 
-
#* cloning of pgsC from A01
 
-
#* ''note: primers were misdesigned but PCR performed anyway to confirm whether sequences were complementary, and to identify PCR parameters''
 
-
# Gel electrophoresis of PCR products
 
-
#* (RESULTS?)
 
-
# Gel purification of PCR product from F1
 
-
# Restriction digest of PCR product (F1) and 1-5A, both with EcoRI, SpeI
 
-
# Gel electrophoresis of PCR product from A01
 
-
#* band visible around 400~500bp, which was correct length -> PCR conditions identified!
 
-
# Transformation of A01, A02, A03
 
-
# Transfer of 012, 013, 014 to solution culture
 
-
#* ''RFP expressed from vector plasmids on 012, 013 plates during refrigeration period, so selection of colonies with inserts now possible''
 
-
 
-
===September 14 (Tue)===
 
-
# Miniprep of A02, 012, 013, 014
 
-
# Restriction digests
 
-
#* 012, 013, 014 with EcoRI, PstI
 
-
#* A02 with EcoRI
 
-
# Gel electrophoresis of digests
 
-
#* A02 was discarded (bad size?)
 
-
#* 012~014: digestions repeated with 1μl each of EcoRI, PstI (previous digestions were 0.5μl each)
 
-
#* gel electrophoresis of repeat digestions again showed bad sizes for all parts
 
-
#** 1-13D terminator part is bad? -> cut check of 1-13D
 
-
#** failure to inactivate restriction enzymes before ligations? -> re-ligation
 
-
# 1-13D cut check with XbaI, PstI
 
-
# Re-ligation (3A assembly)
 
-
#* previously digested 009, 008, 011 inactivated at 80°C for 20min before ligation
 
-
#* 012: 009 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
 
-
#* 013: 008 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/11)
 
-
#* 014: 011 as upstream, 1-13D as downstream, 1-3A as vector (same as on 9/12)
 
-
# Transformation of ligated products (012~014)
 
-
# Gel electrophoresis of yesterday's restriction digests of 1-5A and PCR product from F1 followed by their ligation (1-5A as vector, PCR product as insert)
 
-
# Transfer of A01, A02, A03 to LB liquid culture medium supplanted with 25μl of 2M glucose to prevent leaky expression from Lac promoter (may be affecting growth)
 
-
# Received additional cellulase parts from Karita-sensei: Cel5, Xyn10, Cel44, Man26, Cel8
 
-
#* plasmid DNA resuspended in 10μl MiliQ water each
 
-
#* transformation with 2μl DNA solution
 
-
 
-
===September 15 (Wed)===
 
-
# Miniprep A02, A03 (''E. coli failed to grow in A01'')
 
-
# Cut check of A02, A03 with EcoRI
 
-
#* A02 is ok
 
-
# Transformation of 3-22G
 
-
#* (INFO?)
 
-
# PCR cloning of pgsC from A02
 
-
# Colony check & transfer of cellulase parts from Karita-sensei to solution culture
 
-
# Preparation of lysis buffer for yeast genome DNA extraction according to [insert reference]
 
-
{|
 
-
!Component!!Volume Added!!Final Concentration
 
-
|-
 
-
|Triton X-100||100μl||2%
 
-
|-
 
-
|10%SDS||500μl||1%
 
-
|-
 
-
|5M  NaCl||100μl||100mM
 
-
|-
 
-
|20mM  Tris-HCl(ph8.0)||2.5ml||10mM
 
-
|-
 
-
|0.5M  EDTA(ph8.0)||10μl||1mM
 
-
|-
 
-
|dH2O||1790μl 
 
-
|-
 
-
|'''TOTAL'''||5ml
 
-
|}
 
-
# PCR to clone pgsA, pgsB by 'Megaprimer' method using NEB Phusion polymerase
 
-
 
-
===September 16 (Thu)===
 
-
# Gel electrophoresis to verify yesterday's PCR results
 
-
#* pgsA megaprimer: 750bp -> OK
 
-
#* pgsB megaprimer: 170bp -> OK
 
-
#* pgsC: 450bp -> very faint band?
 
-
# Gel extraction of pgsA, pgsB megaprimers
 
-
# 2nd step of megaprimer PCR for pgsA, pgsB
 
-
#* failure; possible causes:
 
-
#** short annealing step?
 
-
#** low denaturation temperature?
 
-
#** mistakes in procedure?
 
-
# PCR
 
-
#* pgsC using correct (redesigned) primers
 
-
#* pgsA, B megaprimer 2nd step repeat using reaction mix composition modified from OpenWetWare
 
-
#* gel purification of PCR products
 
-
# Miniprep of BglX (temporary designation for part cloned from F1 with faulty primer), Cel5, Cel8, Cel44, Man26, Xyn10
 
-
# Cut check of miniprepped parts with XbaI, PstI
 
-
# Yeast genome DNA extraction (detailed protocol will be provided elsewhere) according to [reference article]
 
-
# Gel electrophoresis
 
-
#* yeast genome DNA
 
-
#* PCR products
 
-
#** pgsC -> OK
 
-
# Restriction digest
 
-
#* pgsC (PCR product) with EcoRI, PstI
 
-
#* 1-1C, 1-3A, 1-5A (vectors) with EcoRI, SpeI
 
-
 
-
===September 17 (Fri)===
 
-
# PCR cloning of ADH1 terminator from yeast genome DNA
 
-
#* 4 simultaneous attempts with varying template concentration, thermocycle settings & timing of polymerase addition (before or after initial denaturation)
 
-
#* gel electrophoresis showed no PCR product obtained for any of the reactions -> ''annealing temperatures were too stringent?''
 
-
# PCR of ADH1 terminator (repeat)
 
-
#* annealing temperature was lowered from 70°C to 60°C
 
-
#* PCR failed again -> ''possible RNA contamination?''
 
-
# PCR of ADH1 terminator (2nd repeat)
 
-
#* genomic DNA treated with RNase (0.1μl added to 1μl genome DNA; incubation at 37°C for 15min) before using as template
 
-
# Gel electrophoresis of yesterday's digests: pgsC, 1-1C, 1-3A, 1-5A
 
-
# Miniprep of 013 and 3-22G
 
-
#* 012, 014 culture solutions have turned red -> picked-up colonies had RFP-carrying vectors, not ligated plasmids; discarded!
 
-
#Cut check of 3-22G with XbaI, PstI; 013 with EcoRI, PstI
 
-
#* (RESULTS?)
 
-
# Ligations
 
-
#* 018: 3A assembly with 004 as upstream, Fcex as downstream, 1-1C as vector
 
-
#* 019: pgsC as insert, 1-1C as vector (''cut at?'')
 
-
# PCR cloning of XynA CBM, pgsA
 
-
# Colony pick-up & transfer to solution culture: 006, 007 (9/10 ligation/transformation) ''more plasmids needed?''
 
-
# Transformation of 004, 005, 008, 009, 018, 019
 
-
 
-
===September 18 (Sat)===
 
-
# PCR cloning of pgsA by overlap extension ''megaprimer method doesn't seem to work so well''
 
-
# Gel electrophoresis of PCR product (after overlap step) of pgsA
 
-
#* correct band seems to be obtained
 
-
# Gel extraction followed by restriction digest of PCR product with EcoRI, PstI
 
-
# Miniprep of 006, 007
 
-
# Restriction digest with EcoRI, PstI followed by gel electrophoresis
 
-
# PCR of pgsB (1st fragment for overlap extension)
 
-
#* ''tried 2 times but couldn't get amplification product!''
 
-
# Transfer to solution culture: 004, 005, 008, 009, 018, 019
 
-
 
-
===September 19 (Sun)===
 
-
# PCR of pgsB (repeat)
 
-
#* again, no product :(
 
-
# PCR of pgsB (4th attempt, including yesterday's)
 
-
#* no product
 
-
# Miniprep of 004, 005, 008, 009, 018, 019
 
-
# Restriction digest of 018 with EcoRI, SpeI; 019 with XbaI, PstI
 
-
# Gel electrophoresis of pgsA, 004, 005, 008, 009, 018, 019
 
-
#* apart from 018 & 019, all are parts digested before; used here to compare with new parts 018, 019
 
-
#* (RESULTS?)
 
-
#* problem with 019?
 
-
# PCR of pgsB 1st fragment (5th attempt)
 
-
#* (RESULTS?)
 
-
# PCR of pgsB - generation of 2nd fragment (170bp) for overlap extension
 
-
# PCR of pgsB - generation of 3rd fragment (1000bp) for overlap extension
 
-
# PCR: Phusion activity check using BglX as template & the primers that generated it
 
-
# PCR: pgsB template check using the outermost primers
 
-
 
-
===September 20 (Mon)===
 
-
# Transfer to solution culture: 004, 005, 006, 007, 008 ,009 ,019
 
-
# PCR: Phusion polymerase & template checks (repeat of 9/19?)
 
-
#* positive control (BglX) was amplified -> Phusion polymerase seems to be working ok
 
-
#* problem with template? primer? thermocycle settings?
 
-
# PCR: primer check
 
-
#* pair of primers for each overlap segment were tested
 
-
#* (RESULTS?)　
 
-
# Digestion of pgsA (PCR product) and 1-1C with EcoRI, PstI
 
-
# Ligation to make new part 020: pgsA as insert, 1-1C as vector
 
-
# Transformation of ligation product
 
-
# PCR of pgsB 1st fragment (''n-th repeat??'')
 
-
* '''Note: MANY MANY rounds of PCR carried out today; due to time constraints they are not described here in detail'''
 
-
 
-
===September 21 (Tue)===
 
-
# Miniprep of yesterday's cultures: 004, 005, 006, 007, 008 ,009; 019 was discarded (turned red)
 
-
# Restriction digest of miniprepped parts with EcoRI, SpeI
 
-
# Gel electrophoresis of digested parts 004, 005, 006, 007, 008, 009, 019 (?)
 
-
#* 005 - OK
 
-
#* 014 - no band visible
 
-
#* 019 - bad length - ''repeat ligation''
 
-
#* 006, 007 - bad lengths; ''repeat colony pick-up & culture?''
 
-
# PCR to synthesize C-terminal half of pgsB (overlap extension method continued)
 
-
#* band of correct size obtained!
 
-
# PCR cloning of Man26B, CelB
 
-
#* gel run failed to turn up bands; repeat with lower annealing temp
 
-
#* repeat run succeeded!
 
-
# Inoculated YPD liquid culture medium with yeast
 
-
# New part 020 (contains pgsA) transferred to solution culture
 
-
# PCR of pgsB (final step - extension of overlapping fragments)
 
-
# Gel electrophoresis to extract PCR products (Man26, CelB) as well as 1-5A for plasmid backbone
 
-
 
-
===September 22 (Wed)===
 
-
# Gel electrophoresis of 1-5A, 1-3A, 1-1C, PCR product (pgsB) followed by extraction/purification
 
-
# Restriction digest of extracted parts with EcoRI, SpeI
 
-
# Miniprep of 020
 
-
# Restriction digest of 020 with XbaI, PstI (for gel run to check & further assembly)
 
-
# Restriction digest of pgsC, pgsA with EcoRI, SpeI
 
-
# Gel electrophoresis of all digested parts above
 
-
#* ''020 was bad; repeat ligation?''
 
-
# Transfer of 006, 007 to solution culture (pick up from new colonies?)
 
-
# Ligations
 
-
#* 019: pgsC (PCR product) into 1-1C vector
 
-
#* 020: pgsA (PCR product) into 1-1C vector
 
-
#* 021: pgsB (PCR product) into 1-1C vector
 
-
#* all using PCR products purified today
 
-
# Transformation of above ligation products
 
-
# Extraction of genome DNA from yeast cultured yesterday
 
-
# PCR cloning of yeast parts from genomic DNA
 
-
#* ADH1 terminator
 
-
#* ADH2 promoter
 
-
#* CYC1 terminator
 
-
#* ENO2 promoter
 
-
#* SUC2 leader sequence
 
-
 
-
===September 23 (Thu)===
 
-
# Gel electrophoresis of yesterday's PCR products followed by extraction
 
-
# Restriction digest of PCR products with EcoRI, SpeI
 
-
#* ENO2, ADH2 incorrect length -> repeat
 
-
# PCR cloning of ENO2 promoter, ADH2 promoter, glr (glutamate racemase)
 
-
# Gel electrophoresis of crude PCR product, extraction & purification from gel
 
-
#* ENO2 promoter, ADH2 promoter, glr obtained!
 
-
# PCR cloning of CelB, Man26B, Cel44A (Cel44A: internal mutations needed; 1st step of overlap extension fragment generation)
 
-
# Gel electrophoresis of CelB, Man26B, Cel44A PCR products
 
-
#* (RESULTS?)
 
-
# Miniprep of 006, 007 followed by restriction digest with EcoRI, SpeI
 
-
# Gel electrophoresis of 006, 007
 
-
#* (RESULTS?)
 
-
# Transfer to solution culture: 019, 020
 
-
#* no white/non-RFP colonies on 021 (pgsB) plate
 
-
#** insert (PCR product) was not digested properly?
 
-
#** problem with gel purification?
 
-
 
-
===September 24 (Fri)===
 
-
# Inoculation of Karita-sensei's cellulase parts into fresh LB (need more plasmid); also, 024 (beta-glucosidase)
 
-
# Extraction from iGEM distribution plates:
 
-
{|
 
-
!ID!!Part Name!!Resistance!!Description
 
-
|-
 
-
|1-6N||<bbpart>BBa_</bbpart>||A,K||T7 promoter
 
-
|-
 
-
|2-2F||<bbpart>BBa_</bbpart>||A||T7 polymerase
 
-
|-
 
-
|1-6I||<bbpart>BBa_</bbpart>||A||tetracycline-repressible promoter
 
-
|}
 
-
# PCR purification of yesterday's Cel44A
 
-
# Miniprep of 019, 020
 
-
# Restriction digest of 019, 020 with XbaI, PstI
 
-
#* inserts of correct lengths obtained!
 
-
# Ligations for 3A assembly
 
-
#* 004: 001 as upstream, 1-2J as downstream, 1-3A as vector
 
-
#* 005: 001 as upstream, 2-22P as downstream, 1-3A as vector
 
-
#* pgsB 10xHC 1-3A ''???''
 
-
# Transformation of ligation products
 
-
# PCR cloning (repeat) of Man26, CelB
 
-
# Gel electrophoresis
 
-
#* (RESULTS?)
 
-
 
-
===September 25 (Sat)===
 
-
# Miniprep of yesterday's solution cultures (cellulase parts-containing plasmids from Karita-sensei)
 
-
# Restriction digest of miniprepped plasmid DNA with XbaI, PstI (''??? these are not biobrick plasmids!'') & gel electrophoresis
 
-
# Transformation of miniprepped parts
 
-
# Restriction digests
 
-
#* 001 with EcoRI, PstI
 
-
#* K1 (''??'') with EcoRI, SpeI
 
-
# Ligations to transfer 001, K1 into 1-1C (Amp-resistance) vector -> designated as 001-2
 
-
#* 025: xylanase (K1) in 1-1C vector
 
-
# PCR cloning of CelB
 
-
# Transformation of 001-2, 025
 
-
 
-
 
-
===September 26 (Sun)===
 
-
# Transfer to culture solution (yesterday's transformations)
 
-
# Miniprep of 1-6N, 2-2F, 1-6I, 021 ''faint hint of red detected''
 
-
# Restriction digest
 
-
#* 1-6N, 1-6I with EcoRI, SpeI
 
-
#* 2-2F, 021 with XbaI, PstI
 
-
# Gel electrophoresis
 
-
#* (RESULTS?)
 
-
# Miniprep of parts moved to solution culture this morning
 
-
# Cut check with XbaI, PstI <- ''??these are not biobrick plasmids!''
 
-
#* Cel8 - ok
 
-
#* Cel44 - ??
 
-
#* Man26 - ok
 
-
#* Xyn10 - ??
 
-
# Ligations of 9/23 restriction digests (ADH1, ADH2, CYC1, ENO2, SUC2, glr) to 1-1C plasmid backbone digested at E, S sites
 
-
#* 026 - ADH1 terminator
 
-
#* 027 - ADH2 promoter
 
-
#* 028 - CYC1 terminator
 
-
#* 029 - ENO2 promoter
 
-
#* 030 - SUC2 leader sequence
 
-
#* 031 - glr (glutamate racemase)
 
-
# Transformation of ligation products
 
-
# Transfer of yesterday's transformations to solution culture: 001-2, 025
 
-
 
-
===September 27 (Mon)===
 
-
# PCR of Man26, CelB
 
-
# PCR of Man48 (??), CBM from XynAcc
 
-
# Restriction digests
 
-
#* 1-2M with EcoRI, SpeI for assembly later
 
-
#* Cel8, Cel44, Cel5 with EcoRI; Xyn10 with PstI (''for checking?'')
 
-
#* 001-2 with EcoRI, SpeI
 
-
#* 025 with XbaI, PstI
 
-
# Gel electrophoresis
 
-
# Ligations
 
-
#* 032: 1-2M as upstream, 019 as downstream, 1-3A as vector
 
-
#* 033: 1-2M as upstream, 020 as downstream, 1-3A as vector
 
-
#* 004: 001-2 as upstream, 1-2J as downstream, 1-3A as vector (''remake using 001-2'')
 
-
#* 005: 001-2 as upstream, 2-22P as downstream, 1-3A as vector (<nowiki>" "</nowiki>)
 
-
# Transformation of ligation products
 
-
# Transfer to solution culture: 026, 027, 028, 029, 030, 031
 
-
 
-
===September 28 (Tue)===
 
-
# Miniprep of 026, 027, 028, 030, 031
 
-
# Restriction digest
 
-
#* 026, 028, 031 with XbaI, PstI
 
-
#* 027, 030 with EcoRI, SpeI
 
-
# Gel electrophoresis
 
-
#* (RESULTS?)
 
-
# Ligation: repeat assemblies of 004, 005, 032, 033 yesterday
 
-
# Transformation of ligation products as well as 3-2P
 
-
{|
 
-
!ID!!Part Name!!Resistance!!Description
 
-
|-
 
-
|3-2P||<bbpart>BBa_</bbpart>||A||??
 
-
|}
 
-
 
-
===September 29 (Wed)===
 
-
# Transfer to solution culture 025, 026, 027, 028, 030, 031
 
-
# Ligations (repeat of 9/26 with different dilution of 1-1C)
 
-
#* 026 - ADH1 terminator
 
-
#* 027 - ADH2 promoter
 
-
#* 028 - CYC1 terminator
 
-
#* 029 - ENO2 promoter
 
-
#* 030 - SUC2 leader sequence
 
-
#* 031 - glr (glutamate racemase)
 
-
# Transformation of 025, 026, 027, 028, 030, 031 (''repeat just in case; previous plates almost all red colonies; cannot pick up colony with correct insert?'')
 
-
 
-
# PCR
 
-
#* pgsB - cloning from plasmid
 
-
#* pgsB - amplification from 021
 
-
#* Man28 - amplification from previous product
 
-
# PCR purification of products
 
-
 
-
# PCR of CelB, XynA-CBM
 
-
 
-
# Miniprep of cultures inoculated this morning (total culture time: 12hr)
 
-
#* 025 -> ok (colorless)
 
-
#* 027 -> turned red; discarded
 
-
#* others: ok?
 
-
# Restriction digest of 026, 028, 031 with XbaI, PstI
 
-
# Transfer to solution culture (9/28 transformations of 004, 005, 032, 033, 3-2P)
 
-
# Gel electrophoresis of PCR products
 
-
 
-
# Restriction digests:
 
-
#* pgsB, Man (??) with EcoRI, SpeI
 
-
#* today's miniprepped plasmids with EcoRI
 
-
#* 1-1C with EcoRI, SpeI
 
-
 
-
===September 30 (Thu)===
 
-
# PCR cloning
 
-
#* CM10
 
-
#* CelB (repeat)
 
-
#* ADH1 terminator, CYC1 terminator, SUC2 leader sequence from yeast genome (repeat)
 
-
#* glr (repeat)
 
-
# PCR purification: CM10, XynA-CBM
 
-
# Restriction digest of all above PCR products with EcoRI, SpeI
 
-
# Gel electrophoresis
 
-
 
-
# Ligation of PCR products to 1-1C backbone
 
-
#* 021: pgsB
 
-
#* 025: XynA-CBM
 
-
#* 026: ADH1 terminator
 
-
#* 028: CYC1 terminator
 
-
#* 031: glr
 
-
#* 034: Man
 
-
#* 035: CM10
 
-
# Transformation of ligation products
 
-
 
-
# PCR cloning of CelB, SUC2, ECO2, ADH2 from genomic DNA
 
-
# Gel electrophoresis
 
-
#* (RESULTS?)
 
-
 
-
# Miniprep of 004, 005, 032, 033, 3-2P (9/29 solution culture)
 
-
# Restriction digest of miniprepped parts
 
-
#* 004, 005, 032 with EcoRI, SpeI (same as earlier today)
 
-
#* 033, 3-2P (same as on 9/29)
 
-
# Gel electrophoresis
 
-
#* (RESULTS?)
 
-
 
-
===TO CHECK/CONFIRM===
 
-
* 'K1' (mentioned on 9/250 -> where did it come from?
 
-
* 9/25 - miniprepped parts (inoculated 9/24) were plasmids from karita sensei? biobrick parts pcr-cloned on 9/23? why where these parts transformed immediately after miniprep?
 
-
* 'CelB, Cel44A' etc vs 'Cel5, Cel8, Cel44' which nomenclature refers to what (received plasmids, pcr product?)
 
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Latest revision as of 03:34, 28 October 2010

Notebook

July / August
Week S M T W T F S
week1 25 26 27 28 29 30 31
week2 1 2 3 4 5 6 7
week3 8 9 10 11 12 13 14
week4 15 16 17 18 19 20 21
week5 22 23 24 25 26 27 28
week6 29 30 31
September
Week S M T W T F S
week6 1 2 3 4
week7 5 6 7 8 9 10 11
week8 12 13 14 15 16 17 18
week9 19 20 21 22 23 24 25
week10 26 27 28 29 30
October
Week S M T W T F S
week10 1 2
week11 3 4 5 6 7 8 9
week12 10 11 12 13 14 15 16
week13 17 18 19 20 21 22 23
week14 24 25 26 27 28 29 30
week15 31



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Retrieved from "http://2010.igem.org/Team:Osaka/Notebook"