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- | __NOTOC__
| + | <html> |
- | ==Calendar== | + | <style type="text/css"> |
- | {|{| class="toccolours" style="float:{{{float|left}}}; width:{{{width|150px}}}; 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="7"|{{{header|July}}} | + | |colspan="8"|{{{header|July / August}}} |
| |- | | |- |
| |- 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 |
Line 14: |
Line 176: |
| |width="14%"|<span style="color:deepskyblue;">S</span> | | |width="14%"|<span style="color:deepskyblue;">S</span> |
| |- | | |- |
- | |colspan ="4"|
| + | |<span style>[[Team:Osaka/week1|week1]]</span> |
- | |<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={{{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={{{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={{{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={{{s25|"color:{{{c25|red}}};"}}}>25</span> | | |<span style={{{s25|"color:{{{c25|red}}};"}}}>25</span> |
| |<span style={{{s26|"color:{{{c26|inherit}}};"}}}>26</span> | | |<span style={{{s26|"color:{{{c26|inherit}}};"}}}>26</span> |
| |<span style={{{s27|"color:{{{c27|inherit}}};"}}}>27</span> | | |<span style={{{s27|"color:{{{c27|inherit}}};"}}}>27</span> |
| |<span style={{{s28|"color:{{{c28|inherit}}};"}}}>28</span> | | |<span style={{{s28|"color:{{{c28|inherit}}};"}}}>28</span> |
- | |[[Team:Osaka/Notebook#July 29 (Thu)|29]]<span style={{{s29|"color:{{{c29|inherit}}};"}}}></span> | + | |[[Team:Osaka/week1#July 29 (Thu)|29]]<span style={{{s29|"color:{{{c29|inherit}}};"}}}></span> |
| |<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span> | | |<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span> |
- | |[[Team:Osaka/Notebook#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|150px}}}; margin-left:1em; text-align:center;"
| + | |
- | |- style="background:{{{color1|#ccccff}}};"
| + | |
- | |colspan="7"|{{{header|August}}}
| + | |
- | |-
| + | |
- | |- style="background:{{{color2|#eeeeff}}};"
| + | |
- | |width="14%"|[[S|<span style="color:red;">S</span>]]
| + | |
- | |width="14%"|[[M]]
| + | |
- | |width="14%"|[[T]]
| + | |
- | |width="14%"|[[W]]
| + | |
- | |width="14%"|[[T]]
| + | |
- | |width="14%"|[[F]]
| + | |
- | |width="14%"|[[S|<span style="color:deepskyblue;">S</span>]]
| + | |
| |- | | |- |
| + | |<span style>[[Team:Osaka/week2|week2]]</span> |
| |<span style={{{s1|"color:{{{c1|red}}};"}}}>1</span> | | |<span style={{{s1|"color:{{{c1|red}}};"}}}>1</span> |
- | |[[Team:Osaka/Notebook#August 2 (Mon)|2]]<span style={{{s2|"color:{{{c2|inherit}}};"}}}></span> | + | |[[Team:Osaka/week2#August 2 (Mon)|2]]<span style={{{s2|"color:{{{c2|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 3 (Tue)|3]]<span style={{{s3|"color:{{{c3|inherit}}};"}}}></span> | + | |[[Team:Osaka/week2#August 3 (Tue)|3]]<span style={{{s3|"color:{{{c3|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 4 (Wed)|4]]<span style={{{s4|"color:{{{c4|inherit}}};"}}}></span> | + | |[[Team:Osaka/week2#August 4 (Wed)|4]]<span style={{{s4|"color:{{{c4|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 5 (Thu)|5]]<span style={{{s5|"color:{{{c5|inherit}}};"}}}></span> | + | |[[Team:Osaka/week2#August 5 (Thu)|5]]<span style={{{s5|"color:{{{c5|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 6 (Fri)|6]]<span style={{{s6|"color:{{{c6|inherit}}};"}}}></span> | + | |[[Team:Osaka/week2#August 6 (Fri)|6]]<span style={{{s6|"color:{{{c6|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 7 (Sat)|7]]<span style={{{s7|"color:{{{c7|deepskyblue}}};"}}}></span> | + | |[[Team:Osaka/week2#August 7 (Sat)|7]]<span style={{{s7|"color:{{{c7|deepskyblue}}};"}}}></span> |
| |- | | |- |
- | |[[Team:Osaka/Notebook#August 8 (Sun)|8]]<span style={{{s8|"color:{{{c8|red}}};"}}}></span> | + | |<span style>[[Team:Osaka/week3|week3]]</span> |
- | |[[Team:Osaka/Notebook#August 9 (Mon)|9]]<span style={{{s9|"color:{{{c9|inherit}}};"}}}></span> | + | |[[Team:Osaka/week3#August 8 (Sun)|8]]<span style={{{s8|"color:{{{c8|red}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 10 (Tue)|10]]<span style={{{s10|"color:{{{c10|inherit}}};"}}}></span> | + | |[[Team:Osaka/week3#August 9 (Mon)|9]]<span style={{{s9|"color:{{{c9|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 11 (Wed)|11]]<span style={{{s11|"color:{{{c11|inherit}}};"}}}></span> | + | |[[Team:Osaka/week3#August 10 (Tue)|10]]<span style={{{s10|"color:{{{c10|inherit}}};"}}}></span> |
| + | |[[Team:Osaka/week3#August 11 (Wed)|11]]<span style={{{s11|"color:{{{c11|inherit}}};"}}}></span> |
| |<span style={{{s12|"color:{{{c12|inherit}}};"}}}>12</span> | | |<span style={{{s12|"color:{{{c12|inherit}}};"}}}>12</span> |
| |<span style={{{s13|"color:{{{c13|inherit}}};"}}}>13</span> | | |<span style={{{s13|"color:{{{c13|inherit}}};"}}}>13</span> |
| |<span style={{{s14|"color:{{{c14|deepskyblue}}};"}}}>14</span> | | |<span style={{{s14|"color:{{{c14|deepskyblue}}};"}}}>14</span> |
| |- | | |- |
| + | |<span style>[[Team:Osaka/week4|week4]]</span> |
| |<span style={{{s15|"color:{{{c15|red}}};"}}}>15</span> | | |<span style={{{s15|"color:{{{c15|red}}};"}}}>15</span> |
- | |[[Team:Osaka/Notebook#August 16 (Mon)|16]]<span style={{{s16|"color:{{{c16|inherit}}};"}}}></span> | + | |[[Team:Osaka/week4#August 16 (Mon)|16]]<span style={{{s16|"color:{{{c16|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 17 (Tue)|17]]<span style={{{s17|"color:{{{c17|inherit}}};"}}}></span> | + | |[[Team:Osaka/week4#August 17 (Tue)|17]]<span style={{{s17|"color:{{{c17|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 18 (Wed)|18]]<span style={{{s18|"color:{{{c18|inherit}}};"}}}></span> | + | |[[Team:Osaka/week4#August 18 (Wed)|18]]<span style={{{s18|"color:{{{c18|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 19 (Thu)|19]]<span style={{{s19|"color:{{{c19|inherit}}};"}}}></span> | + | |[[Team:Osaka/week4#August 19 (Thu)|19]]<span style={{{s19|"color:{{{c19|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 20 (Fri)|20]]<span style={{{s20|"color:{{{c20|inherit}}};"}}}></span> | + | |[[Team:Osaka/week4#August 20 (Fri)|20]]<span style={{{s20|"color:{{{c20|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 21 (Sat)|21]]<span style={{{s21|"color:{{{c21|deepskyblue}}};"}}}></span> | + | |[[Team:Osaka/week4#August 21 (Sat)|21]]<span style={{{s21|"color:{{{c21|deepskyblue}}};"}}}></span> |
| |- | | |- |
- | |[[Team:Osaka/Notebook#August 22 (Sun)|22]]<span style={{{s22|"color:{{{c22|red}}};"}}}></span> | + | |<span style>[[Team:Osaka/week5|week5]]</span> |
- | |[[Team:Osaka/Notebook#August 23 (Mon)|23]]<span style={{{s23|"color:{{{c23|inherit}}};"}}}></span> | + | |[[Team:Osaka/week5#August 22 (Sun)|22]]<span style={{{s22|"color:{{{c22|red}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 24 (Tue)|24]]<span style={{{s24|"color:{{{c24|inherit}}};"}}}></span> | + | |[[Team:Osaka/week5#August 23 (Mon)|23]]<span style={{{s23|"color:{{{c23|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 25 (Wed)|25]]<span style={{{s25|"color:{{{c25|inherit}}};"}}}></span> | + | |[[Team:Osaka/week5#August 24 (Tue)|24]]<span style={{{s24|"color:{{{c24|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 26 (Thu)|26]]<span style={{{s26|"color:{{{c26|inherit}}};"}}}></span> | + | |[[Team:Osaka/week5#August 25 (Wed)|25]]<span style={{{s25|"color:{{{c25|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 27 (Fri)|27]]<span style={{{s27|"color:{{{c27|inherit}}};"}}}></span> | + | |[[Team:Osaka/week5#August 26 (Thu)|26]]<span style={{{s26|"color:{{{c26|inherit}}};"}}}></span> |
- | |[[Team:Osaka/Notebook#August 28 (Sat)|28]]<span style={{{s28|"color:{{{c28|deepskyblue}}};"}}}></span> | + | |[[Team:Osaka/week5#August 27 (Fri)|27]]<span style={{{s27|"color:{{{c27|inherit}}};"}}}></span> |
| + | |[[Team:Osaka/week5#August 28 (Sat)|28]]<span style={{{s28|"color:{{{c28|deepskyblue}}};"}}}></span> |
| |- | | |- |
- | |<span style={{{s29|"color:{{{c29|red}}};"}}}>29</span> | + | |<span style>[[Team:Osaka/week6|week6]]</span> |
- | |<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span> | + | |[[Team:Osaka/week6#August 29 (Sun)|29]]<span style={{{s29|"color:{{{c29|red}}};"}}}></span> |
- | |<span style={{{s31|"color:{{{c31|inherit}}};"}}}>31</span> | + | |[[Team:Osaka/week6#August 30 (Mon)|30]]<span style={{{s30|"color:{{{c30|inherit}}};"}}}></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 |
Line 119: |
Line 243: |
| |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 |
Line 171: |
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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> |
| |- | | |- |
- | |<span style={{{s10|"color:{{{c10|red}}};"}}}>10</span> | + | |<span style>[[Team:Osaka/week12|week12]]</span> |
- | |<span style={{{s11|"color:{{{c11|inherit}}};"}}}>11</span> | + | |[[Team:Osaka/week12#October 10 (Sun)|10]]<span style={{{s10|"color:{{{c10|red}}};"}}}></span> |
- | |<span style={{{s12|"color:{{{c12|inherit}}};"}}}>12</span> | + | |[[Team:Osaka/week12#October 11 (Mon)|11]]<span style={{{s11|"color:{{{c11|inherit}}};"}}}></span> |
- | |<span style={{{s13|"color:{{{c13|inherit}}};"}}}>13</span> | + | |[[Team:Osaka/week12#October 12 (Tue)|12]]<span style={{{s12|"color:{{{c12|inherit}}};"}}}></span> |
- | |<span style={{{s14|"color:{{{c14|inherit}}};"}}}>14</span> | + | |[[Team:Osaka/week12#October 13 (Wed)|13]]<span style={{{s13|"color:{{{c13|inherit}}};"}}}></span> |
- | |<span style={{{s15|"color:{{{c15|inherit}}};"}}}>15</span> | + | |[[Team:Osaka/week12#October 14 (Thu)|14]]<span style={{{s14|"color:{{{c14|inherit}}};"}}}></span> |
- | |<span style={{{s16|"color:{{{c16|deepskyblue}}};"}}}>16</span> | + | |[[Team:Osaka/week12#October 15 (Fri)|15]]<span style={{{s15|"color:{{{c15|inherit}}};"}}}></span> |
| + | |[[Team:Osaka/week12#October 16 (Sat)|16]]<span style={{{s16|"color:{{{c16|deepskyblue}}};"}}}></span> |
| |- | | |- |
- | |<span style={{{s17|"color:{{{c17|red}}};"}}}>17</span> | + | |<span style>[[Team:Osaka/week13|week13]]</span> |
- | |<span style={{{s18|"color:{{{c18|inherit}}};"}}}>18</span> | + | |[[Team:Osaka/week13#October 17 (Sun)|17]]<span style={{{s17|"color:{{{c17|red}}};"}}}></span> |
- | |<span style={{{s19|"color:{{{c19|inherit}}};"}}}>19</span> | + | |[[Team:Osaka/week13#October 18 (Mon)|18]]<span style={{{s18|"color:{{{c18|inherit}}};"}}}></span> |
- | |<span style={{{s20|"color:{{{c20|inherit}}};"}}}>20</span> | + | |[[Team:Osaka/week13#October 19 (Tue)|19]]<span style={{{s19|"color:{{{c19|inherit}}};"}}}></span> |
- | |<span style={{{s21|"color:{{{c21|inherit}}};"}}}>21</span> | + | |[[Team:Osaka/week13#October 20 (WEd)|20]]<span style={{{s20|"color:{{{c20|inherit}}};"}}}></span> |
- | |<span style={{{s22|"color:{{{c22|inherit}}};"}}}>22</span> | + | |[[Team:Osaka/week13#October 21 (Thu)|21]]<span style={{{s21|"color:{{{c21|inherit}}};"}}}></span> |
- | |<span style={{{s23|"color:{{{c23|deepskyblue}}};"}}}>23</span> | + | |[[Team:Osaka/week13#October 22 (Fri)|22]]<span style={{{s22|"color:{{{c22|inherit}}};"}}}></span> |
| + | |[[Team:Osaka/week13#October 23 (Sat)|23]]<span style={{{s23|"color:{{{c23|deepskyblue}}};"}}}></span> |
| |- | | |- |
- | |<span style={{{s24|"color:{{{c24|red}}};"}}}>24</span> | + | |<span style>[[Team:Osaka/week14|week14]]</span> |
- | |<span style={{{s25|"color:{{{c25|inherit}}};"}}}>25</span> | + | |[[Team:Osaka/week14#October 24 (Sun)|24]]<span style={{{s24|"color:{{{c24|red}}};"}}}></span> |
- | |<span style={{{s26|"color:{{{c26|inherit}}};"}}}>26</span> | + | |[[Team:Osaka/week14#October 25 (Mon)|25]]<span style={{{s25|"color:{{{c25|inherit}}};"}}}></span> |
- | |<span style={{{s27|"color:{{{c27|inherit}}};"}}}>27</span> | + | |[[Team:Osaka/week14#October 26 (Tue)|26]]<span style={{{s26|"color:{{{c26|inherit}}};"}}}></span> |
| + | |[[Team:Osaka/week14#October 27 (Wed)|27]]<span style={{{s27|"color:{{{c27|inherit}}};"}}}></span> |
| |<span style={{{s28|"color:{{{c28|inherit}}};"}}}>28</span> | | |<span style={{{s28|"color:{{{c28|inherit}}};"}}}>28</span> |
| |<span style={{{s29|"color:{{{c29|inherit}}};"}}}>29</span> | | |<span style={{{s29|"color:{{{c29|inherit}}};"}}}>29</span> |
| |<span style={{{s30|"color:{{{c30|deepskyblue}}};"}}}>30</span> | | |<span style={{{s30|"color:{{{c30|deepskyblue}}};"}}}>30</span> |
| |- | | |- |
| + | |<span style>[[Team:Osaka/week15|week15]]</span> |
| |[[{{{prefix|}}}10.31{{{suffix|}}}|<span style={{{s31|"color:{{{c31|red}}};"}}}>31</span>]] | | |[[{{{prefix|}}}10.31{{{suffix|}}}|<span style={{{s31|"color:{{{c31|red}}};"}}}>31</span>]] |
| |colspan="6"| | | |colspan="6"| |
- | |- style="background:{{{color3|#eeeeff}}};"
| |
- | |colspan="7"|{{{footer|}}}
| |
- | |}<noinclude>
| |
- | {| class="toccolours" style="float:{{{float|left}}}; width:{{{width|150px}}}; margin-left:1em; text-align:center;"
| |
- | |- style="background:{{{color1|#ccccff}}};"
| |
- | |colspan="7"|{{{header|November}}}
| |
- | |-
| |
- | |- style="background:{{{color2|#eeeeff}}};"
| |
- | |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>
| |
- | |-
| |
- | |colspan ="1"|
| |
- | |<span style={{{s1|"color:{{{c1|inherit}}};"}}}>1</span>
| |
- | |<span style={{{s2|"color:{{{c2|inherit}}};"}}}>2</span>
| |
- | |<span style={{{s3|"color:{{{c3|inherit}}};"}}}>3</span>
| |
- | |<span style={{{s4|"color:{{{c4|inherit}}};"}}}>4</span>
| |
- | |<span style={{{s5|"color:{{{c5|inherit}}};"}}}>5</span>
| |
- | |<span style={{{s6|"color:{{{c6|deepskyblue}}};"}}}>6</span>
| |
- | |-
| |
- | |<span style={{{s7|"color:{{{c7|red}}};"}}}>7</span>
| |
- | |<span style={{{s8|"color:{{{c8|inherit}}};"}}}>8</span>
| |
- | |<span style={{{s9|"color:{{{c9|inherit}}};"}}}>9</span>
| |
- | |<span style={{{s10|"color:{{{c10|inherit}}};"}}}>10</span>
| |
- | |<span style={{{s11|"color:{{{c11|inherit}}};"}}}>11</span>
| |
- | |<span style={{{s12|"color:{{{c12|inherit}}};"}}}>12</span>
| |
- | |<span style={{{s13|"color:{{{c13|deepskyblue}}};"}}}>13</span>
| |
- | |-
| |
- | |<span style={{{s14|"color:{{{c14|red}}};"}}}>14</span>
| |
- | |<span style={{{s15|"color:{{{c15|inherit}}};"}}}>15</span>
| |
- | |<span style={{{s16|"color:{{{c16|inherit}}};"}}}>16</span>
| |
- | |<span style={{{s17|"color:{{{c17|inherit}}};"}}}>17</span>
| |
- | |<span style={{{s18|"color:{{{c18|inherit}}};"}}}>18</span>
| |
- | |<span style={{{s19|"color:{{{c19|inherit}}};"}}}>19</span>
| |
- | |<span style={{{s20|"color:{{{c20|deepskyblue}}};"}}}>20</span>
| |
- | |-
| |
- | |<span style={{{s21|"color:{{{c21|red}}};"}}}>21</span>
| |
- | |<span style={{{s22|"color:{{{c22|inherit}}};"}}}>22</span>
| |
- | |<span style={{{s23|"color:{{{c23|inherit}}};"}}}>23</span>
| |
- | |<span style={{{s24|"color:{{{c24|inherit}}};"}}}>24</span>
| |
- | |<span style={{{s25|"color:{{{c25|inherit}}};"}}}>25</span>
| |
- | |<span style={{{s26|"color:{{{c26|inherit}}};"}}}>26</span>
| |
- | |<span style={{{s27|"color:{{{c27|deepskyblue}}};"}}}>27</span>
| |
- | |-
| |
- | |<span style={{{s28|"color:{{{c28|red}}};"}}}>28</span>
| |
- | |<span style={{{s29|"color:{{{c29|inherit}}};"}}}>29</span>
| |
- | |<span style={{{s30|"color:{{{c30|inherit}}};"}}}>30</span>
| |
- | |colspan="4"|
| |
| |- style="background:{{{color3|#eeeeff}}};" | | |- style="background:{{{color3|#eeeeff}}};" |
| |colspan="7"|{{{footer|}}} | | |colspan="7"|{{{footer|}}} |
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- | ----
| |
- |
| |
- |
| |
- | ==Notebook==
| |
- | ===July 29 (Thu)===
| |
- | Attendance: Torigata, Takino, Teoh, Yasumoto, Kakuda, Saka, Tamura
| |
- | # Safety lecture for junior members.
| |
- | # Preparation of LB agar plates (26 Amp, 25 Kan, 25 Cam).
| |
- |
| |
- | ===July 31 (Sat)===
| |
- | Attendance: Miyatake, Hirayama, Torigata, Teoh, Tadashi, Yasumoto, Kakuda, Saka
| |
- | # Meeting
| |
- | #* Summer project schedule
| |
- | #* List of genes to clone
| |
- |
| |
- | ===August 2 (Mon)===
| |
- | Attendance: Torigata, Takino, Teoh, Tadashi, Yasumoto, Saka
| |
- | # Culture medium preparation
| |
- | #* LB agar plates (49 antibiotic-less plates)
| |
- | #* LB liquid medium (500 ml)
| |
- | # Competent cells preparation - Nojima Method
| |
- | #* SOB medium (MgCl2 not yet added) -> stored at 4˚C
| |
- | #* TB buffer -> stored at 4˚C
| |
- | #* Single-colony streaking of E. coli (strain DH5α) on 2 LB agar plates -> 37˚C incubation o/n
| |
- | Tomorrow we shall prepare the remainder of the reagents and inoculate SOB with o/n-incubated E. coli.
| |
- |
| |
- | ===August 3 (Tue)===
| |
- | Attendance: Nakamura, Kakuda, Saka, Yasumoto, Teoh
| |
- | # Competent cells preparation (continued)
| |
- | #* Preparation of glucose solution for making SOC medium.
| |
- | #* Inoculation of pre-culture SOB from o/n-incubated agar plate colonies.
| |
- | #* (Night) Transfer from pre-culture to growth culture.
| |
- |
| |
- | ===August 4 (Wed)===
| |
- | Attendance: Nakamura, Saka, Kakuda, Yasumoto, Torigata, Teoh
| |
- | # OD measurements throughout the day till required OD (0.3~0.7) was obtained.
| |
- | # Completion of competent cells according to protocol.
| |
- |
| |
- | ===August 5 (Thu)===
| |
- | Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino
| |
- | # Transformation of Registry parts (See Table 1).
| |
- | Note: 'ID' is an internal identifier used by wet work members to simplify labeling etc. In the case of iGEM distribution parts it usually refers to the plate location.
| |
- | {|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
| |
- | |+Table 1
| |
- | !ID!!Part Name!!Resistance!!Description
| |
- | |-
| |
- | |2-20J||<bbpart>BBa_K118023</bbpart>||A||''C. fermi'' endocellulase Cen A coding
| |
- | |-
| |
- | |2-20H||<bbpart>BBa_K118022</bbpart>||A||''C. fermi'' exocellulase Cex coding
| |
- | |-
| |
- | |1-2M||<bbpart>BBa_B0034</bbpart>||A||RBS
| |
- | |-
| |
- | |1-13D||<bbpart>BBa_B0010</bbpart>||A||terminator
| |
- | |-
| |
- | |1-1D||<bbpart>BBa_R0010</bbpart>||A||promoter
| |
- | |-
| |
- | |1-18F||<bbpart>BBa_E1010</bbpart>||K||RFP coding
| |
- | |}
| |
- |
| |
- | ===August 6 (Fri)===
| |
- | Attendance: Nakamura, Saka, Yasumoto, Takino, Teoh
| |
- | # Colony check
| |
- | #* All transformed cells produced colonies!
| |
- | #* Non-transformed cells (negative controls) did not grow on Amp, Kan or Cam plates -> confirmed lack of natural antibiotic resistance
| |
- | # Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C
| |
- |
| |
- | ===August 7 (Sat)===
| |
- | Attendance: Nakamura, Saka, Yasumoto, Takino
| |
- | # Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
| |
- | # Transformation of construction plasmids (See Table 2)
| |
- | # Meeting
| |
- | {|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
| |
- | |+Table 2
| |
- | !ID!!Part Name!!Resistance!!Description
| |
- | |-
| |
- | |1-1C||<bbpart>pSB1A3</bbpart>||A||construction plasmid containing mRFP coding device (<bbpart>BBa_J04450</bbpart>)
| |
- | |-
| |
- | |1-3A||<bbpart>pSB1C3</bbpart>||C||<nowiki>(" ")</nowiki>
| |
- | |-
| |
- | |1-5A||<bbpart>pSB1K3</bbpart>||K||<nowiki>(" ")</nowiki>
| |
- | |}
| |
- |
| |
- | ===August 8 (Sun)===
| |
- | Attendance: Nakamura, Yasumoto
| |
- | # Colony check
| |
- | #*All parts successfully transformed
| |
- | # Transfer to LB culture medium
| |
- |
| |
- | ===August 9 (Mon)===
| |
- | # Miniprep of 1-1C
| |
- | # Restriction digests of 2-20H, 2-20J, 1-1D, 1-18F, 1-1C, 1-13D, 1-2M
| |
- | #* (WHICH ENZYMES?)
| |
- | # Gel electrophoresis of digests ("cut check")
| |
- | #* 2-20H, 2-20J, 1-1C -> OK
| |
- | #* 1-1D, 1-18F, 1-13D, 1-2M -> not cut (single band, MW approx. equal to vector + insert)
| |
- | # Transfer of 1-3A, 1-5A colonies to solution culture (repeat)
| |
- | #*''Yesterday's inoculated culture mediums contained the wrong antibiotics!''
| |
- | # Transformation of <bbpart>BBa_I13521</bbpart>, <bbpart>BBa_I13522</bbpart>, <bbpart>BBa_I13600</bbpart>, <bbpart>BBa_K204031</bbpart>, <bbpart>BBa_K204051</bbpart>, <bbpart>BBa_K204032</bbpart> from last year's stock
| |
- |
| |
- | ===August 10 (Tue)===
| |
- | # Miniprep of 1-3A, 1-5A
| |
- | # Restriction digests of 1-3A, 1-5A
| |
- | # Gel electrophoresis
| |
- | ## 1st run: 1-3A, 1-5A (newly miniprepped), 1-1D, 1-18F, 1-13D, 1-2M (repeat)
| |
- | ##* 1-3A, 1-5A -> OK; others -> not cut (''again'')
| |
- | ## 2nd run: 1-1D, 1-18F, 1-13D, 1-2M (2nd repeat)
| |
- | ##* all parts not cut
| |
- |
| |
- | ===August 11 (Wed)===
| |
- | # Miniprep of last year's parts transformed on Monday
| |
- | # Gel electrophoresis of 1-1D, 1-18F, 1-13D, 1-2M (3rd repeat!!!)
| |
- | #* all 4 not cut... AGAIN
| |
- | #* so far all restriction digests involving XbaI seem to have failed; problem with enzyme stock?
| |
- | #* will try with different set of restriction enzymes next week
| |
- | # Sent miniprepped last year's parts to ECUST team in Shanghai, China
| |
- |
| |
- | ===August 16 (Mon)===
| |
- | # Restriction digests
| |
- | #* 1-13D, 1-2M, 1-1D, 1-18F with EcoRI, PstI to check for point mutation(s) affecting XbaI site
| |
- | #* 2-20J with XbaI, PstI to check/confirm XbaI activity
| |
- | # Gel electrophoresis of digests
| |
- | #* (RESULTS?)
| |
- | # Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D, 1-2M, 1-1D, 1-18F
| |
- | #* 3ml LB liquid medium; 3μl Amp or 0.6μl Kan solution added
| |
- |
| |
- | ===August 17 (Tue)===
| |
- | # Mini-prep of 1-13D, 1-2M, 1-1D, 1-18F parts inoculated yesterday (2 of each)
| |
- | # 'Cut check' (restriction digest + gel electrophoresis to confirm insert) of the miniprepped parts:
| |
- | #* 1-1D, 1-18F with EcoRI, SpeI
| |
- | #* 1-13D, 1-2M with XbaI, PstI
| |
- | #* (RESULTS?)
| |
- | # Transformation of secretion tag parts using 25μl of competent cells each(See Table 3)
| |
- | {|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
| |
- | |+Table 3
| |
- | !ID!!Part Name!!Resistance!!Description
| |
- | |-
| |
- | |2-22P||<bbpart>BBa_K103006</bbpart>||A||OmpA outer membrane protein + linker
| |
- | |-
| |
- | |1-2J||<bbpart>BBa_J32015</bbpart>||A,K||PelB leader sequence
| |
- | |}
| |
- |
| |
- | ===August 18 (Wed)===
| |
- | iGEM Japan Meet-Up in Kyoto
| |
- | Attendance: Nakamura, Yasumoto, Saka, Kakuda
| |
- |
| |
- | ===August 19 (Thu)===
| |
- | # Transfer of 2-22P, 1-2J to solution culture
| |
- | # Gel electrophoresis of digests from 'cut check' products from Tuesday
| |
- | #* repeat run, but each digest together with undigested plasmid DNA)
| |
- | #* 2% agarose gel instead of the usual 1%
| |
- | #* (RESULTS?)
| |
- | # Gel electrophoresis of 1-1D digest only
| |
- | #* (RESULT?)
| |
- | # Multiple restriction digests of 1-1D to check for problems at restriction sites
| |
- | #* tried the following: EcoRI only; SpeI only; EcoRI + SpeI
| |
- | # Night: miniprep of 2-22P, 1-2J inoculated in the morning
| |
- |
| |
- | ===August 20 (Fri)===
| |
- | # Gel electrophoresis of 1-1D and its digests
| |
- | #* (RESULTS?)
| |
- | # 'Cut check' of parts miniprepped the night before
| |
- | #* both 2-22P & 1-2J cut with XbaI, PstI
| |
- | #* enzyme inactivation at 80°C, 20min
| |
- | #* (RESULTS?)
| |
- | # Restriction digest of 2-20J (WHICH ENZYMES?)
| |
- | # Ligation according to 3A assembly method: 2-20J or 2-20H + 2-22P or 1-2J; 1-3A as vector
| |
- | #* reaction mixture: 2μl of each plasmid, 2μl ligase buffer, 1μl T4 DNA ligase, water to make 20μl total
| |
- | #* reaction at room temperature for 10min; ligase inactivation at 80°C for 20min
| |
- | # Transformation of ligated parts using 50μl of competent cells each; 2μl ligation product; 150μl Chloramphenicol spread on agar plates before inoculation with pre-incubation mix
| |
- |
| |
- | ===August 21 (Sat)===
| |
- | # Transfer of white (non-RFP) colonies from yesterday's transformation to 3ml LB liquid medium added with 1μl Cam solution
| |
- | #* ''we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded''
| |
- | # 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector
| |
- | #* ligation product designated as 001; Chloramphenicol resistance
| |
- | #* same ligation mix composition as yesterday's
| |
- | # Transformation of 001 with pre-incubation for 1.5hr instead of 1hr
| |
- |
| |
- | ===August 22 (Sun)===
| |
- | # Transfer of 001 to culture solution; incubation at 30°C (''why??'')
| |
- | # Transformation of the following parts (See Table 4)
| |
- | #* O/N incubation at 37°C as per normal
| |
- | {|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
| |
- | |+Table 4
| |
- | !ID!!Part Name!!Resistance!!Description
| |
- | |-
| |
- | |2-4A||<bbpart>BBa_J63005</bbpart>||A||yeast ADH1 promoter
| |
- | |-
| |
- | |F1||N/A||A||beta-galactosidase from Edinburgh team
| |
- | |-
| |
- | |F2||N/A||C||RBS + F1
| |
- | |-
| |
- | |F3||N/A||C||Lac promoter + RBS + F1
| |
- | |}
| |
- |
| |
- | ===August 23 (Mon)===
| |
- | # Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
| |
- | # Miniprep & 'Cut check' of 001
| |
- | #* cut at EcoRI, SpeI
| |
- | #* gel run with DNA ladder, digested plasmid, undigested plasmid
| |
- | #* (RESULTS?)
| |
- | # Transfer of 3 more colonies of 001 to liquid solution (''to store as glycerol stock - see Tue notes'')
| |
- | # Transformation of the following registry parts (See Table5)
| |
- | {|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
| |
- | |+Table 5
| |
- | !ID!!Part Name!!Resistance!!Description
| |
- | |-
| |
- | |2-2O||<bbpart>J63003</bbpart>||A||yeast Kozak sequence
| |
- | |-
| |
- | |3-11I||<bbpart>K105027</bbpart>||A||'cyc100' minimal promoter
| |
- | |}
| |
- |
| |
- | ===August 24 (Tue)===
| |
- | # Colony check of part transformed yesterday: both 2-2O & 3-11I produced >100 colonies
| |
- | #* transfer to solution culture
| |
- | # Miniprep of 2-4A, F1, F2, F3 followed by 'cut check' with EcoRI, SpeI
| |
- | #* (RESULTS)
| |
- | # Transfer of F1 to solution culture (''why?'')
| |
- | # Preparation of glycerol stock of cell culture containing 001 (''why?'')
| |
- | #* 200ml of culture solution mixed with 100ml of 50% glycerol
| |
- | #* stored at -80°C
| |
- |
| |
- | ===August 25 (Wed)===
| |
- | # Miniprep of parts in solution culture: 2-2O, 3-11I, F1
| |
- | # Cut check of 3-11I & F1 with EcoRI, SpeI
| |
- | #* (RESULTS?)
| |
- |
| |
- | ===August 26 (Thu)===
| |
- | # Transformation of <bbpart>BBa_K204022</bbpart>, <bbpart>BBa_K204025</bbpart>, and <bbpart>BBa_K204040</bbpart> to send to Shanghai ECUST team in China.
| |
- |
| |
- | ===August 27 (Fri)===
| |
- | # Transfer of yesterday's transformed parts (all produced colonies) to solution culture
| |
- | # Transformation of the following parts (See Table 6)
| |
- | #* ''using competent cells opened on 8/20''
| |
- | # Preparation of YPD yeast culture medium with the following recipe (See Table 7)
| |
- | #* pH was adjusted to 5.8
| |
- | #* autoclaved before use
| |
- | #* 12.5g (2.5%) agar added to 500ml and 21 YPD agar plates were prepared
| |
- | # Preparation of 41 LB agar plates from pre-mixed broth powder and 1.5% agar
| |
- | {|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
| |
- | |+Table 6
| |
- | !ID!!Part Name!!Resistance!!Description
| |
- | |-
| |
- | |1-1K||<bbpart>BBa_J63010</bbpart>||A||Protein fusion vector (Silver standard)
| |
- | |-
| |
- | |1-1I||<bbpart>BBa_J63009</bbpart>||A||Low copy protein fusion vector (Silver standard)
| |
- | |-
| |
- | |3-3G||<bbpart>BBa_K157013</bbpart>||A||15aa glycine-serine linker (Freiburg standard)
| |
- | |}
| |
- | {|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000; padding:5px;" cellpadding="5"
| |
- | |+Table 7
| |
- | |MiliQ water||1 liter
| |
- | |-
| |
- | |Bacto tryptone||20.0g||2%
| |
- | |-
| |
- | |Bacto yeast extract||10.0g||1%
| |
- | |-
| |
- | |Glucose||20.0g||2%
| |
- | |}
| |
- |
| |
- | ===August 28 (Sat)===
| |
- | # Miniprep of parts in solution culture
| |
- | # Restriction digest (for cut check) - 37°C for 30min
| |
- | #* 2-4A & 3-11I with EcoRI, SpeI
| |
- | #* 2-2O with XbaI, PstI
| |
- | #* K204022, K204025, K204040 wih EcoRI, PstI
| |
- | # Gel electrophoresis of digests
| |
- | #* Plasmids not detected for 2-4A & 3-11I - ''mistake during miniprep? culture duration too long, plasmid loss occurred?''
| |
- | # Transfer of the following parts to solution culture
| |
- | #* 1-1K, 1-1I, 3-3G (yesterday's transformations)
| |
- | #* 2-4A, 3-11I (repeat pick-up from 10/22, 10/23 plates)
| |
- |
| |
- | ===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
| |
- | # 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)
| |
- | # 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?
| |
- | # Gel electrophoresis of CelB, Man26B, Cel44A PCR products
| |
- |
| |
- | ===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
| |
- | #* 025: xylanase (K1) in 1-1C vector
| |
- | # PCR cloning of CelB
| |
- | # Transformation of 001 (new), 025
| |
| | | |
| | | |
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