Team:ZJU-China/September.html

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href="https://2011.igem.org/Team:ZJU-China/August.html">August<br />
href="https://2011.igem.org/Team:ZJU-China/August.html">August<br />
</a> <a href="https://2011.igem.org/Team:ZJU-China/September.html">September<br />
</a> <a href="https://2011.igem.org/Team:ZJU-China/September.html">September<br />
-
</a></div>
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</a><a href="https://2011.igem.org/Team:ZJU-China/October.html">October</a></div>
<h4>Protocol</h4>
<h4>Protocol</h4>
Line 192: Line 190:
cellpadding="1">
cellpadding="1">
-
<td width="260">鈥?Meeting. Summarize all the experiment work so
+
<td width="260">▪Meeting. Summarize all the experiment work so
far.</td>
far.</td>
-
<td width="297">鈥?Cut: 22M(X+P), 1C3(E+P)</td>
+
<td width="297">▪Cut: 22M(X+P), 1C3(E+P)</td>
Line 219: Line 217:
<table width="563" border="0" cellspacing="0" cellpadding="1">
<table width="563" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="361">鈥?Cut vgb+YFP+tetR, fdhF+RFP+tetR.<br />
+
<td width="361">▪Cut vgb+YFP+tetR, fdhF+RFP+tetR.<br />
-
鈥?Run the digestion results</td>
+
▪Run the digestion results</td>
-
<td width="198">鈥?Culture the positive results</td>
+
<td width="198">▪Culture the positive results</td>
</tr>
</tr>
</table>
</table>
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<tr>
<tr>
-
<td width="171">鈥?Miniprep: Y#4, R#2/3/4/5<br />
+
<td width="171">▪Miniprep: Y#4, R#2/3/4/5<br />
-
鈥?Cut Y#4, R#2/3/4/5</td>
+
▪Cut Y#4, R#2/3/4/5</td>
-
<td width="250">鈥un the gel.<br />
+
<td width="250">▪Run the gel.<br />
-
鈥?Send R#3,Y#4 sample to sequencing</td>
+
▪Send R#3,Y#4 sample to sequencing</td>
-
<td width="145">鈥?Hypoxia culture: RFP3, RFP4<br />
+
<td width="145">▪Hypoxia culture: RFP3, RFP4<br />
-
鈥?Preserve Y#4, R#2/3/4/5</td>
+
▪Preserve Y#4, R#2/3/4/5</td>
</tr>
</tr>
</table>
</table>
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<table width="572" border="0" cellspacing="0" cellpadding="1">
<table width="572" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="92">鈥?Phusion PCR</td>
+
<td width="92">▪Phusion PCR</td>
-
<td width="276">鈥?Hypoxia Culture: R, Y<br />
+
<td width="276">▪Hypoxia Culture: R, Y<br />
-
鈥?Run the PCR results. Bands are confirmed right.</td>
+
▪Run the PCR results. Bands are confirmed right.</td>
-
<td width="198">鈥?Purification: vgb, YFP, tetR.</td>
+
<td width="198">▪Purification: vgb, YFP, tetR.</td>
</tr>
</tr>
</table>
</table>
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<table width="569" border="0" cellspacing="0" cellpadding="1">
<table width="569" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="152">鈥?Culture: YFP, RFP, A25<br />
+
<td width="152">▪Culture: YFP, RFP, A25<br />
-
鈥?Culture in micro-oxygen: YFP, RFP, A25</td>
+
▪Culture in micro-oxygen: YFP, RFP, A25</td>
-
<td width="149">鈥?Culture in slope medium: RFP, YFP, A25<br />
+
<td width="149">▪Culture in slope medium: RFP, YFP, A25<br />
-
鈥?Hypoxia culture: RFP, YFP, A25</td>
+
▪Hypoxia culture: RFP, YFP, A25</td>
-
<td width="262">鈥?Check the fluorescence.<br />
+
<td width="262">▪Check the fluorescence.<br />
-
鈥?Neither RFP in hypoxia or oxygen condition. Little YFP both in
+
▪Neither RFP in hypoxia or oxygen condition. Little YFP both in
hypoxia and oxygen.</td>
hypoxia and oxygen.</td>
</tr>
</tr>
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cellspacing="0">
cellspacing="0">
<tr>
<tr>
-
<td width="285">鈥?Miniprep: RFP3, YFP4</td>
+
<td width="285">▪Miniprep: RFP3, YFP4</td>
-
<td width="331">鈥?Cut: RFP3, YFP4, !C3.</td>
+
<td width="331">▪Cut: RFP3, YFP4, !C3.</td>
</tr>
</tr>
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cellpadding="1">
cellpadding="1">
-
<td width="191">鈥?Run the gel: Yu's PCR results, 1C3, RFP3, YFP4<br />
+
<td width="191">▪Run the gel: Yu's PCR results, 1C3, RFP3, YFP4<br />
-
鈥?Purification: RFP3, YFP4</td>
+
▪Purification: RFP3, YFP4</td>
-
<td>鈥?Ligation: RFP3+1C3, YFP4+1C3<br />
+
<td>▪Ligation: RFP3+1C3, YFP4+1C3<br />
-
鈥?Hypoxia culture: RFP3, YFP4, A25.</td>
+
▪Hypoxia culture: RFP3, YFP4, A25.</td>
-
<td>鈥?Transform the ligation results.<br />
+
<td>▪Transform the ligation results.<br />
-
鈥?Confirmed RFP expression only in hypoxia condition.</td>
+
▪Confirmed RFP expression only in hypoxia condition.</td>
</table>
</table>
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<table id="intable" width="600" border="0" cellspacing="0"
<table id="intable" width="600" border="0" cellspacing="0"
cellpadding="1">
cellpadding="1">
-
<td width="414">鈥?Check the RFP, YFP plates.</td>
+
<td width="414">▪Check the RFP, YFP plates.</td>
-
<td width="182">鈥?Colony PCR</td>
+
<td width="182">▪Colony PCR</td>
</table>
</table>
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<table width="609" border="0" cellspacing="0" cellpadding="1">
<table width="609" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="607">鈥?Culture YFP1/2, RFP3/4</td>
+
<td width="607">▪Culture YFP1/2, RFP3/4</td>
</tr>
</tr>
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<tr>
<tr>
-
<td width="297">鈥?Preserve the strain of RFP1, YFP1/2 in 1C3.<br />
+
<td width="297">▪Preserve the strain of RFP1, YFP1/2 in 1C3.<br />
-
鈥?Miniprep the remains.</td>
+
▪Miniprep the remains.</td>
-
<td width="140">鈥?Cut: YFP1/2, RFP1</td>
+
<td width="140">▪Cut: YFP1/2, RFP1</td>
-
<td width="154">鈥?Run the digestion results.<br />
+
<td width="154">▪Run the digestion results.<br />
-
鈥?Culture RFP2</td>
+
▪Culture RFP2</td>
</tr>
</tr>
</table>
</table>
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<table width="612" border="0" cellspacing="0" cellpadding="1">
<table width="612" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="335">鈥?Miniprep: RFP2, YFP2, RFP1</td>
+
<td width="335">▪Miniprep: RFP2, YFP2, RFP1</td>
-
<td width="273">鈥?Cut them and run the gel.</td>
+
<td width="273">▪Cut them and run the gel.</td>
</tr>
</tr>
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<table width="610" border="0" cellspacing="0" cellpadding="1">
<table width="610" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="161">鈥?Cut YFP(1A3), RFP(1A3) with S+E</td>
+
<td width="161">▪Cut YFP(1A3), RFP(1A3) with S+E</td>
-
<td width="162">鈥?Purification: RFP(1A3), YFP(1A3)</td>
+
<td width="162">▪Purification: RFP(1A3), YFP(1A3)</td>
-
<td width="148">鈥?Ligation: RFP+1C3, YFP+1C3</td>
+
<td width="148">▪Ligation: RFP+1C3, YFP+1C3</td>
-
<td width="131">鈥?Transform the ligation results.</td>
+
<td width="131">▪Transform the ligation results.</td>
</tr>
</tr>
</table>
</table>
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<table width="608" border="0" cellspacing="0" cellpadding="1">
<table width="608" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="244">鈥?Check the plates.</td>
+
<td width="244">▪Check the plates.</td>
-
<td width="164">鈥?Culture lig RFY+YFP+IC3<br />
+
<td width="164">▪Culture lig RFY+YFP+IC3<br />
-
鈥?Culture 1K3</td>
+
▪Culture 1K3</td>
-
<td width="194">鈥?Colony PCR: RFP(1C3)锛?YFP(1C3)</td>
+
<td width="194">▪Colony PCR: RFP(1C3), YFP(1C3)</td>
</tr>
</tr>
</table>
</table>
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cellpadding="1">
cellpadding="1">
<tr>
<tr>
-
<td width="191">鈥?Colony PCR: 1A3, 1K3, lig RFP+YFP+1C3.</td>
+
<td width="191">▪Colony PCR: 1A3, 1K3, lig RFP+YFP+1C3.</td>
-
<td>鈥?Run the PCR results.</td>
+
<td>▪Run the PCR results.</td>
-
<td>鈥?Miniprep: lig RFP+YFP+1C3</td>
+
<td>▪Miniprep: lig RFP+YFP+1C3</td>
</tr>
</tr>
</table>
</table>
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<table id="intable3" border="0" cellspacing="0" cellpadding="1">
<table id="intable3" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td>鈥?Transform 2M, 2I, 6I, 23L.</td>
+
<td>▪Transform 2M, 2I, 6I, 23L.</td>
-
<td>鈥?Purification: 1K3</td>
+
<td>▪Purification: 1K3</td>
</tr>
</tr>
</table>
</table>
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<table width="600" border="0" cellspacing="0" cellpadding="1">
<table width="600" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td>鈥?Miniprep: 20H, 20J, 22B<br />
+
<td>▪Miniprep: 20H, 20J, 22B<br />
-
鈥?Cut: 1K3 with E+P, lig3 with S+E,<br />
+
▪Cut: 1K3 with E+P, lig3 with S+E,<br />
12I(CFP) with P+X,</td>
12I(CFP) with P+X,</td>
-
<td>鈥?Gel excision and /or purification:<br />
+
<td>▪Gel excision and /or purification:<br />
1K3, lig3, 12I(CFP)<br />
1K3, lig3, 12I(CFP)<br />
-
鈥?Culture: <br />
+
▪Culture: <br />
1I, 1K, 3L, 5E, 7C<br />
1I, 1K, 3L, 5E, 7C<br />
-
鈥?Run the gel.<br />
+
▪Run the gel.<br />
-
鈥?Miniprep: <br />
+
▪Miniprep: <br />
RFP+YFP+1C3.<br />
RFP+YFP+1C3.<br />
</td>
</td>
-
<td>鈥?Run the gel of<br />
+
<td>▪Run the gel of<br />
1K3, lig3, CFP again.<br />
1K3, lig3, CFP again.<br />
-
鈥?Gel excision and purification<br />
+
▪Gel excision and purification<br />
-
鈥?Ligation:<br />
+
▪Ligation:<br />
Lig3+CFP+1K3</td>
Lig3+CFP+1K3</td>
</tr>
</tr>
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<table width="618" border="0" cellspacing="0" cellpadding="1">
<table width="618" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td>鈥?Preserve: 1I, 1K, 3C, 5E, 7C<br />
+
<td>▪Preserve: 1I, 1K, 3C, 5E, 7C<br />
-
鈥?Transform the ligation results</td>
+
▪Transform the ligation results</td>
-
<td>鈥?Colony PCR: 2M, 2I锛?I锛?3L<br />
+
<td>▪Colony PCR: 2M, 2I,6I,23L<br />
-
鈥?Cut: 20H, 20J, 22B with X+P</td>
+
▪Cut: 20H, 20J, 22B with X+P</td>
-
<td>鈥?Miniprep: 2M-1, 2I-1, 6I-1, 23L-1<br />
+
<td>▪Miniprep: 2M-1, 2I-1, 6I-1, 23L-1<br />
-
鈥?Cut: 2M, 2I with E+S</td>
+
▪Cut: 2M, 2I with E+S</td>
</tr>
</tr>
</table>
</table>
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<table width="600" border="0" cellspacing="0" cellpadding="1">
<table width="600" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td>鈥?Colony PCR: RFP+YFP+1K3</td>
+
<td>▪Colony PCR: RFP+YFP+1K3</td>
-
<td>鈥?Cut 13K with E+S, <br />
+
<td>▪Cut 13K with E+S, <br />
10I with P+X, 1C3 with E+P<br />
10I with P+X, 1C3 with E+P<br />
-
鈥?Run the gel: <br />
+
▪Run the gel: <br />
13K, 10I, 1C3, 2I, 2M, 20J, 22H, 22B</td>
13K, 10I, 1C3, 2I, 2M, 20J, 22H, 22B</td>
-
<td>鈥?Ligation: 13K+10I+1C3<br />
+
<td>▪Ligation: 13K+10I+1C3<br />
-
鈥?Culture: YFP(Amp), CFP(Amp)</td>
+
▪Culture: YFP(Amp), CFP(Amp)</td>
</tr>
</tr>
</table>
</table>
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<table width="620" border="0" cellspacing="0" cellpadding="1">
<table width="620" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="169">鈥?Order primers for 20H, 20J<br />
+
<td width="169">▪Order primers for 20H, 20J<br />
-
鈥?Culture 20H-1, 20J-1, 22B-1</td>
+
▪Culture 20H-1, 20J-1, 22B-1</td>
-
<td width="225">鈥?Plate the ligation results<br />
+
<td width="225">▪Plate the ligation results<br />
-
鈥?Culture RFP+YFP+CFP+1K3</td>
+
▪Culture RFP+YFP+CFP+1K3</td>
-
<td width="220">鈥?Colony PCR: 3A, 1C3<br />
+
<td width="220">▪Colony PCR: 3A, 1C3<br />
-
鈥?Preserve: 1K3, RFP+YFP+1C3.</td>
+
▪Preserve: 1K3, RFP+YFP+1C3.</td>
</tr>
</tr>
</table>
</table>
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<table width="620" border="0" cellspacing="0" cellpadding="1">
<table width="620" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="120">鈥?Miniprep: 20J, 20H, RFP+YFP+CFP+1K3 1A3]<br />
+
<td width="120">▪Miniprep: 20J, 20H, RFP+YFP+CFP+1K3 1A3]<br />
-
鈥?Run the gel of PCR results</td>
+
▪Run the gel of PCR results</td>
-
<td>鈥?Cut RFP+YFP+CFP+1K3 with E+P<br />
+
<td>▪Cut RFP+YFP+CFP+1K3 with E+P<br />
-
鈥?Colony PCR: 13K+10I+1C3<br />
+
▪Colony PCR: 13K+10I+1C3<br />
-
鈥?PCR: fdhF+rush+Vgb+tetR</td>
+
▪PCR: fdhF+rush+Vgb+tetR</td>
<td></td>
<td></td>
-
<td>鈥?Preserve: 20H, 20J, 22B<br />
+
<td>▪Preserve: 20H, 20J, 22B<br />
-
鈥?Run the gel: 20H, 20J, 22B<br />
+
▪Run the gel: 20H, 20J, 22B<br />
-
鈥?Cut the bands and purification.</td>
+
▪Cut the bands and purification.</td>
-
<td>鈥?Run the gel: 13K+10I+1C3, RFP+YFP+CFP+1K3<br />
+
<td>▪Run the gel: 13K+10I+1C3, RFP+YFP+CFP+1K3<br />
-
鈥?Culture: 13K+10I+1C3<br />
+
▪Culture: 13K+10I+1C3<br />
-
鈥?Grad PCR: fdhF.</td>
+
▪Grad PCR: fdhF.</td>
</tr>
</tr>
</table>
</table>
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cellpadding="1">
cellpadding="1">
<tr>
<tr>
-
<td width="191">鈥?Miniprep: 13K+10I+1C3, 1C3<br />
+
<td width="191">▪Miniprep: 13K+10I+1C3, 1C3<br />
-
鈥?Run the gel of digestion results.</td>
+
▪Run the gel of digestion results.</td>
-
<td width="97">鈥?Cut: 13K+10I+1C3, 1A3, fdhF</td>
+
<td width="97">▪Cut: 13K+10I+1C3, 1A3, fdhF</td>
-
<td width="112">鈥?Run the gel: 20H, 20J, rush+vgb, RYC+1K3,
+
<td width="112">▪Run the gel: 20H, 20J, rush+vgb, RYC+1K3,
fdhF, 13K+10I, 1A3</td>
fdhF, 13K+10I, 1A3</td>
-
<td width="94">鈥?Cut and purification: fdhF, 13K+10I, 1A3<br />
+
<td width="94">▪Cut and purification: fdhF, 13K+10I, 1A3<br />
-
鈥?Culture: CFP, YFP</td>
+
▪Culture: CFP, YFP</td>
-
<td width="88">鈥?Purification: Rush PCR<br />
+
<td width="88">▪Purification: Rush PCR<br />
-
鈥?Cut: 22B with X, rush PCR results with S</td>
+
▪Cut: 22B with X, rush PCR results with S</td>
</tr>
</tr>
</table>
</table>
Line 547: Line 545:
cellpadding="1">
cellpadding="1">
<tr>
<tr>
-
<td width="189">鈥?Run the 20H, 20J from PCR results.<br />
+
<td width="189">▪Run the 20H, 20J from PCR results.<br />
-
鈥?Transform: fdhF+13K+10I+1A3</td>
+
▪Transform: fdhF+13K+10I+1A3</td>
-
<td width="122">鈥?Purification: 20B, Rush<br />
+
<td width="122">▪Purification: 20B, Rush<br />
-
鈥?Ligation: 22B+Rush</td>
+
▪Ligation: 22B+Rush</td>
-
<td width="169">鈥?Purification: ligation results<br />
+
<td width="169">▪Purification: ligation results<br />
-
鈥?Cut: the ligation results with X+S</td>
+
▪Cut: the ligation results with X+S</td>
-
<td width="113">鈥?PCR 20H<br />
+
<td width="113">▪PCR 20H<br />
-
鈥?Self-ligation: rush+22M</td>
+
▪Self-ligation: rush+22M</td>
</tr>
</tr>
</table>
</table>
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<table width="603" border="0" cellspacing="0" cellpadding="1">
<table width="603" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="276">鈥?Colony PCR: fdhF+13K+10I+1A3 on plates</td>
+
<td width="276">▪Colony PCR: fdhF+13K+10I+1A3 on plates</td>
-
<td width="177">鈥?Run the gel of PCR results</td>
+
<td width="177">▪Run the gel of PCR results</td>
-
<td width="144">鈥?Culture the positive results.</td>
+
<td width="144">▪Culture the positive results.</td>
</tr>
</tr>
</table>
</table>
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<table width="611" border="0" cellspacing="0" cellpadding="1">
<table width="611" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="130">鈥?Miniprep: fdhF+13K+10I+1A3<br />
+
<td width="130">▪Miniprep: fdhF+13K+10I+1A3<br />
-
鈥?Preserve the above.</td>
+
▪Preserve the above.</td>
-
<td width="202">鈥?Cut and run the results: fdhF+13K+10I+1A3,
+
<td width="202">▪Cut and run the results: fdhF+13K+10I+1A3,
13K+10I+1C3</td>
13K+10I+1C3</td>
-
<td width="162">鈥?Cut: RFP with E+S, YFP with X+P, 1K3 with
+
<td width="162">▪Cut: RFP with E+S, YFP with X+P, 1K3 with
E+P, 1C3 with E+P</td>
E+P, 1C3 with E+P</td>
-
<td width="109">鈥?Purification the digestion results.</td>
+
<td width="109">▪Purification the digestion results.</td>
</tr>
</tr>
</table>
</table>
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<table width="600" border="0" cellspacing="0" cellpadding="1">
<table width="600" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="376">鈥?Ligate: RFP+1A3/ YFP+1C3/1K3, RFP+1A3/1C3</td>
+
<td width="376">▪Ligate: RFP+1A3/ YFP+1C3/1K3, RFP+1A3/1C3</td>
-
<td width="220">鈥?Transform the ligation results.</td>
+
<td width="220">▪Transform the ligation results.</td>
</tr>
</tr>
</table>
</table>
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<table width="604" border="0" cellspacing="0" cellpadding="1">
<table width="604" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="197">鈥?Order primers for 20H, 20J<br />
+
<td width="197">▪Order primers for 20H, 20J<br />
-
鈥?Culture 20H-1, 20J-1, 22B-1</td>
+
▪Culture 20H-1, 20J-1, 22B-1</td>
-
<td width="200">鈥?Plate the ligation results<br />
+
<td width="200">▪Plate the ligation results<br />
-
鈥?Culture RFP+YFP+CFP+1K3</td>
+
▪Culture RFP+YFP+CFP+1K3</td>
-
<td width="201">鈥?Colony PCR: 3A, 1C3<br />
+
<td width="201">▪Colony PCR: 3A, 1C3<br />
-
鈥?Preserve: 1K3, RFP+YFP+1C3.</td>
+
▪Preserve: 1K3, RFP+YFP+1C3.</td>
</tr>
</tr>
</table>
</table>
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<table width="613" border="0" cellspacing="0" cellpadding="1">
<table width="613" border="0" cellspacing="0" cellpadding="1">
<tr>
<tr>
-
<td width="121">鈥?Miniprep: 20J, 20H, RFP+YFP+CFP+1K3 1A3<br />
+
<td width="121">▪Miniprep: 20J, 20H, RFP+YFP+CFP+1K3 1A3<br />
-
鈥?Run the gel of PCR results</td>
+
▪Run the gel of PCR results</td>
-
<td width="116">鈥?Cut RFP+YFP+CFP+1K3 with E+P<br />
+
<td width="116">▪Cut RFP+YFP+CFP+1K3 with E+P<br />
-
鈥?PCR: fdhF+rush+Vgb+tetR<br />
+
▪PCR: fdhF+rush+Vgb+tetR<br />
-
鈥?Colony PCR: 13K+10I+1C3</td>
+
▪Colony PCR: 13K+10I+1C3</td>
-
<td width="66">鈥?Preserve: 20H, 20J, 22B<br />
+
<td width="66">▪Preserve: 20H, 20J, 22B<br />
-
鈥?Run the gel: 20H, 20J, 22B<br />
+
▪Run the gel: 20H, 20J, 22B<br />
-
鈥?Cut the bands and purification.</td>
+
▪Cut the bands and purification.</td>
-
<td width="112">鈥?Run the gel: 13K+10I+1C3, RFP+YFP+CFP+1K3<br />
+
<td width="112">▪Run the gel: 13K+10I+1C3, RFP+YFP+CFP+1K3<br />
-
鈥?Culture: 13K+10I+1C3<br />
+
▪Culture: 13K+10I+1C3<br />
-
鈥?Grad PCR: fdhF.</td>
+
▪Grad PCR: fdhF.</td>
</tr>
</tr>
</table>
</table>
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-
<p>Directly use DH5伪 fdhF+RFP colony to inoculate silicone tube set
+
<p>Directly use DH5α fdhF+RFP colony to inoculate silicone tube set
with 5ml LB. rest for 12h for biofilm attachment.</p>
with 5ml LB. rest for 12h for biofilm attachment.</p>
</div>
</div>
Line 672: Line 670:
<h1>15th Sep</h1>
<h1>15th Sep</h1>
<p>The silicone tube set with 3h of attachment achieves optimum
<p>The silicone tube set with 3h of attachment achieves optimum
-
biofilm formation. Bubbling sets glass slides are froze in -80鈩?for
+
biofilm formation. Bubbling sets glass slides are froze in -80鈩for
later observation with laser confocal. Biofilm formed on glass slide is
later observation with laser confocal. Biofilm formed on glass slide is
-
about 10渭m thick.</p>
+
about 10μm thick.</p>
</div>
</div>
<div class="block" id="nsheet">
<div class="block" id="nsheet">
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first pressed by a covering slide so that it would stick to it and
first pressed by a covering slide so that it would stick to it and
peeled off from the filter for observation.<br />
peeled off from the filter for observation.<br />
-
Biofilm on glass slide is about 8.6渭m thick. Biofilm formed on gas-solid
+
Biofilm on glass slide is about 8.6μm thick. Biofilm formed on gas-solid
interface is too concentrated in bacteria and culture medium that the
interface is too concentrated in bacteria and culture medium that the
florescence of too strong to see any clear structure.</p>
florescence of too strong to see any clear structure.</p>
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CSS</a> <a href="http://ung.igem.org/Team_Wikis?year=2011"> Team Wikis</a> <a
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CSS</a> <a href="http://ung.igem.org/Team_Wikisyear=2011"> Team Wikis</a> <a
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Latest revision as of 18:41, 28 October 2011

Notebook - September

Lab Notes

July August
 September
October

Protocol

Protocol

Brainstorm

Brainstorm

Lab Notes - September

Biobrick Group

Week9

Day Note
Aug.29th Monday
▪Meeting. Summarize all the experiment work so far. ▪Cut: 22M(X+P), 1C3(E+P)
Aug.30th Tuesday
Aug.31st Wednesday
▪Cut vgb+YFP+tetR, fdhF+RFP+tetR.
▪Run the digestion results		 ▪Culture the positive results
Sept.1st Thursday
▪Miniprep: Y#4, R#2/3/4/5
▪Cut Y#4, R#2/3/4/5		 ▪Run the gel.
▪Send R#3,Y#4 sample to sequencing		 ▪Hypoxia culture: RFP3, RFP4
▪Preserve Y#4, R#2/3/4/5
Sept.2nd Friday
▪Phusion PCR ▪Hypoxia Culture: R, Y
▪Run the PCR results. Bands are confirmed right.		 ▪Purification: vgb, YFP, tetR.
Sept.3rd Saturday
▪Culture: YFP, RFP, A25
▪Culture in micro-oxygen: YFP, RFP, A25		 ▪Culture in slope medium: RFP, YFP, A25
▪Hypoxia culture: RFP, YFP, A25		 ▪Check the fluorescence.
▪Neither RFP in hypoxia or oxygen condition. Little YFP both in hypoxia and oxygen.
Sept.4th Sunday
▪Miniprep: RFP3, YFP4 ▪Cut: RFP3, YFP4, !C3.

Week10

Day Note
Sept.5th Monday
▪Run the gel: Yu's PCR results, 1C3, RFP3, YFP4
▪Purification: RFP3, YFP4		 ▪Ligation: RFP3+1C3, YFP4+1C3
▪Hypoxia culture: RFP3, YFP4, A25.		 ▪Transform the ligation results.
▪Confirmed RFP expression only in hypoxia condition.
Sept.6th Tuesday
▪Check the RFP, YFP plates. ▪Colony PCR
Sept.7th Wednesday
▪Culture YFP1/2, RFP3/4
Sept.8th Thurday
▪Preserve the strain of RFP1, YFP1/2 in 1C3.
▪Miniprep the remains.		 ▪Cut: YFP1/2, RFP1 ▪Run the digestion results.
▪Culture RFP2
Sept.9th Friday
▪Miniprep: RFP2, YFP2, RFP1 ▪Cut them and run the gel.
Sept.10th Saturday
▪Cut YFP(1A3), RFP(1A3) with S+E ▪Purification: RFP(1A3), YFP(1A3) ▪Ligation: RFP+1C3, YFP+1C3 ▪Transform the ligation results.
Sept.11th Sunday
▪Check the plates. ▪Culture lig RFY+YFP+IC3
▪Culture 1K3		 ▪Colony PCR: RFP(1C3), YFP(1C3)

 

Week11

Day Note
Sept.12th Monday
▪Colony PCR: 1A3, 1K3, lig RFP+YFP+1C3. ▪Run the PCR results. ▪Miniprep: lig RFP+YFP+1C3
Sept.13th Tuesday
▪Transform 2M, 2I, 6I, 23L. ▪Purification: 1K3
Sept.14th Wednesday
▪Miniprep: 20H, 20J, 22B
▪Cut: 1K3 with E+P, lig3 with S+E,
12I(CFP) with P+X,		 ▪Gel excision and /or purification:
1K3, lig3, 12I(CFP)
▪Culture:
1I, 1K, 3L, 5E, 7C
▪Run the gel.
▪Miniprep:
RFP+YFP+1C3.
 ▪Run the gel of
1K3, lig3, CFP again.
▪Gel excision and purification
▪Ligation:
Lig3+CFP+1K3
Sept.15th Thursday
▪Preserve: 1I, 1K, 3C, 5E, 7C
▪Transform the ligation results		 ▪Colony PCR: 2M, 2I,6I,23L
▪Cut: 20H, 20J, 22B with X+P		 ▪Miniprep: 2M-1, 2I-1, 6I-1, 23L-1
▪Cut: 2M, 2I with E+S
Sept.16th Friday
▪Colony PCR: RFP+YFP+1K3 ▪Cut 13K with E+S,
10I with P+X, 1C3 with E+P
▪Run the gel:
13K, 10I, 1C3, 2I, 2M, 20J, 22H, 22B		 ▪Ligation: 13K+10I+1C3
▪Culture: YFP(Amp), CFP(Amp)
Sept.17th Saturday
▪Order primers for 20H, 20J
▪Culture 20H-1, 20J-1, 22B-1		 ▪Plate the ligation results
▪Culture RFP+YFP+CFP+1K3		 ▪Colony PCR: 3A, 1C3
▪Preserve: 1K3, RFP+YFP+1C3.
Sept.18th Sunday
▪Miniprep: 20J, 20H, RFP+YFP+CFP+1K3 1A3]
▪Run the gel of PCR results		 ▪Cut RFP+YFP+CFP+1K3 with E+P
▪Colony PCR: 13K+10I+1C3
▪PCR: fdhF+rush+Vgb+tetR		 ▪Preserve: 20H, 20J, 22B
▪Run the gel: 20H, 20J, 22B
▪Cut the bands and purification.		 ▪Run the gel: 13K+10I+1C3, RFP+YFP+CFP+1K3
▪Culture: 13K+10I+1C3
▪Grad PCR: fdhF.

 

Week12

Day Note
Sept.19th Monday
▪Miniprep: 13K+10I+1C3, 1C3
▪Run the gel of digestion results.		 ▪Cut: 13K+10I+1C3, 1A3, fdhF ▪Run the gel: 20H, 20J, rush+vgb, RYC+1K3, fdhF, 13K+10I, 1A3 ▪Cut and purification: fdhF, 13K+10I, 1A3
▪Culture: CFP, YFP		 ▪Purification: Rush PCR
▪Cut: 22B with X, rush PCR results with S
Sept.20th Tuesday
▪Run the 20H, 20J from PCR results.
▪Transform: fdhF+13K+10I+1A3		 ▪Purification: 20B, Rush
▪Ligation: 22B+Rush		 ▪Purification: ligation results
▪Cut: the ligation results with X+S		 ▪PCR 20H
▪Self-ligation: rush+22M
Sept.21st Wednesday
▪Colony PCR: fdhF+13K+10I+1A3 on plates ▪Run the gel of PCR results ▪Culture the positive results.
Sept.22nd Thursday
▪Miniprep: fdhF+13K+10I+1A3
▪Preserve the above.		 ▪Cut and run the results: fdhF+13K+10I+1A3, 13K+10I+1C3 ▪Cut: RFP with E+S, YFP with X+P, 1K3 with E+P, 1C3 with E+P ▪Purification the digestion results.
Sept.23rd Friday
▪Ligate: RFP+1A3/ YFP+1C3/1K3, RFP+1A3/1C3 ▪Transform the ligation results.
Sept. 24th Saturday
▪Order primers for 20H, 20J
▪Culture 20H-1, 20J-1, 22B-1		 ▪Plate the ligation results
▪Culture RFP+YFP+CFP+1K3		 ▪Colony PCR: 3A, 1C3
▪Preserve: 1K3, RFP+YFP+1C3.
Sept.25th Sunday
▪Miniprep: 20J, 20H, RFP+YFP+CFP+1K3 1A3
▪Run the gel of PCR results		 ▪Cut RFP+YFP+CFP+1K3 with E+P
▪PCR: fdhF+rush+Vgb+tetR
▪Colony PCR: 13K+10I+1C3		 ▪Preserve: 20H, 20J, 22B
▪Run the gel: 20H, 20J, 22B
▪Cut the bands and purification.		 ▪Run the gel: 13K+10I+1C3, RFP+YFP+CFP+1K3
▪Culture: 13K+10I+1C3
▪Grad PCR: fdhF.


Biofilm Group

3rd Sep

Directly use DH5α fdhF+RFP colony to inoculate silicone tube set with 5ml LB. rest for 12h for biofilm attachment.

4th Sep

Add 50mlLB+Amp and start circulation.

6th Sep

Biofilm with direct inoculation is optimum. Expressed red florescence but only one small fraction of the biofilm remained after cutting off the part of the tube.

12th Sep

4 silicone tube sets with biofilm attachement time of 3h, 5h, 7.5h and 10h; 4 bubbling sets.

15th Sep

The silicone tube set with 3h of attachment achieves optimum biofilm formation. Bubbling sets glass slides are froze in -80鈩for later observation with laser confocal. Biofilm formed on glass slide is about 10μm thick.

16th Sep

New sets with silicone tube, a mixture of RFP, CFP and YFP expressing e.coli. Attachment 3h.

17th Sep

Glass slide with bubbling set with the same mixture of e.coli.
Place two filter film on solid culture medium and placed the mixture of e.coli on the filter to form gas-solid interface biofilm.

20th Sep

Observation of glass slide and gas-solid biofilm under laser confocal. Glass slide are observed directly and gas-solid biofilm is first pressed by a covering slide so that it would stick to it and peeled off from the filter for observation.
Biofilm on glass slide is about 8.6μm thick. Biofilm formed on gas-solid interface is too concentrated in bacteria and culture medium that the florescence of too strong to see any clear structure.

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