IGEM:Tsinghua/2007/Projects/RAP

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<title>RAP</title>
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<h1>Introduction</h1>
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<h1>RAP</h1>
+
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+
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<h2>Introduction</h2>
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<table width="100%">
<table width="100%">
<tr><td>
<tr><td>
-
<h3>Naturally oscillation works with pulses in a single operon.</h3></td></tr>
+
<h2>Designed oscillator simulating the natural oscillations of pulses in a single operon.</h2></td></tr>
<tr><td>
<tr><td>
Referring to the expression oscillators, the Elowitz oscillator is the first and  
Referring to the expression oscillators, the Elowitz oscillator is the first and  
-
the only successful one so far, in which three operons expression three  
+
the only typically successful one so far, in which three operons express three  
different transcriptional repressors, lacI, lambda C1 and tetR, respectively.  
different transcriptional repressors, lacI, lambda C1 and tetR, respectively.  
The three transcriptional repressors in the Elowitz oscillators inhibit one another to generate  
The three transcriptional repressors in the Elowitz oscillators inhibit one another to generate  
oscillations. However, this is a relatively complex system, and protein  
oscillations. However, this is a relatively complex system, and protein  
-
accumulation has been observed on a single cell scale during the oscillating  
+
accumulation has been observed at the single-cell scale during the oscillating  
-
cycles. While the natural oscillations usually works pulses with a stage when  
+
cycles. While the natural oscillations usually works in pulses including a stage when  
-
the signal is reset to zero. For example, in processes of the active potential,  
+
the signal is reset to zero. For example, in the process of the active  
-
the cardiac cycles and the muscle contraction, the neural signal is triggered  
+
potential, the cardiac cycles and the muscle contraction, the neural/electric signal is triggered  
-
with the state of &quot;zero&quot;, and then amplify itself until an mechanism of
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at the state of &quot;zero&quot;, and then amplified until a feedback inhibiting  
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inhibiting is reinforced to blocked the amplification. At last, the signal fall
+
mechanism is reinforced to blocked the amplification. At last, as the inhibition
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to the state of &quot;zero&quot;. To understand the natural oscillations, herein we  
+
dominates, the signal falls
 +
to the state of &quot;zero&quot;, which can be thought as the &quot;reset&quot;. To understand  
 +
and simulate the natural oscillations, we  
propose a method to allow the oscillator being reset to &quot;zero&quot; after each cycle  
propose a method to allow the oscillator being reset to &quot;zero&quot; after each cycle  
and find that it is an still simpler method to generate oscillations. A  
and find that it is an still simpler method to generate oscillations. A  
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<tr><td>
<tr><td>
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<h3>Fast-degrading T7 RNA polymerase.</h3></td></tr>
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<h2>Fast-degrading T7 RNA polymerase.</h2></td></tr>
<tr><td>
<tr><td>
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Since fast-degrading transcriptional repressors are available, such as the lacI,  
+
Fast-degrading transcriptional repressors are available, such as the lacI,  
lambda C1 and tetR, because those protein are not conservative in their  
lambda C1 and tetR, because those protein are not conservative in their  
C-terminus and ready to be fused with a degrading marker. While the  
C-terminus and ready to be fused with a degrading marker. While the  
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fast-degrading RNA polymerase is the very problem in this project. Most RNA  
+
fast-degrading RNA polymerase is the key in this project. Most RNA  
polymerases consist of multiple subunits, except for T odd number RNA  
polymerases consist of multiple subunits, except for T odd number RNA  
polymerase, SP6 RNA polymerase and so on. Those RNA polymerases are very  
polymerase, SP6 RNA polymerase and so on. Those RNA polymerases are very  
conservative at their C-terminus and can not be engineered to fuse a C-terminus  
conservative at their C-terminus and can not be engineered to fuse a C-terminus  
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degrading marker. However, fortunately their N-terminus are not that  
+
degrading marker. Fortunately their N-terminus are not that conservative and can  
-
conservative and can be engineered to get some N-terminus fusions. Some  
+
be engineered to get some N-terminus fusions. Some degrading marker of E. coli  
-
degrading marker of E. coli can be identified on the N-terminus, such as the  
+
can be identified on the N-terminus, such as the degrading marker recognized by  
-
degrading marker recognized by Lon and other heat shock proteases. The UmuD  
+
Lon and other heat shock proteases. UmuD  
-
degrades quickly in E. coli and its N-terminal 30 amino residues were reported  
+
degrades quickly in <i>E. coli</i> and its N-terminal 30 amino residues were reported  
to work as a N-terminal degrading marker. Therefore, we try to construct a fast  
to work as a N-terminal degrading marker. Therefore, we try to construct a fast  
degrading T7 RNA polymerase with UmuD N-terminal degrading marker.</td></tr>
degrading T7 RNA polymerase with UmuD N-terminal degrading marker.</td></tr>
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<tr><td>
<tr><td>
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<h3>Model and simulation</h3></td></tr>
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<h2>Model and simulation</h2></td></tr>
<tr><td>
<tr><td>
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</table>
</table>
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<h2>Strains &amp; Plasmids</h2>
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<h1>Strains &amp; Plasmids</h1>
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<table width="100%"><tr><td><h3>Strains</h3></td></tr>
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<table width="100%"><tr><td><h2>Strains</h2></td></tr>
<tr><td>
<tr><td>
<table  border="1" cellspacing="0" cellpadding="0" width="100%" style="border-collapse: collapse; border: medium none">
<table  border="1" cellspacing="0" cellpadding="0" width="100%" style="border-collapse: collapse; border: medium none">
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</td></tr></table>
</td></tr></table>
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<table width="100%"><tr><td><h3>Plasmids</h3></td></tr>
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<table width="100%"><tr><td><h2>Plasmids</h2></td></tr>
<tr><td>
<tr><td>
<table  border="1" cellspacing="0" cellpadding="0" width="100%" style="border-collapse: collapse; border: medium none">
<table  border="1" cellspacing="0" cellpadding="0" width="100%" style="border-collapse: collapse; border: medium none">
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pZS1-lTLrLLtCL</td>
pZS1-lTLrLLtCL</td>
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
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A kindly gift from Prof. Michael B. Elowitz</td>
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Prof. Michael B. Elowitz</td>
</tr>
</tr>
<tr>
<tr>
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pJBA110</td>
pJBA110</td>
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
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A kindly gift from Prof. Michael B. Elowitz</td>
+
Prof. Michael B. Elowitz</td>
</tr>
</tr>
<tr>
<tr>
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pJBA111</td>
pJBA111</td>
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
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A kindly gift from Prof. Michael B. Elowitz</td>
+
Prof. Michael B. Elowitz</td>
</tr>
</tr>
<tr>
<tr>
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pJBA112</td>
pJBA112</td>
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
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A kindly gift from Prof. Michael B. Elowitz</td>
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Prof. Michael B. Elowitz</td>
</tr>
</tr>
</table>
</table>
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<h2>Flowchart</h2>
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<h1>Materials</h1>
 +
 
 +
<table width="100%"><tr><td><h2>Reagents</h2></td></tr>
 +
<tr><td>
 +
<table  border="1" cellspacing="0" cellpadding="0" width="100%" style="border-collapse: collapse; border: medium none">
 +
<tr>
 +
<td width="466" valign="top" style="border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Reagents</td>
 +
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Source</td>
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</tr>
 +
<tr>
 +
<td width="466" valign="top" style="border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Mouse-anti-T7 RNA Polymerase monoclonal antibodies.</td>
 +
<td valign="top" style="border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Novagen</td>
 +
</tr>
 +
</table>
 +
</td></tr></table>
 +
 
 +
<p> </p>
 +
 
 +
 
 +
<h1>Flowchart</h1>
<p>[[Image:RAPv1.4.gif]]
<p>[[Image:RAPv1.4.gif]]
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<h2>Eperiment Records</h2>
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<h1>Experiment Records</h1>
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<h3>Thread 1: RAP Ampilifier.</h3>
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<h2>Thread 1: RAP repressor.</h2>
<table border="1" cellspacing="0" cellpadding="0" width="1247" style="width: 935.15pt; border-collapse: collapse; border: medium none">
<table border="1" cellspacing="0" cellpadding="0" width="1247" style="width: 935.15pt; border-collapse: collapse; border: medium none">
<tr>
<tr>
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
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Nubmer</td>
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Number</td>
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
Date</td>
Date</td>
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<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
Operator</td>
Operator</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
 +
 </td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
 +
 </td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
 +
 </td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
 +
 </td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
 +
 </td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
 +
 </td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
 +
 </td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
 +
Lijun Zhou</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="22">
 +
 </td>
</tr>
</tr>
</table>
</table>
-
<h3>Thread 2: Fast-degrading T7 RNA polymerase.</h3>
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<h2>Thread 2: RAP Amplifier.</h2>
<table border="1" cellspacing="0" cellpadding="0" width="1247" style="width: 935.15pt; border-collapse: collapse; border: medium none">
<table border="1" cellspacing="0" cellpadding="0" width="1247" style="width: 935.15pt; border-collapse: collapse; border: medium none">
<tr>
<tr>
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
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Nubmer</td>
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Number</td>
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
Date</td>
Date</td>
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<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
Operator</td>
Operator</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
1</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-23</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
PCR clone the pSC101 ori from pBHR91.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Success</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract of the PCR product.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-23</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-23</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
1</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2-2</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract the PCR product and insert the product into pEASY-B cloning
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vector. Transformation with blue/white screening.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Pick clones.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-24</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-24</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2-2</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Get the white clones and perform PCR screening.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Three clones were found correct.</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Have the correct clones sequenced. </td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-25</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
4</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-25</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2-2</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Send the correct clones for sequencing.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Wait for the sequencing results.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
5</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-29</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
4</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2-2</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
The sequencing result was analyzed. </td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
The sequence is correct.</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Synthesize the RAP promoter and insert the RAP promoter into the vector.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-29</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6-29</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
5</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2-3</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Synthesize the RAP promoter.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Wait for the synthesis result.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi</td>
</tr>
</tr>
</table>
</table>
-
<h3>Thread 3: RAP repressor.</h3>
+
<h2>Thread 3: Fast-degrading T7 RNA polymerase.</h2>
<table border="1" cellspacing="0" cellpadding="0" width="1247" style="width: 935.15pt; border-collapse: collapse; border: medium none">
<table border="1" cellspacing="0" cellpadding="0" width="1247" style="width: 935.15pt; border-collapse: collapse; border: medium none">
<tr>
<tr>
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
-
Nubmer</td>
+
Number</td>
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
Date</td>
Date</td>
Line 254: Line 424:
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
Operator</td>
Operator</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
1</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-11</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
PCR clone the T7 RNA polymerase gene from a previously construct vector
 +
containing T7 RNA polymerase gene.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Success</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract of the PCR product.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-11</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-11</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
1</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract the PCR product and insert the product into pEASY-B cloning
 +
vector. Transformation with blue/white screening.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Pick clones.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-12</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-12</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Pick the white clones and perform PCR screening.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Three clones were found correct.</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Have the correct clones sequenced. </td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-13</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
4</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-13</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
2 clones were sent for sequencing.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Wait for the sequencing results.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
5</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-19</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
4</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
The sequencing result was analyzed. </td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
All the clones have the same mutations. The template may contains
 +
mutations.</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Try a different template to clone the T7 RNAP gene.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-19</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-20</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
5</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Clone the T7 RNA polymerase gene from E. coli BL21(DE3) culture.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Failed.</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract the genome DNA from E. coli BL21(DE3).</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-23</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-23</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract the E. coli BL21(DE3) genome DNA.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Success.</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
PCR clone the T7 RNA polymerase from the E. coli BL21(DE3) genome DNA.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-23</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
8</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-23</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
PCR clone the T7 RNA polymerase from the E. coli BL21(DE3) genome DNA.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Success</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract of the PCR product.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-23</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
9</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-23</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
8</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract the PCR product and insert the product into pEASY-B cloning
 +
vector. Transformation with blue/white screening.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Pick clones.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-24</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
10</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-24</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
9</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Get the white clones and perform PCR screening.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Three clones were found correct.</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Have the correct clones sequenced. </td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-25</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
11</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-25</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
10</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Send the correct clones for sequencing.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Wait for the sequencing results.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Die Hu, Tian Fang</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
12</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-29</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
11</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
The sequencing result was analyzed. </td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
There were two mutations at the restriction site in the primer.</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Synthesize another pair of primers for cloning.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-29</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Chun Li, Qin Zhou</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
13</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
7-29</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
12</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Synthesize another pair of primers for cloning.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Wait for the primer and perform PCR cloning again.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Chun Li, Qin Zhou</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
14</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
8-2</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
13</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
PCR clone the T7 RNA polymerase from the E. coli BL21(DE3) genome DNA.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Success</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract of the PCR product.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
8-2</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Chun Li, Qin Zhou</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
15</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
8-2</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
14</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Extract the PCR product and insert the product into pEASY-B cloning
 +
vector. Transformation with blue/white screening.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Pick clones.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
8-3</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Chun Li, Qin Zhou</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
16</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
8-3</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
15</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Get the white clones and perform PCR screening.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Three clones were found correct.</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Have the correct clones sequenced. </td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
8-5</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Chun Li, Qin Zhou</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
17</td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
8-5</td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
16</td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
3-1</td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
6 clones were sent for sequencing.</td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Wait for the sequencing results.</td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
-</td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
Zhenyu Shi, Chun Li, Qin Zhou</td>
 +
 +
</tr>
 +
<tr>
 +
<td width="47" valign="top" style="width: 35.05pt; border: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
 </td>
 +
<td valign="top" style="width: 76px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
 </td>
 +
<td valign="top" style="width: 57px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
 </td>
 +
<td valign="top" style="width: 92px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
 </td>
 +
<td valign="top" style="width: 245px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
 </td>
 +
<td valign="top" style="width: 142px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
 </td>
 +
<td valign="top" style="width: 150px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
 </td>
 +
<td valign="top" style="width: 220px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
 </td>
 +
<td valign="top" style="width: 96px; border-left: medium none; border-right: 1.0pt solid black; border-top: 1.0pt solid black; border-bottom: 1.0pt solid black; padding-left: 5.4pt; padding-right: 5.4pt; padding-top: 0cm; padding-bottom: 0cm" height="21">
 +
 </td>
</tr>
</tr>
</table>
</table>
 +
 +
<h1>Project Staff</h1>
 +
 +
<table width="100%">
 +
<tr>
 +
<td><h2>Dry Experiment</h2></td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<table width="100%">
 +
<tr>
 +
<td width="297">Project Proposal</td><td>Zhenyu Shi, Peng Dong</td>
 +
</tr>
 +
<tr>
 +
<td width="297">Mathametical Simulation</td><td>Xinyu Zhao</td>
 +
</tr>
 +
</table>
 +
</td>
 +
 +
<tr>
 +
<td><h2>Wet Experiment</h2></td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<table width="100%">
 +
<tr>
 +
<td width="297">Experiment Design & Management</td><td>Zhenyu Shi</td>
 +
</tr>
 +
<tr>
 +
<td width="297">Experiment Operation</td><td>Chen Chen, Lijun Zhou, Peng
 +
Dong, Xinyu Zhao, Yexing Liu, Zhenyu Shi, Zhou Yu.</td>
 +
</tr>
 +
</table>
 +
</td>
 +
</tr>
 +
</table>

Revision as of 07:47, 6 August 2007

Contents

Introduction

Designed oscillator simulating the natural oscillations of pulses in a single operon.

Referring to the expression oscillators, the Elowitz oscillator is the first and the only typically successful one so far, in which three operons express three different transcriptional repressors, lacI, lambda C1 and tetR, respectively. The three transcriptional repressors in the Elowitz oscillators inhibit one another to generate oscillations. However, this is a relatively complex system, and protein accumulation has been observed at the single-cell scale during the oscillating cycles. While the natural oscillations usually works in pulses including a stage when the signal is reset to zero. For example, in the process of the active potential, the cardiac cycles and the muscle contraction, the neural/electric signal is triggered at the state of "zero", and then amplified until a feedback inhibiting mechanism is reinforced to blocked the amplification. At last, as the inhibition dominates, the signal falls to the state of "zero", which can be thought as the "reset". To understand and simulate the natural oscillations, we propose a method to allow the oscillator being reset to "zero" after each cycle and find that it is an still simpler method to generate oscillations. A fast-degrading DNA polymerase and a fast-degrading transcriptional repressor are engaged in this system. This oscillation works in four stages: (1)Triggering: the RNA polymerase gene is expressed by constructive promoters. (2)Amplication: the RNA transcripts the gene coding itself. (3) Inhibiting: the RNA transcripts a transcriptional repressor, which has longer degrading half-life than the RNA polymerase, that blocks the transcription of the RNA polymerase gene. (4) Resetting: the transcription of the RNA polymerase is blocked and until the transcriptional repressor degrades to a concentration below a specific level.

And we found that this oscillator can be constructed in a single operon.

Fast-degrading T7 RNA polymerase.

Fast-degrading transcriptional repressors are available, such as the lacI, lambda C1 and tetR, because those protein are not conservative in their C-terminus and ready to be fused with a degrading marker. While the fast-degrading RNA polymerase is the key in this project. Most RNA polymerases consist of multiple subunits, except for T odd number RNA polymerase, SP6 RNA polymerase and so on. Those RNA polymerases are very conservative at their C-terminus and can not be engineered to fuse a C-terminus degrading marker. Fortunately their N-terminus are not that conservative and can be engineered to get some N-terminus fusions. Some degrading marker of E. coli can be identified on the N-terminus, such as the degrading marker recognized by Lon and other heat shock proteases. UmuD degrades quickly in E. coli and its N-terminal 30 amino residues were reported to work as a N-terminal degrading marker. Therefore, we try to construct a fast

degrading T7 RNA polymerase with UmuD N-terminal degrading marker.

Model and simulation

Ongoing.

Strains & Plasmids

Strains

Strain Source
E. coli TOP10 Transgen biotech company, Beijing.
E. coli MC4100 GCSC of Yale
E. coli JM109 Prof. Chen's lab collection
E. coli BL21(DE3) Transgen biotech company, Beijing.

Plasmids

Plasmid Source
pEASY-Blunt Transgen biotech company, Beijing.
pET15b Prof. Chen's lab collection
pBluescript II SK(-) Prof. Chen's lab collection
pMD18-T Takara, Japan.
pBHR91 Prof. Chen's lab collection
pZS1-lTLrLLtCL Prof. Michael B. Elowitz
pJBA110 Prof. Michael B. Elowitz
pJBA111 Prof. Michael B. Elowitz
pJBA112 Prof. Michael B. Elowitz


Materials

Reagents

Reagents Source
Mouse-anti-T7 RNA Polymerase monoclonal antibodies. Novagen

 


Flowchart

Image:RAPv1.4.gif


Experiment Records

Thread 1: RAP repressor.

Number Date Inherit Chart Index Notes Results Plan Expected Time Operator
              Lijun Zhou  

Thread 2: RAP Amplifier.

Number Date Inherit Chart Index Notes Results Plan Expected Time Operator
1 6-23 - 2-1 PCR clone the pSC101 ori from pBHR91. Success Extract of the PCR product. 6-23 Zhenyu Shi
2 6-23 1 2-2

Extract the PCR product and insert the product into pEASY-B cloning

vector. Transformation with blue/white screening.
- Pick clones. 6-24 Zhenyu Shi
3 6-24 2 2-2 Get the white clones and perform PCR screening. Three clones were found correct. Have the correct clones sequenced. 6-25 Zhenyu Shi
4 6-25 3 2-2 Send the correct clones for sequencing. - Wait for the sequencing results. - Zhenyu Shi
5 6-29 4 2-2 The sequencing result was analyzed. The sequence is correct. Synthesize the RAP promoter and insert the RAP promoter into the vector. 6-29 Zhenyu Shi
6 6-29 5 2-3 Synthesize the RAP promoter. - Wait for the synthesis result. - Zhenyu Shi

Thread 3: Fast-degrading T7 RNA polymerase.

Number Date Inherit Chart Index Notes Results Plan Expected Time Operator
1 7-11 - 3-1

PCR clone the T7 RNA polymerase gene from a previously construct vector

containing T7 RNA polymerase gene.
Success Extract of the PCR product. 7-11 Zhenyu Shi, Die Hu, Tian Fang
2 7-11 1 3-1

Extract the PCR product and insert the product into pEASY-B cloning

vector. Transformation with blue/white screening.
- Pick clones. 7-12 Zhenyu Shi, Die Hu, Tian Fang
3 7-12 2 3-1 Pick the white clones and perform PCR screening. Three clones were found correct. Have the correct clones sequenced. 7-13 Zhenyu Shi, Die Hu, Tian Fang
4 7-13 3 3-1 2 clones were sent for sequencing. - Wait for the sequencing results. - Zhenyu Shi, Die Hu, Tian Fang
5 7-19 4 3-1 The sequencing result was analyzed.

All the clones have the same mutations. The template may contains

mutations.
Try a different template to clone the T7 RNAP gene. 7-19 Zhenyu Shi, Die Hu, Tian Fang
6 7-20 5 3-1 Clone the T7 RNA polymerase gene from E. coli BL21(DE3) culture. Failed. Extract the genome DNA from E. coli BL21(DE3). 7-23 Zhenyu Shi, Die Hu, Tian Fang
7 7-23 6 3-1 Extract the E. coli BL21(DE3) genome DNA. Success. PCR clone the T7 RNA polymerase from the E. coli BL21(DE3) genome DNA. 7-23 Zhenyu Shi, Die Hu, Tian Fang
8 7-23 7 3-1 PCR clone the T7 RNA polymerase from the E. coli BL21(DE3) genome DNA. Success Extract of the PCR product. 7-23 Zhenyu Shi, Die Hu, Tian Fang
9 7-23 8 3-1

Extract the PCR product and insert the product into pEASY-B cloning

vector. Transformation with blue/white screening.
- Pick clones. 7-24 Zhenyu Shi, Die Hu, Tian Fang
10 7-24 9 3-1 Get the white clones and perform PCR screening. Three clones were found correct. Have the correct clones sequenced. 7-25 Zhenyu Shi, Die Hu, Tian Fang
11 7-25 10 3-1 Send the correct clones for sequencing. - Wait for the sequencing results. - Zhenyu Shi, Die Hu, Tian Fang
12 7-29 11 3-1 The sequencing result was analyzed. There were two mutations at the restriction site in the primer. Synthesize another pair of primers for cloning. 7-29 Zhenyu Shi, Chun Li, Qin Zhou
13 7-29 12 3-1 Synthesize another pair of primers for cloning. - Wait for the primer and perform PCR cloning again. - Zhenyu Shi, Chun Li, Qin Zhou
14 8-2 13 3-1 PCR clone the T7 RNA polymerase from the E. coli BL21(DE3) genome DNA. Success Extract of the PCR product. 8-2 Zhenyu Shi, Chun Li, Qin Zhou
15 8-2 14 3-1

Extract the PCR product and insert the product into pEASY-B cloning

vector. Transformation with blue/white screening.
- Pick clones. 8-3 Zhenyu Shi, Chun Li, Qin Zhou
16 8-3 15 3-1 Get the white clones and perform PCR screening. Three clones were found correct. Have the correct clones sequenced. 8-5 Zhenyu Shi, Chun Li, Qin Zhou
17 8-5 16 3-1 6 clones were sent for sequencing. - Wait for the sequencing results. - Zhenyu Shi, Chun Li, Qin Zhou
                 

Project Staff

Dry Experiment

Project ProposalZhenyu Shi, Peng Dong
Mathametical SimulationXinyu Zhao

Wet Experiment

Experiment Design & ManagementZhenyu Shi
Experiment OperationChen Chen, Lijun Zhou, Peng Dong, Xinyu Zhao, Yexing Liu, Zhenyu Shi, Zhou Yu.
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