IGEM:Tsinghua/2007/Projects/RAP: Difference between revisions
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<h1>Introduction</h1> | |||
<h1> | |||
<table width="100%"> | <table width="100%"> | ||
<tr><td> | <tr><td> | ||
< | <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 | 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 | accumulation has been observed at the single-cell scale during the oscillating | ||
cycles. While the natural oscillations usually works pulses | cycles. While the natural oscillations usually works in pulses including a stage when | ||
the signal is reset to zero. For example, in | 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 | ||
at the state of "zero", and then amplified until a feedback inhibiting | |||
mechanism is reinforced to blocked the amplification. At last, as the inhibition | |||
to the state of "zero". To understand the natural oscillations, | 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 | 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 | and find that it is an still simpler method to generate oscillations. A | ||
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<tr><td> | <tr><td> | ||
< | <h2>Fast-degrading T7 RNA polymerase.</h2></td></tr> | ||
<tr><td> | <tr><td> | ||
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 | ||
fast-degrading RNA polymerase is the | 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 | ||
degrading marker. | 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. | 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> | ||
< | <h2>Model and simulation</h2></td></tr> | ||
<tr><td> | <tr><td> | ||
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</table> | </table> | ||
< | <h1>Strains & Plasmids</h1> | ||
<table width="100%"><tr><td>< | <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> | ||
<table width="100%"><tr><td>< | <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"> | ||
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"> | ||
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"> | ||
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"> | ||
Prof. Michael B. Elowitz</td> | |||
</tr> | </tr> | ||
</table> | </table> | ||
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<h2>Flowchart</ | <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> | |||
</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> | |||
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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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< | <h1>Experiment Records</h1> | ||
< | <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> | ||
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Number</td> | |||
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Date</td> | Date</td> | ||
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Operator</td> | Operator</td> | ||
</tr> | |||
<tr> | |||
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</td> | |||
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</td> | |||
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</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> | |||
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</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> | |||
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</td> | |||
</tr> | </tr> | ||
</table> | </table> | ||
< | <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> | ||
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Number</td> | |||
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Date</td> | Date</td> | ||
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Operator</td> | Operator</td> | ||
</tr> | |||
<tr> | |||
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1</td> | |||
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6-23</td> | |||
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-</td> | |||
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2-1</td> | |||
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PCR clone the pSC101 ori from pBHR91.</td> | |||
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Success</td> | |||
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Extract of the PCR product.</td> | |||
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6-23</td> | |||
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Zhenyu Shi</td> | |||
</tr> | |||
<tr> | |||
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2</td> | |||
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6-23</td> | |||
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1</td> | |||
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2-2</td> | |||
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Extract the PCR product and insert the product into pEASY-B cloning | |||
vector. Transformation with blue/white screening.</td> | |||
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-</td> | |||
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Pick clones.</td> | |||
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6-24</td> | |||
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Zhenyu Shi</td> | |||
</tr> | |||
<tr> | |||
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3</td> | |||
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6-24</td> | |||
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2</td> | |||
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2-2</td> | |||
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Get the white clones and perform PCR screening.</td> | |||
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Three clones were found correct.</td> | |||
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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> | ||
< | <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"> | ||
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 255: | Line 425: | ||
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> | |||
</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> | </tr> | ||
</table> | </table> | ||
</td> | |||
</tr> | |||
</table> |
Revision as of 04:47, 6 August 2007
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 | ||||||||||
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Plasmids | ||||||||||||||||||||
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Materials
Reagents | ||||
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Flowchart
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 |
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Wet Experiment |
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