IGEM:Tsinghua/2007/Projects/RAP

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<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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1</td>
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<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">
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7-15</td>
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<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">
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-</td>
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<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">
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1-4</td>
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<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">
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Inoculate E. coli harboring pET15b.</td>
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<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>
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<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">
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Extract plasmids</td>
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<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">
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7-16</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">
 +
Yexing Liu</td>
 +
 +
</tr>
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<tr>
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<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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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="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">
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7-16</td>
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<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">
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1</td>
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<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">
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1-4</td>
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<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">
 +
Extract the pET15b plasmid, which was the digested by XagI and KspAI,</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>
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<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">
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Extract digestion products.</td>
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<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">
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7-17</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">
 +
Yexing Liu</td>
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 +
</tr>
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<tr>
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<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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3</td>
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<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">
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7-17</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 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">
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2</td>
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<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">
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1-4</td>
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<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">
 +
The digestion products were extracted and blunted with Klewnow fragment
 +
exo(-) from MBI, then ligated and transformed.</td>
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<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>
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<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">
 +
Check the plates.</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">
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7-18</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">
 +
Yexing Liu</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">
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4</td>
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<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">
 +
7-18</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">
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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="22">
<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">
 +
1-4</td>
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<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">
 +
6 clones were picked from the plates and cultured.</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>
 +
<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">
 +
Extract the plasmids and perform restriction analysis.</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">
 +
7-18</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">
 +
Yexing Liu</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">
 +
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="22">
 +
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="22">
 +
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="22">
 +
1-5</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 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">
 +
Extract plasmid from the 6 cultures and perform restriction analysis.</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">
 +
4 of the 6 clones were found to be correct, which were then named as
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pET15b-LacI(-).</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">
 +
Clone the lacI and EGFP genes.</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">
 +
-</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">
 +
Yexing Liu</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">
 +
6</td>
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<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">
 +
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="22">
 +
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="22">
 +
1-5</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">
 +
Inoculate E.coli harboring plasmid pET-15b without lacI in 50ml LB added
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50μl Amp. On the shaker 37 degrees over night.</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">
 +
Extract plasmids</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">
 +
8-6</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">
 +
Lijun 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="22">
 +
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="22">
 +
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="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">
 +
1-5</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">
 +
Inoculate three strain of E. coli harboring plasmid pZS1-lTLrLLtCL.</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">
 +
Extract plasmids</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">
 +
8-6</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">
 +
Lijun 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="22">
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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="22">
 +
8-6</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">
 +
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="22">
 +
1-1</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="22">
 +
Extract the pET15-b plasmid, which was then digested NcoI and BamHI.<p>
 +
The digestion products were extracted and kept at -20 degrees.</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">
 +
Wait for ligation.</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">
 +
8-6</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">
 +
Lijun 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="22">
 +
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="22">
 +
8-6</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">
 +
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="22">
 +
1-1,1-2,1-3</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="22">
 +
Extract the plasmid from the three oscillator plasmid strain's culture.<p>
 +
Use 3 pairs of different primers to clone three different versions of
 +
lacI from the three plasmid.</p>
 +
<p>The PCR products were ligated with pEASY-B cloning vector.</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">
 +
Check the plates.</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">
 +
8-7</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">
 +
Lijun 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="22">
 +
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="22">
 +
8-7</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">
 +
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="22">
 +
1-3</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="22">
 +
From each of the 3 plates, 10 clones were picked and verified by colony
 +
PCR. 4 of each strains 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="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">
 +
Wait 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="22">
 +
-</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">
 +
Lijun 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="22">
 +
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="22">
 +
8-7</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">
 +
1-6</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">
 +
Inoculate the strains harboring plasmid - pJBA110, pJBA111, pJBA112.</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>
 +
<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">
 +
PCR clone three different versions of EGFPs from the three different
 +
plasmids.</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">
 +
8-8</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">
 +
Lijun 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="22">
 +
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="22">
 +
8-8</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">
 +
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="22">
 +
1-6</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">
 +
Extract the plasmids - pJBA110, pJBA111, pJBA112.<p>User primers to
 +
clone different version of EGFPs.</p>
 +
<p>The PCR products were ligated with pEASY-B cloning vector.</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 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>
<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 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">
 +
Check the plates by colony PCR.</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">
 +
8-9</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">
 +
Lijun 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="22">
 +
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="22">
 +
8-9</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">
 +
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="22">
 +
1-6</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">
 +
From each of the 3 plates, 10 clones were picked and verified by colony
 +
PCR. 4 of each strains 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="22">
 </td>
 </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">
 +
Waiting 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="22">
<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">
 +
-</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">
Lijun Zhou</td>
Lijun 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="22">
 +
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="22">
 +
8-10</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">
 +
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="22">
 +
1-3</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="22">
 +
Culture the three different lacI strains.</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">
 +
There were correct clones of all the 3 kinds of lacI clones.</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">
 +
Extract the plasmids and ligate with pET15b-LacI(-).</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">
 +
8-11</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="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">
 +
Lijun 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="22">
 +
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="22">
 +
8-11</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="22">
 +
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="22">
 +
1-7</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">
 +
Extract the plasmids from the 3 different strains.<p>The plasmids were
 +
then enzyme digested with NcoI and BamHI and ligated with the digested
 +
pET15b-LacI(-), and then transformed.</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>
 +
<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">
 +
Check the plates.</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">
 +
8-12</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="22">
 +
Lijun 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="22">
 +
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="22">
 +
8-12</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="22">
 +
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="22">
 +
1-7</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">
 +
From each of the three plates, 10 clones were picked and checked by PCR.<p>
 +
The corrected clones were then cultured and verified by enzyme
 +
digestion.</p>
 +
<p>Extract more of the three plasmids, and then digest with BamHI and
 +
EcoRI.</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">
 +
The correct clones were named pETLacI0, pETLacI1, and pETLacI2.</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">
 +
Wait for EGFPs.</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">
 +
-</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">
 +
Lijun 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="22">
 +
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="22">
 +
8-13</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="22">
 +
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="22">
 +
1-7</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">
 +
Culture the three different EGFP strains.</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">
 +
There were correct clones of all the 3 kinds of EGFPs.</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">
 +
Extract the plamids.</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">
 +
8-14</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">
 +
Lijun 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="22">
 +
18</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">
 +
8-14</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">
 +
17</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">
 +
1-7</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">
 +
Extract the three different EGFP strains and digest them with BamHI and
 +
EcoRI.<p>The digestion products of the three pETLacIs and three EGFPs
 +
were extracted, ligated and transformed.</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">
 +
Check for the plates.</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">
 +
8-15</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">
 +
Lijun 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="22">
 +
19</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">
 +
8-15</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">
 +
18</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">
 +
1-7</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">
 +
From each of all the 9 plates, 10 clones were picked for PCR
 +
verification.</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">
 +
Each of the 9 constructions turn out to give positive clones.</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">
 +
 </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">
 +
Lijun Zhou</td>
</tr>
</tr>
Line 398: Line 797:
<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 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>
 +
<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">
 +
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">
 +
8-15</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">
 +
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">
 +
The sequence was reduced and sent for synthesis again.<p> </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 Augct company claimed that they failed to synthesize the whole
 +
sequences.</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>
 +
<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">
 +
9-2</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">
 +
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">
 +
 </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 sequence was sent to synthesize in Invitrogen.</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 augct company again failed to synthesize the reduced sequence.</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">
 +
9-15</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">
 +
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">
 +
9-3</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">
 +
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">
 +
 </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">
 +
Invitrogen failed to prodive the synthesized sequennce in time.</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.</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">
 +
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">
 +
10-10</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">
 +
 </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">
 +
Invitrogen finally provided the synthesized promoter.</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 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>
Zhenyu Shi</td>
Line 404: Line 888:
</table>
</table>
-
<h2>Thread 3: Fast-degrading T7 RNA polymerase.</h2>
+
<h2>Thread 3.1: 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">
Line 778: Line 1,262:
Have the correct clones sequenced. </td>
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">
<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>
+
8-6</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 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">
Chun Li, Qin Zhou,
Chun Li, Qin Zhou,
Line 788: Line 1,272:
17</td>
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">
<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>
+
8-6</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 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>
16</td>
Line 808: Line 1,292:
<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">
 +
18</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">
 +
8-10</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">
 +
17</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">
 +
Analyze the sequencing results.</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 clones were found to have correct sequence.</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">
 +
Construct the pT7Blue.</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">
 +
8-10</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">
 +
Chun Li, Qin Zhou, 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">
 +
19</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">
 +
8-10</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">
 +
18</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">
 +
3-2</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">
 +
PCR clone the part of the pBluescript II SK(-) plasmid.<p>The PCR
 +
products were purified, phosphorated, ligated and then transformed.</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>
 +
<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">
 +
Check the transformants.</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">
 +
8-11</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">
 +
Chun Li, Qin Zhou, 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">
 +
20</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">
 +
8-11</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">
 +
19</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">
 +
3-2</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">
 +
8 clones were picked and cultured.<p>Plasmids were extracted and
 +
restriction-analyzed.</p>
 +
<p>The correct T7 RNA gene clone was 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">
 +
2 clone were found to be 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">
 +
Ligate the T7 RNAP gene into pT7Blue.</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">
 +
8-12</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">
 +
Chun Li, Qin Zhou, 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">
 +
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">
 +
8-12</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">
 +
20</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">
 +
3-3</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">
 +
The plasmid of T7 RNA gene clone and pT7Blue were extracted and digested
 +
by SpeI and BamHI.<p>The digestion products were gel purified, ligated
 +
and transformed.</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>
 +
<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">
 +
Check the plates.</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">
 +
8-13</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">
 +
Chun Li, Qin Zhou, 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">
 +
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="21">
 +
8-13</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">
<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">
 +
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">
 +
3-3</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">
 +
6 clones were picked from the plates and cultured.<p>Extract the
 +
plasmids and perform enzyme digestion.</p>
 +
<p>Extract more of the pT7BlueT7 plasmid and digest it with NcoI and
 +
SpeI.</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">
 +
4 clones were found to be correct, which were named as pT7BlueT7</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">
 +
Clone the Xis tag.</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">
 +
8-13</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">
 +
Chun Li, Qin Zhou, 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">
 +
23</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">
 +
8-13</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">
 +
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="21">
 +
3-5</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 xis tag from the E. coli genome DNA.<p>The PCR products
 +
were then digested with NcoI and SpeI. </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>
 +
<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 products and ligate.</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">
 +
8-14</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">
 +
Yexing Liu</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">
 +
24</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">
 +
8-14</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">
 +
23</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 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">
 +
3-6</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 digestion products were purified, ligated and transformed.</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>
 +
<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">
 +
Check the plates.</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">
 +
8-15</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">
 +
Yexing Liu</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">
 +
25</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">
 +
8-15</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">
 +
24</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">
 +
3-7</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 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 picked from the plates and cultured.<p>Plasmids were
 +
extracted from the 6 cultures and restriction analyzed.</p>
 +
<p>The 4 possibly correct 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">
 +
4 clones were suspected to be 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">
 +
Check the activity of the T7 RNAP gene with xis marker.</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">
 +
8-16</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">
 +
Yexing Liu</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">
 +
26</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">
 +
8-16</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">
 +
25</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">
 +
3-7</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 plasmids from the 4 clones was co-transformed with pACYCDuet-Lux
 +
into E. coli TOP10.<p>The pBluescript II SK(-) plasmid and pACYCDuet-Lux
 +
plasmid were also transformed into E. coli TOP10 as a control.</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>
 +
<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">
 +
Test the luciferase activity.</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">
 +
8-17</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">
 +
Yexing Liu</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">
 +
27</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">
 +
8-17</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">
 +
26</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">
 +
3-4</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">
 +
One clone from every transformation was picked and cultured in 200ml
 +
shaking flask.<p>The five cultures were certrifuged and resuspended with
 +
Tris buffer, And the resuspended E. coli was lyzed by supersonic
 +
treatments.</p>
 +
<p>Substrates was added to the the lyzed product to test luciferase
 +
activity.</p>
 +
<p> </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 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 data of the four clones were 323, 36, 102, 301. The control was only&nbsp;
 +
13.</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">
 +
Yexing Liu</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">
 +
28</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">
 +
8-18</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">
 +
25,27</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">
 +
3-7</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>
 +
<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 sequencing result confirmed that the clone with luciferase data 102
 +
does not contains mutations. Other three clones all contains mutations
 +
in the xis regeion. </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 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>
Line 829: Line 1,545:
</table>
</table>
-
<h1>Project Staff</h1>
+
<p> </p>
 +
 +
<h2>Thread 3.2: Luciferase Reporter.</h2>
-
<table width="100%">
+
<table border="1" cellspacing="0" cellpadding="0" width="1247" style="width: 935.15pt; border-collapse: collapse; border: medium none">
-
<tr>
+
-
<td><h2>Dry Experiment</h2></td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
<table width="100%">
+
<tr>
<tr>
-
<td width="297">Project Proposal</td><td>Zhenyu Shi, Peng Dong</td>
+
<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">
 +
Date</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">
 +
Inherit</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">
 +
Chart Index</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">
 +
Notes</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">
 +
Results</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">
 +
Plan</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">
 +
Expected Time</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">
 +
Operator</td>
 +
 
</tr>
</tr>
<tr>
<tr>
-
<td width="297">Mathametical Simulation</td><td>Xinyu Zhao</td>
+
<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">
 +
6-15</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">
 +
3-4</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">
 +
Culture the E. coli strain harboring pGL3-Basic and the E. coli strain
 +
harboring pGL3-Basic.</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">
 +
Extract of the plasmids.</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">
 +
6-16</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>
+
<tr>
-
</td>
+
<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">
 +
6-16</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">
 +
3-4</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 plasmid. Digest the plasmids with NcoI and BamHI.<p>The
 +
digestion products were purified, ligated and transformed.</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">
 +
Check the plate.</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">
 +
6-17</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><h2>Wet Experiment</h2></td>
+
-
</tr>
+
-
<tr>
+
-
<td>
+
-
<table width="100%">
+
<tr>
<tr>
-
<td width="297">Experiment Design & Management</td><td>Zhenyu Shi</td>
+
<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">
 +
6-17</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">
 +
3-4</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">
 +
Four of the colonies on the plates were picked and cultured.<p>Extract
 +
the plasmids and digest them.</p>
 +
<p>The pACYCDuet-Lux plasmid was transformed into E. coli BL21(DE3).</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 four clones were correct, which were named as pACYCDuet-Lux.</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">
 +
Check the luciferase activity.</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">
 +
6-18</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>
<tr>
<tr>
-
<td width="297">Experiment Operation</td><td>Chen Chen, Lijun Zhou, Peng
+
<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">
-
Dong, Xinyu Zhao, Yexing Liu, Zhenyu Shi, Zhou Yu.</td>
+
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">
 +
6-18</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">
 +
3-4</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">
 +
One of the transformants was picked and cultured in 200ml shaking flask.
 +
A control E. coli BL21(DE3) strain was also cultured. IPTG was added at
 +
the 6 hour.<p>The two cultures were certrifuged and resuspended with
 +
Tris buffer, And the resuspended E. coli was lyzed by supersonic
 +
treatments.</p>
 +
<p>Substrates was added to the the lyzed product to test luciferase
 +
activity.</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 luminance of sample from E. coli BL21(DE3) harboring pACYCDuet-Lux
 +
was 123, while the luminance from the control sample was only 17.<p>The
 +
plasmid pACYCDuet-Lux can serve as a T7 RNAP reporter.</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">
 +
Zhou Yu</td>
 +
 
</tr>
</tr>
</table>
</table>
-
</td>
 
-
</tr>
 
-
</table>
 

Revision as of 06:51, 25 October 2007

Contents

Introduction

Synthetic oscillator in a single operon that simulates the natural oscillations.

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
1 7-15 - 1-4 Inoculate E. coli harboring pET15b.   Extract plasmids 7-16 Yexing Liu
2 7-16 1 1-4 Extract the pET15b plasmid, which was the digested by XagI and KspAI,   Extract digestion products. 7-17 Yexing Liu
3 7-17 2 1-4

The digestion products were extracted and blunted with Klewnow fragment

exo(-) from MBI, then ligated and transformed.
  Check the plates. 7-18 Yexing Liu
4 7-18 3 1-4 6 clones were picked from the plates and cultured.   Extract the plasmids and perform restriction analysis. 7-18 Yexing Liu
5 7-19 4 1-5 Extract plasmid from the 6 cultures and perform restriction analysis.

4 of the 6 clones were found to be correct, which were then named as

pET15b-LacI(-).
Clone the lacI and EGFP genes. - Yexing Liu
6 8-5 5 1-5

Inoculate E.coli harboring plasmid pET-15b without lacI in 50ml LB added

50μl Amp. On the shaker 37 degrees over night.
- Extract plasmids 8-6 Lijun Zhou
7 8-5 - 1-5 Inoculate three strain of E. coli harboring plasmid pZS1-lTLrLLtCL. - Extract plasmids 8-6 Lijun Zhou
8 8-6 6 1-1 Extract the pET15-b plasmid, which was then digested NcoI and BamHI.

The digestion products were extracted and kept at -20 degrees.</td>

-</td>

Wait for ligation.</td>

8-6</td>

Lijun Zhou</td>

</tr>

9</td>

8-6</td>

7</td>

1-1,1-2,1-3</td>

Extract the plasmid from the three oscillator plasmid strain's culture.<p> Use 3 pairs of different primers to clone three different versions of

lacI from the three plasmid.

The PCR products were ligated with pEASY-B cloning vector.</td>

-</td>

Check the plates.</td>

8-7</td>

Lijun Zhou</td>

</tr>

10</td>

8-7</td>

9</td>

1-3</td>

From each of the 3 plates, 10 clones were picked and verified by colony PCR. 4 of each strains were sent for sequencing.</td>

-</td>

Wait the sequencing results.</td>

-</td>

Lijun Zhou</td>

</tr>

11</td>

8-7</td>

-</td>

1-6</td>

Inoculate the strains harboring plasmid - pJBA110, pJBA111, pJBA112.</td>

 </td>

PCR clone three different versions of EGFPs from the three different plasmids.</td>

8-8</td>

Lijun Zhou</td>

</tr>

12</td>

8-8</td>

11</td>

1-6</td>

Extract the plasmids - pJBA110, pJBA111, pJBA112.<p>User primers to

clone different version of EGFPs.

The PCR products were ligated with pEASY-B cloning vector.</td>

 </td>

Check the plates by colony PCR.</td>

8-9</td>

Lijun Zhou</td>

</tr>

13</td>

8-9</td>

12</td>

1-6</td>

From each of the 3 plates, 10 clones were picked and verified by colony PCR. 4 of each strains were sent for sequencing.</td>

 </td>

Waiting for the sequencing results.</td>

-</td>

Lijun Zhou</td>

</tr>

14</td>

8-10</td>

10</td>

1-3</td>

Culture the three different lacI strains.</td>

There were correct clones of all the 3 kinds of lacI clones.</td>

Extract the plasmids and ligate with pET15b-LacI(-).</td>

8-11</td>

Lijun Zhou</td>

</tr>

15</td>

8-11</td>

14</td>

1-7</td>

Extract the plasmids from the 3 different strains.<p>The plasmids were then enzyme digested with NcoI and BamHI and ligated with the digested pET15b-LacI(-), and then transformed.</td>

 </td>

Check the plates.</td>

8-12</td>

Lijun Zhou</td>

</tr>

16</td>

8-12</td>

15</td>

1-7</td>

From each of the three plates, 10 clones were picked and checked by PCR.<p> The corrected clones were then cultured and verified by enzyme

digestion.

Extract more of the three plasmids, and then digest with BamHI and EcoRI.</td>

The correct clones were named pETLacI0, pETLacI1, and pETLacI2.</td>

Wait for EGFPs.</td>

-</td>

Lijun Zhou</td>

</tr>

17</td>

8-13</td>

13</td>

1-7</td>

Culture the three different EGFP strains.</td>

There were correct clones of all the 3 kinds of EGFPs.</td>

Extract the plamids.</td>

8-14</td>

Lijun Zhou</td>

</tr>

18</td>

8-14</td>

17</td>

1-7</td>

Extract the three different EGFP strains and digest them with BamHI and EcoRI.<p>The digestion products of the three pETLacIs and three EGFPs were extracted, ligated and transformed.</td>

 </td>

Check for the plates.</td>

8-15</td>

Lijun Zhou</td>

</tr>

19</td>

8-15</td>

18</td>

1-7</td>

From each of all the 9 plates, 10 clones were picked for PCR verification.</td>

Each of the 9 constructions turn out to give positive clones.</td>

 </td>

 </td>

Lijun Zhou</td>

</tr> </table>

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
7 8-15 6 2-3 The sequence was reduced and sent for synthesis again.

 </td>

The Augct company claimed that they failed to synthesize the whole sequences.</td>

Wait for the synthesis result.</td>

-</td>

Zhenyu Shi</td>

</tr>

8</td>

9-2</td>

7</td>

 </td>

The sequence was sent to synthesize in Invitrogen.</td>

The augct company again failed to synthesize the reduced sequence.</td>

 </td>

9-15</td>

Zhenyu Shi</td>

</tr>

9</td>

9-3</td>

8</td>

 </td>

-</td>

Invitrogen failed to prodive the synthesized sequennce in time.</td>

Wait.</td>

-</td>

Zhenyu Shi</td>

</tr>

10</td>

10-10</td>

9</td>

 </td>

 </td>

Invitrogen finally provided the synthesized promoter.</td>

 </td>

 </td>

Zhenyu Shi</td>

</tr> </table>

Thread 3.1: 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

Die Hu, Tian Fang,

Zhenyu Shi
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

Die Hu, Tian Fang,

Zhenyu Shi
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

Die Hu, Tian Fang,

Zhenyu Shi
4 7-13 3 3-1 2 clones were sent for sequencing. - Wait for the sequencing results. -

Die Hu, Tian Fang,

Zhenyu Shi
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

Die Hu, Tian Fang,

Zhenyu Shi
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

Die Hu, Tian Fang,

Zhenyu Shi
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

Die Hu, Tian Fang,

Zhenyu Shi
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

Die Hu, Tian Fang,

Zhenyu Shi
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

Die Hu, Tian Fang,

Zhenyu Shi
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

Die Hu, Tian Fang,

Zhenyu Shi
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

Chun Li, Qin Zhou,

Zhenyu Shi
13 7-29 12 3-1 Synthesize another pair of primers for cloning. - Wait for the primer and perform PCR cloning again. -

Chun Li, Qin Zhou,

Zhenyu Shi
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

Chun Li, Qin Zhou,

Zhenyu Shi
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

Chun Li, Qin Zhou,

Zhenyu Shi
16 8-3 15 3-1 Get the white clones and perform PCR screening. Three clones were found correct. Have the correct clones sequenced. 8-6

Chun Li, Qin Zhou,

Zhenyu Shi
17 8-6 16 3-1 6 clones were sent for sequencing. - Wait for the sequencing results. -

Chun Li, Qin Zhou,

Zhenyu Shi
18 8-10 17 3-1 Analyze the sequencing results. All clones were found to have correct sequence. Construct the pT7Blue. 8-10 Chun Li, Qin Zhou, Zhenyu Shi
19 8-10 18 3-2 PCR clone the part of the pBluescript II SK(-) plasmid.

The PCR products were purified, phosphorated, ligated and then transformed.</td>

 </td>

Check the transformants.</td>

8-11</td>

Chun Li, Qin Zhou, Zhenyu Shi</td>

</tr>

20</td>

8-11</td>

19</td>

3-2</td>

8 clones were picked and cultured.<p>Plasmids were extracted and

restriction-analyzed.

The correct T7 RNA gene clone was culture.</td>

2 clone were found to be correct.</td>

Ligate the T7 RNAP gene into pT7Blue.</td>

8-12</td>

Chun Li, Qin Zhou, Zhenyu Shi</td>

</tr>

21</td>

8-12</td>

20</td>

3-3</td>

The plasmid of T7 RNA gene clone and pT7Blue were extracted and digested by SpeI and BamHI.<p>The digestion products were gel purified, ligated and transformed.</td>

 </td>

Check the plates.</td>

8-13</td>

Chun Li, Qin Zhou, Zhenyu Shi</td>

</tr>

22</td>

8-13</td>

21</td>

3-3</td>

6 clones were picked from the plates and cultured.<p>Extract the

plasmids and perform enzyme digestion.

Extract more of the pT7BlueT7 plasmid and digest it with NcoI and SpeI.</td>

4 clones were found to be correct, which were named as pT7BlueT7</td>

Clone the Xis tag.</td>

8-13</td>

Chun Li, Qin Zhou, Zhenyu Shi</td>

</tr>

23</td>

8-13</td>

22</td>

3-5</td>

PCR clone the xis tag from the E. coli genome DNA.<p>The PCR products were then digested with NcoI and SpeI. </td>

 </td>

Extract the products and ligate.</td>

8-14</td>

Yexing Liu</td>

</tr>

24</td>

8-14</td>

23</td>

3-6</td>

The digestion products were purified, ligated and transformed.</td>

 </td>

Check the plates.</td>

8-15</td>

Yexing Liu</td>

</tr>

25</td>

8-15</td>

24</td>

3-7</td>

6 clones were picked from the plates and cultured.<p>Plasmids were

extracted from the 6 cultures and restriction analyzed.

The 4 possibly correct clones were sent for sequencing.</td>

4 clones were suspected to be correct.</td>

Check the activity of the T7 RNAP gene with xis marker.</td>

8-16</td>

Yexing Liu</td>

</tr>

26</td>

8-16</td>

25</td>

3-7</td>

The plasmids from the 4 clones was co-transformed with pACYCDuet-Lux into E. coli TOP10.<p>The pBluescript II SK(-) plasmid and pACYCDuet-Lux plasmid were also transformed into E. coli TOP10 as a control.</td>

 </td>

Test the luciferase activity.</td>

8-17</td>

Yexing Liu</td>

</tr>

27</td>

8-17</td>

26</td>

3-4</td>

One clone from every transformation was picked and cultured in 200ml shaking flask.<p>The five cultures were certrifuged and resuspended with Tris buffer, And the resuspended E. coli was lyzed by supersonic

treatments.

Substrates was added to the the lyzed product to test luciferase activity.

 </td>

The data of the four clones were 323, 36, 102, 301. The control was only  13.</td>

Wait for the sequencing results.</td>

-</td>

Yexing Liu</td>

</tr>

28</td>

8-18</td>

25,27</td>

3-7</td>

 </td>

The sequencing result confirmed that the clone with luciferase data 102 does not contains mutations. Other three clones all contains mutations in the xis regeion. </td>

 </td>

 </td>

 </td>

</tr> </table>

<p> 

Thread 3.2: Luciferase Reporter.

Number Date Inherit Chart Index Notes Results Plan Expected Time Operator
1 6-15 - 3-4

Culture the E. coli strain harboring pGL3-Basic and the E. coli strain

harboring pGL3-Basic.
  Extract of the plasmids. 6-16 Zhenyu Shi
2 6-16 1 3-4 Extract the plasmid. Digest the plasmids with NcoI and BamHI.

The digestion products were purified, ligated and transformed.</td>

 </td>

Check the plate.</td>

6-17</td>

Zhenyu Shi</td>

</tr>

3</td>

6-17</td>

2</td>

3-4</td>

Four of the colonies on the plates were picked and cultured.<p>Extract

the plasmids and digest them.

The pACYCDuet-Lux plasmid was transformed into E. coli BL21(DE3).</td>

All four clones were correct, which were named as pACYCDuet-Lux.</td>

Check the luciferase activity.</td>

6-18</td>

Zhenyu Shi</td>

</tr>

4</td>

6-18</td>

3</td>

3-4</td>

One of the transformants was picked and cultured in 200ml shaking flask. A control E. coli BL21(DE3) strain was also cultured. IPTG was added at the 6 hour.<p>The two cultures were certrifuged and resuspended with Tris buffer, And the resuspended E. coli was lyzed by supersonic

treatments.

Substrates was added to the the lyzed product to test luciferase activity.</td>

The luminance of sample from E. coli BL21(DE3) harboring pACYCDuet-Lux was 123, while the luminance from the control sample was only 17.<p>The plasmid pACYCDuet-Lux can serve as a T7 RNAP reporter.</td>

 </td>

 </td>

Zhou Yu</td>

</tr>

</table>

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