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| - | {{:Team:DTU-Denmark/Templates/Standard_page_begin|Methods}}
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| - | == Lab ==
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| - | \section{Overview}
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| - | Our project is a proof of concept project, showing that the \textit{E. coli} sRNA \textit{sroB} and the intergenic sRNA in the \textit{chbBCARG} gene can be used to control gene expression by targeting the \textit{ybfM} Shine-Delgarno, and that these sRNAs can be rationally designed to targeted other Shine-Delgarnos
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| - | The experimental part of the project can be broken into 3 distinct parts, which combine to form the complete project:
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| - | \begin{itemize}
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| - | \item Construction of plasmids
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| - | \item Strain construction
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| - | \item Improving the \textit{araBAD} promoter
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| - | \end{itemize}
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| - | \paragraph{Construction of plasmids}
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| - | necessary for testing our system involves taking the native system from \textit{E. coli}, as well as a slightly modified system and putting them on plasmids that let us both control the expression of these components and measure the output of the system.
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| - | \paragraph{Strain construction}
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| - | involves deleting the original genes from the chromosome of a \textit{E. coli} W3110 strain. Since we use the original genes, these need to be deleted from the chromosome to prevent them from interfering with our measurements.
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| - | \paragraph{Improving the \textit{araBAD} promoter}
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| - | entails expanding the dynamic range of this promoter by modifying the -10 and -35 sequence of the promoter, as well as randomly changing the nucleotide sequence around and in between these sequences. Since the \textit{araBAD} promoter is used in our project improving this promoter could lead to even finer control of our system.
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| - |
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| - | == Notebook ==
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| - | <html>
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| - | <table width="673px" border="0">
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| - | <tr>
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| - | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;">
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| - | <b>Day</b>
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| - | </td>
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| - | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Monday</b>
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| - | </td>
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| - | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Tuesday</b>
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| - | </td>
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| - | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Wednesday</b>
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| - | </td>
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| - | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Thursday</b>
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| - | </td>
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| - | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Friday</b>
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| - | </td>
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| - | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Saturday</b>
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | <td style="background-color:#FFCC66;width:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Morning</b>
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| - | </td>
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| - |
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| - | <td style="background-color:#e7e7e7;color:#555555;height:100px;width:200px;text-align:left;">
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| - | <ul>
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| - | <li>transform pBAD18 into competent cells DH5α and grow them in LB media overnight </li>
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| - | </ul>
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| - | </td>
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| - |
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| - | <td style="background-color:#e7e7e7;color:#555555;height:100px;width:200px;text-align:left;">
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| - | <ul>
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| - | <li>extract plasmid pBAD18.</li>
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| - | <li>Digest pBAD18 with EcoRI and XbaI restriction enzyme at 37℃ in incubator for 2 hours. Gel analysis.</li>
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| - | </ul>
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| - | </td>
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| - |
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| - | <td style="background-color:#e7e7e7;color:#555555;height:100px;width:200px;text-align:left;">
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| - | <ul>
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| - | <li>Restriction digest of 5' dif XP with XbaI and PstI </li>
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| - | </ul>
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| - | </td>
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| - | <td style="background-color:#e7e7e7;color:#555555;height:100px;width:200px;text-align:left;">
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| - | <ul>
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| - | <li>Restriction digest of 5' dif XP with XbaI and PstI </li>
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| - | </ul>
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| - | </td>
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| - |
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| - | <td style="background-color:#e7e7e7;color:#555555;height:100px;width:200px;text-align:left;">
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| - | <ul>
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| - | <li>Restriction digest of 5' dif XP with XbaI and PstI </li>
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| - | </ul>
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| - | </td>
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| - | <td style="background-color:#e7e7e7;color:#555555;height:100px;width:200px;text-align:left;">
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| - | </td>
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| - | </tr>
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| - |
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| - | <td style="background-color:#FFCC66;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Afternoon</b>
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| - | </td>
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| - | <td style="background-color:#e7e7e7;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;">
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| - |
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| - | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:center;">
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| - | </td>
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| - |
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| - | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:center;">
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| - | </td>
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| - |
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| - | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:center;">
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| - | </td>
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| - |
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| - | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:left;color:#555555;">
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| - | <ul>
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| - | <b>Start assembly of PyrD vector</b>
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| - | <li>Overnight annealing of 5' Ins ( synthesized oligos )</li>
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| - | </ul>
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| - | </td>
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| - |
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| - | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:left;color:#555555;">
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| - | <ul>
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| - | <li>Gel analysis of resultant products from 5' dif XP digest</li>
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| - | <li>PCR purification of cut 5' dif XP</li>
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| - | <li></li>
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| - | </ul>
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| - | </td>
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| - | </tr>
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| - | </table>
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| - | </html>
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| - |
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| - | {{:Team:DTU-Denmark/Templates/Standard_page_end}}
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