Team:DTU-Denmark/Technical stuff lab
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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. | 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. | ||
- | + | == Notebook == | |
+ | <html> | ||
+ | <table width="850px" border="0"> | ||
+ | <tr> | ||
+ | <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>Day</b> | ||
+ | </td> | ||
+ | <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> | ||
+ | </td> | ||
+ | <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> | ||
+ | </td> | ||
+ | <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> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Thursday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Friday</b> | ||
+ | </td> | ||
+ | <td style="background-color:#FFFF99;text-align:center; font-family: helvetica, arial, sans-serif;color:#555555;"><b>Saturday</b> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="background-color:#FFCC66;width:100px;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;"><b>Morning</b> | ||
+ | </td> | ||
- | + | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:top;"> | |
+ | </td> | ||
- | + | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:top;"> | |
+ | </td> | ||
- | + | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:top;"> | |
+ | </td> | ||
- | + | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:top;"> | |
+ | </td> | ||
- | + | <td style="background-color:#e7e7e7;color:#555555;height:100px;width:200px;text-align:left;"> | |
+ | <ul> | ||
+ | <li>Restriction digest of 5' dif XP with XbaI and PstI </li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;color:#555555;height:100px;width:200px;text-align:left;"> | ||
+ | </td> | ||
+ | </tr> | ||
- | + | <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> | |
+ | </td> | ||
+ | <td style="background-color:#e7e7e7;text-align:center;font-family: helvetica, arial, sans-serif;color:#555555;font-family: helvetica, arial, sans-serif;color:#555555;"> | ||
+ | |||
+ | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:center;"> | ||
+ | </td> | ||
- | + | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:center;"> | |
+ | </td> | ||
- | + | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:center;"> | |
+ | </td> | ||
- | + | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:left;color:#555555;"> | |
+ | <ul> | ||
+ | <b>Start assembly of PyrD vector</b> | ||
+ | <li>Overnight annealing of 5' Ins ( synthesized oligos )</li> | ||
+ | </ul> | ||
+ | </td> | ||
- | + | <td style="background-color:#e7e7e7;height:100px;width:200px;text-align:left;color:#555555;"> | |
- | + | <ul> | |
- | + | <li>Gel analysis of resultant products from 5' dif XP digest</li> | |
- | + | <li>PCR purification of cut 5' dif XP</li> | |
- | + | <li></li> | |
- | + | </ul> | |
- | + | </td> | |
- | + | </tr> | |
- | + | </table> | |
- | + | </html> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
{{:Team:DTU-Denmark/Templates/Standard_page_end}} | {{:Team:DTU-Denmark/Templates/Standard_page_end}} |
Revision as of 11:35, 19 September 2011
Methods
Lab
\section{Overview} 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
The experimental part of the project can be broken into 3 distinct parts, which combine to form the complete project: \begin{itemize} \item Construction of plasmids \item Strain construction \item Improving the \textit{araBAD} promoter \end{itemize} \paragraph{Construction of plasmids} 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. \paragraph{Strain construction} 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. \paragraph{Improving the \textit{araBAD} promoter} 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.
Notebook
Day | Monday | Tuesday | Wednesday | Thursday | Friday | Saturday |
Morning |
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Afternoon |
|
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