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Team:Bielefeld-CeBiTec/Notebook/Journal/Biosafety/Jun
From 2014.igem.org
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<td style="padding:30px; width:400px"><h1> June </h1></td> | <td style="padding:30px; width:400px"><h1> June </h1></td> | ||
<td width="100px" align="right"><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Biosafety/Jul"><img src="https://static.igem.org/mediawiki/2014/9/9b/Bielefeld-CeBiTec_2014-08-14_Arrow-right.png" width="50px"> </a> </td> | <td width="100px" align="right"><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Biosafety/Jul"><img src="https://static.igem.org/mediawiki/2014/9/9b/Bielefeld-CeBiTec_2014-08-14_Arrow-right.png" width="50px"> </a> </td> | ||
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| - | <div id="text"> | + | <div id="text" style="text-align:left"> |
<div class="tab" id="Week1"> | <div class="tab" id="Week1"> | ||
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 1 06/02 - 06/08</a></h6> |
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
| - | <li>Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Deletion_alanine_racemase" target="_blank">Primer</a> for the deletion of the whole coding sequence of the | + | <ul> |
| + | <li> | ||
| + | Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Deletion_alanine_racemase" target="_blank">Primer</a> for the deletion of the whole coding sequence of the constitutive alanine racemase (<i>alr</i>) and the catabolic alanine racemase (<i>dadX</i>) from <i>E. coli</i> using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge Red/ET-System</a>. | ||
| + | |||
| + | |||
| + | <ul> | ||
| + | <li>alr: <a href="http://parts.igem.org/Part:BBa_K1465403" target="_blank">BBa_K1465403</a> and <a href="http://parts.igem.org/Part:BBa_K1465404" target="_blank">BBa_K1465404</a></li> | ||
| + | <li>dadX: <a href="http://parts.igem.org/Part:BBa_K1465405" target="_blank">BBa_K1465405</a> and <a href="http://parts.igem.org/Part:BBa_K1465406" target="_blank">BBa_K1465406</a></li> | ||
| + | </ul> | ||
| + | </li> | ||
</li> | </li> | ||
| + | </ul> | ||
</div> | </div> | ||
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| - | <div id="text"> | + | <div id="text" style="text-align:left"> |
<div class="tab" id="Week2"> | <div class="tab" id="Week2"> | ||
<div class="show"> | <div class="show"> | ||
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<div class="hide"> | <div class="hide"> | ||
| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 2 06/09 - 06/15</a></h6> |
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
| + | <ul> | ||
| + | <li> | ||
| + | Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Integration_alr" target="_blank">Primer</a> for the integration of the konstitutive alanine racemase <i>alr</i> into the pSB1C3-Backbone. | ||
| + | |||
| + | </li> | ||
| + | </ul> | ||
</div> | </div> | ||
</div> | </div> | ||
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| - | <div id="text"> | + | <div id="text" style="text-align:left"> |
<div class="tab" id="Week3"> | <div class="tab" id="Week3"> | ||
<div class="show"> | <div class="show"> | ||
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<div class="hide"> | <div class="hide"> | ||
| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 3 06/16 - 06/22</a></h6> |
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
| + | <ul> | ||
| + | <li> | ||
| + | Transformation of the <i>E. coli</i> strains KRX (<a href="http://www.promega.de/resources/protocols/technical-bulletins/101/single-step-krx-competent-cells-protocol" target="_blank">Promega</a>) and DH5alpha (<a href="http://tools.lifetechnologies.com/content/sfs/manuals/11319019.pdf" target="_blank">Invitrogen</a>) with the plasmid pRedET containg the Recombinase using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge RedET-System protocol</a>. | ||
| + | </li> | ||
| + | </ul> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px"> | <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px"> | ||
| - | <div id="text"> | + | <div id="text" style="text-align:left"> |
<div class="tab" id="Week4"> | <div class="tab" id="Week4"> | ||
<div class="show"> | <div class="show"> | ||
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<div class="hide"> | <div class="hide"> | ||
| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 4 06/23 - 06/29</a></h6> |
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
| + | <ul> | ||
| + | <li> | ||
| + | Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of <i>alr</i> using the primer <a href="http://parts.igem.org/Part:BBa_K1465403" target="_blank">BBa_K1465403</a> and <a href="http://parts.igem.org/Part:BBa_K1465404" target="_blank">BBa_K1465404</a> | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul> | ||
| + | <li> | ||
| + | Purification using the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugationgel" target="_blank">gel extraction clean-up kit</a> from Promega. | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <ul> | ||
| + | <li> | ||
| + | Transformation of the oligonucleotide containing the flanking sites for the deletion of <i>alr</i>. The successful replacement of the constitutive alanine racemase was verified via kanamycin selection and | ||
| + | |||
| + | <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#alr_Ec_control1" target="_blank">alr_Ec_control1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#alr_Ec_control2" target="_blank">alr_Ec_control2</a>) | ||
| + | |||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (3004 bp)</li> | ||
| + | </ul> | ||
| + | </li> | ||
| + | |||
| + | <li> | ||
| + | Resulting in the genotype KRX <i>kan:alr</i>, DH5aplha <i>kan:alr</i> repsectivly. | ||
| + | </li> | ||
| + | </ul> | ||
</div> | </div> | ||
</div> | </div> | ||
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| - | <div id="text"> | + | <div id="text" style="text-align:left"> |
<div class="tab" id="Week5"> | <div class="tab" id="Week5"> | ||
<div class="show"> | <div class="show"> | ||
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<div class="hide"> | <div class="hide"> | ||
| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 5 06/30 - 07/06</a></h6> |
</div> | </div> | ||
| - | <div class="content"> | + | <div class="content" style="margin-right:10%; margin-left:10%"> |
| + | <ul> | ||
| + | <li> | ||
| + | The successfull deletion of the chromosomal kanamycin resistance was established by the plasmid Flp705 as described in the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge RedET-System protocol</a>.<br> | ||
| + | The plasmid is removed by a temperature shift and the removal of the kanamycin resistance is verified by streaking colonies in parallel on LB-kanamycin and LB agar. | ||
| + | </li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li> | ||
| + | Resulting in the genotype KRX <i>∆ alr</i>, DH5aplha <i>∆ alr</i> repsectivly. | ||
| + | </li> | ||
| + | </ul> | ||
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 02:36, 9 October 2014
June |
 |
-
Design of the Primer for the deletion of the whole coding sequence of the constitutive alanine racemase (alr) and the catabolic alanine racemase (dadX) from E. coli using the Genebridge Red/ET-System.
- alr: BBa_K1465403 and BBa_K1465404
- dadX: BBa_K1465405 and BBa_K1465406
- Design of the Primer for the integration of the konstitutive alanine racemase alr into the pSB1C3-Backbone.
- Transformation of the E. coli strains KRX (Promega) and DH5alpha (Invitrogen) with the plasmid pRedET containg the Recombinase using the Genebridge RedET-System protocol.
- Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of alr using the primer BBa_K1465403 and BBa_K1465404
- Purification using the gel extraction clean-up kit from Promega.
-
Transformation of the oligonucleotide containing the flanking sites for the deletion of alr. The successful replacement of the constitutive alanine racemase was verified via kanamycin selection and
Colony PCR (alr_Ec_control1, alr_Ec_control2)
- Annealing temperature: 55 °C
- Bands as expected (3004 bp)
- Resulting in the genotype KRX kan:alr, DH5aplha kan:alr repsectivly.
-
The successfull deletion of the chromosomal kanamycin resistance was established by the plasmid Flp705 as described in the Genebridge RedET-System protocol.
The plasmid is removed by a temperature shift and the removal of the kanamycin resistance is verified by streaking colonies in parallel on LB-kanamycin and LB agar.
- Resulting in the genotype KRX ∆ alr, DH5aplha ∆ alr repsectivly.
