Team:Bielefeld-CeBiTec/Notebook/Journal/Biosafety/Jun

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<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>
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                <a href="#Week1">Week 1 &nbsp;&nbsp;&nbsp; 06/02 - 06/08</a>
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                 <h6><a  style="font-size:24px" href="#">Week 1 &nbsp;&nbsp;&nbsp; 06/02 - 06/08</a></h6>
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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>.
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                <a href="#Week2">Week 2 &nbsp;&nbsp;&nbsp; 06/09 - 06/15</a>
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                 <h6><a  style="font-size:24px" href="#">Week 2 &nbsp;&nbsp;&nbsp; 06/09 - 06/15</a></h6>
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<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.
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                <a href="#Week3">Week 3 &nbsp;&nbsp;&nbsp; 06/16 - 06/22</a>
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                 <h6><a  style="font-size:24px" href="#">Week 3 &nbsp;&nbsp;&nbsp; 06/16 - 06/22</a></h6>
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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>.
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                <a href="#Week4">Week 4 &nbsp;&nbsp;&nbsp; 06/23 - 06/29</a>
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                 <h6><a  style="font-size:24px" href="#">Week 4 &nbsp;&nbsp;&nbsp; 06/23 - 06/29</a></h6>
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Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of <i>alr</i> and 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.
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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>)

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		<li>Annealing temperature: 55 °C</li>
		<li>Bands as expected (3004 bp)</li>
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Resulting in the genotype KRX <i>kan:alr</i>, DH5aplha <i>kan:alr</i> repsectivly.
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                <a href="#Week5">Week 5 &nbsp;&nbsp;&nbsp; 06/30 - 07/06</a>
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                 <h6><a  style="font-size:24px" href="#">Week 5 06/30 - 07/06</a></h6>
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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>.
</li>. 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. 
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Resulting in the genotype KRX <i>∆ alr</i>, DH5aplha <i>∆ alr</i> repsectivly.
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