Team:ULB-Brussels/mat

From 2011.igem.org

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<h1>Strain:</h1>
 
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<h2>E. coli strain:<u></u></h2>
 
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<ul>
 
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  <li>MC1061&nbsp;: <em>F−&nbsp;araD139, Δ(ara-leu)7696,galE15, galK16, Δ(lac)X74, rpsL (Strr), hsdR2 (rK−mK+), mcrA mcrB1</em></li>
 
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  <li>DG1(Delphi genetics):&nbsp;<em>mcrA&nbsp;Δ (mrr-hsdRMS-mcrBC, modification-, <br />
 
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    restriction-)&nbsp;F80lacZDM15&nbsp;Δ lacX74 recA1 araD139&nbsp;Δ (ara-leu)7697 galU galK rpsL endA1 nupG</em></li>
 
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  <li>TOP10 (Invitrogen)&nbsp;: <em>F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-</em></li>
 
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  <li>MG1655&nbsp;: <em>F-&nbsp;λ-&nbsp;ilvG- rfb-50 rph-1</em></li>
 
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  <li>MG1655 <em>ΔtldD::frt-cm-frt</em></li>
 
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</ul>
 
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<h2>Saccharomyces cerevisae strain:</h2>
 
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<ul>
 
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  <li>23344C&nbsp;:<em>ura3</em></li>
 
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</ul>
 
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<h1>Plasmid:</h1>
 
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<p>pCP20:<br />
 
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pCP20 has the Flp recombinase gene repressed by CI857ts which is not functional at a temperature higher or equal to 42°C<strong></strong></p>
 
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<h2>pFL44S :</h2>
 
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<p>pFL44S is a shuttle plasmid between yeast and bacteria. It has the ampicillin resistance gene and the bacterial origin of replication colE1. For selection in yeast it has the <em>ura3</em> gene. It has also the 2micron yeast origin of replication. </p>
 
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<h2>pKD46:</h2>
 
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<p>pKD46 expresses the Red system under control of the well-regulated promoter pBAD (induced by arabinose and repressed by glucose). The replication origin of the plasmid is also thermo sensitive. It is not replicated at a temperature higher or equal to 42°C.</p>
 
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<h2>pSB1A3:</h2>
 
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<p>pSB1A3 is one of the standard iGEM plasmid which has a resistance gene to ampicillin. It is composed of four unique restriction sites:</p>
 
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<ul>
 
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  <li>EcoRI (prefix)</li>
 
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  <li>XbaI (prefix)</li>
 
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  <li>SpeI (suffix)</li>
 
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  <li>PstI (suffix)</li>
 
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</ul>
 
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<h2>PCR Topo® XL plasmid (Invitrogen)</h2>
 
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<p>This plasmid is used in the Topo® XL PCR Cloning Kit. For the cloning the plasmid is linearized and topoisomerase l is activated. The vector includes the following features:</p>
 
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<ul>
 
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  <li><em>ccdB</em> gene for positive selection</li>
 
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  <li>Kanamycin and Zeocin™ resistance genes</li>
 
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  <li>EcoRI sites flanking the PCR product&nbsp;insertion site for easy excision of inserts</li>
 
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  <li>M13 forward&nbsp;and reverse primer sites for sequencing</li>
 
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</ul>
 
<h1>Primers</h1>
<h1>Primers</h1>
<p>The following primers were produced by Sigma-Aldrich (option desalt)</p>
<p>The following primers were produced by Sigma-Aldrich (option desalt)</p>
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<p>In green is the frt’ sequence which is not homologous with the chloramphenicol gene.</p>
<p>In green is the frt’ sequence which is not homologous with the chloramphenicol gene.</p>
<ul>
<ul>
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   <li><strong>topo-frt’-cm-frt’-for:</strong> <u>tccggcaaaaaagggcaag</u>gtgtcaccaccctgcccttttCgccagtgtgctggatttc</li>
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   <li><strong>topo-frt’-cm-frt’-for:</strong> tccggcaaaaaagggcaaggtgtcaccaccctgcccttttCgccagtgtgctggatttc</li>
   <li><strong>topo-frt’-cm-frt’-rev:</strong> TGCAGCGGCCGCTACTAGTACTCTAGAAGCGGCCGCGAATGATGGATATCTGCAGATTTC </li>
   <li><strong>topo-frt’-cm-frt’-rev:</strong> TGCAGCGGCCGCTACTAGTACTCTAGAAGCGGCCGCGAATGATGGATATCTGCAGATTTC </li>
</ul>
</ul>
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<h2>Biobrick frt’-cm-frt’ sequencing (primer M13 as described in Topo® XL PCR Kit)</h2>
<h2>Biobrick frt’-cm-frt’ sequencing (primer M13 as described in Topo® XL PCR Kit)</h2>
<ul>
<ul>
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   <li><strong>FRT’-cm-FRT’-SEQ-FOR: 5’-</strong>Gaggaaacagctatga-3’</li>
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   <li><strong>FRT’-cm-FRT’-SEQ-FOR: </strong></li>
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   <li><strong>FRT’-cm-FRT’-SEQ-REV:</strong> 5’-gaccggcagcaaatg-3’<strong></strong></li>
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</ul>
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<p>5’-Gaggaaacagctatga-3’</p>
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<ul>
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   <li><strong>FRT’-cm-FRT’-SEQ-REV:</strong></li>
</ul>
</ul>
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<p>&nbsp;</p>
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<p> 5’-gaccggcagcaaatg-3’<strong></strong></p>
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<p>&nbsp;</p>
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Revision as of 12:58, 21 September 2011

Materials & Method: page1

Primers

The following primers were produced by Sigma-Aldrich (option desalt)

Yeast cloning:

  • PKD46-FOR:

5’-  gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggctgccacctgcatcgatttat-3’

  • PKD46-REV:

5’-cgtgagttttcgttccactgagcgtcagaccccgtagaaagagttttcgttccactgagc-3’

  • PFL-FOR:

5’-Gcctttttacggttcctggc-3’

  • PFL-REV:

5’-tttctacgggggtctgacgc-3’

  • PCP-FOR:

5’-tggctcttgtatctatcagtgaagcatcaagactaacaaatcagccaaacgtctcttcag-3’

  • PCP-REV:

5’-ggggctgtatgcacaaagcatcttctgttgagttaagaacttatatgcgtctatttatgtagg-3’
In green is the 40 homologous nucleotides need for the yeast cloning.

Yeast cloning verification:

  • FLP/CI-FOR:

5’-Acatggcgagttttgacgag-3’

  • FLP/CI-REV:

5’-accacactagagaacatactg-3’

pINDEL sequencing:

  • pID-seq1:

5’-aggatcttcacctagatcctt-3’

  • pID-seq2:

 5’-gatgggctagtcaatgataatta-3’

  • pID-seq3:

5’-ccgttacgtaggtaggaatc-3’

  • pID-seq4:

5’-agatggggatggggcagtc-3’

  • pID-seq5:

5’-gatttcggatcaacgttcttaat-3’

  • pID-seq6:

5’-caatcactttcgtctactcc-3’

  • pID-seq7:

 5’-ccagatatttcgccgcgac-3’

  • pID-seq8:

5’-cggggccagcaaaaaatcca-3’

  • pID-seq9:

5’-ccctgatttttcaccacccc-3’

  • pID-seq10:

5’-cttccgaaaatgcaacgcga-3’

Biobrick frt’-cm-frt’

  • frt’-cm-frt’-for: 5’-Gaagttcctatactttttagagaataggaacttcgttgatcgggcacgtaagagg-3’
  • frt’-cm-frt’-rev :5’-GAAGTTCCTATTCTCTAAAAAGTATAGGAACTTCTTATTACGCCCCGCCCTGCC-3’

In green is the frt’ sequence which is not homologous with the chloramphenicol gene.

  • topo-frt’-cm-frt’-for: tccggcaaaaaagggcaaggtgtcaccaccctgcccttttCgccagtgtgctggatttc
  • topo-frt’-cm-frt’-rev: TGCAGCGGCCGCTACTAGTACTCTAGAAGCGGCCGCGAATGATGGATATCTGCAGATTTC

In red is the iGEM restriction sites (prefix and suffix) and in green is the mutation made to suppress the EcoRI restriction site (in the Topo® XL PCR plasmid).

Biobrick frt’-cm-frt’ sequencing (primer M13 as described in Topo® XL PCR Kit)

  • FRT’-cm-FRT’-SEQ-FOR:

5’-Gaggaaacagctatga-3’

  • FRT’-cm-FRT’-SEQ-REV:

 5’-gaccggcagcaaatg-3’

iGEM ULB Brussels Team - Contact us

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