Difference between revisions of "Team:HSHL/Design"

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<h1>Design</h1>
 
<h1>Design</h1>
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<p> The HMA3 Sequence is with 2619 bp quite big which make it a little bit difficult for us to actually isolate this sequence out of the <i>A. halleri</i>. That is the reason why we decided to get it synthesized by IDT.
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</div>
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 +
<div class="column third_size">
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<h3>1st idea - Working with the whole sequence</h3>
 +
<p> The HMA3 Sequence is with 2619 bp quite big which make it a little bit difficult for us to actually isolate this sequence out of the <i>A. halleri</i>. That is the reason why we decided to get it synthesized by <a href="https://eu.idtdna.com/pages"  target="_blank">IDT</a>.
 
</p>
 
</p>
  
 
<p>First option was to use the whole sequence (2631 bp). A CaMV 35S (universal promoter, <u>Ca</u>uliflower <u>M</u>osaic <u>V</u>irus) is used as a promoter with a length of 345 bp. </p>
 
<p>First option was to use the whole sequence (2631 bp). A CaMV 35S (universal promoter, <u>Ca</u>uliflower <u>M</u>osaic <u>V</u>irus) is used as a promoter with a length of 345 bp. </p>
  
−
<p>For our Biobrick we decided to use the Vector called pSB1C3 with a chloramphenicol resistance. By including the sequence of the restriction sites of EcoRI at the beginning and PstI at the end of our sequence, which are the same restriction sites for the plasmide, we can insert our sequence into our targeted vector. In our specific case EcoRI would cut about two times into our Gene HMA3 for example. To make sure the restriction enzyme will not cut several times into our sequence we had to change some codons in the original sequence of our Gene HMA3 (you can see the changes of the codons below in red). </p>
+
<p>For our Biobrick we decided to use the Vector called pSB1C3 with a chloramphenicol resistance. By including the sequence of the restriction sites of EcoRI at the beginning and PstI at the end of our sequence, which are the same restriction sites for the plasmide, we can insert our sequence into our targeted vector. In our specific case EcoRI would cut about two times into our Gene HMA3 for example. To make sure the restriction enzyme will not cut several times into our sequence we had to change some codons in the original sequence of our Gene HMA3 (you can see the changes of the codons below in red). </p>
 
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       <td> CaMV 35S promoter</td>  
 
       <td> CaMV 35S promoter</td>  
 
       <td> 245</td>
 
       <td> 245</td>
−
       <td> black</td>  
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       <td> letters</td>  
 
   </tr>  
 
   </tr>  
 
   <tr>  
 
   <tr>  
 
       <td> AhHMA3 cDNA</td>  
 
       <td> AhHMA3 cDNA</td>  
 
       <td> 2.274</td>
 
       <td> 2.274</td>
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       <td> <span class="highlightGreen">green</span></td>  
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       <td> <span class="highlightGreen">highlighted</span></td>  
 
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   <tr>  
 
   <tr>  
 
       <td> restriction site of EcoRI (G/AATTC)</td>  
 
       <td> restriction site of EcoRI (G/AATTC)</td>  
 
       <td> 6 </td>
 
       <td> 6 </td>
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       <td> <span class="highlightYellow">yellow</span></td>  
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       <td> <span class="highlightYellow">highlighted</span></td>  
 
   </tr>   
 
   </tr>   
 
   <tr>  
 
   <tr>  
 
       <td> restriction site of PstI (CTGCA/G)</td>  
 
       <td> restriction site of PstI (CTGCA/G)</td>  
 
       <td> 6 </td>
 
       <td> 6 </td>
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       <td> <span class="highlightLightBlue">light blue</span></td>  
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<h3>2nd idea - Separating the sequence into 4 parts</h3>
 +
<p> The second option is to separate our whole promoter and gene sequence in 4 fragment parts. You can see the parts in the table below.
 +
 +
 +
<table>
 +
  <tr>
 +
      <th> </th>
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      <th> Restriction site at the</th> 
 +
  </tr>
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  <tr>
 +
      <td>
 +
          <table>
 +
                <tr>
 +
                    <th> Sequence</th>
 +
                    <th> Length [bp] </th> 
 +
                </tr>
 +
          </table>
 +
      </td>
 +
      <td>
 +
          <table>
 +
                <tr>
 +
                    <th> beginning of the sequence</th>
 +
                    <th> end of the sequence</th> 
 +
                </tr>
 +
          </table>
 +
      </td>
 +
  </tr>
 +
<tr>
 +
      <td>
 +
          <table>
 +
                <tr>
 +
                    <th> IDT <br> 1. Part sequence</th>
 +
                    <th> 614 </th> 
 +
                </tr>
 +
          </table>
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      </td>
 +
      <td>
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          <table>
 +
                <tr>
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                    <th> EcorRI: G/AATTC</th>
 +
                    <th> HindIII: A/AGCTT</th> 
 +
                </tr>
 +
          </table>
 +
      </td>
 +
  </tr>
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<tr>
 +
      <td>
 +
          <table>
 +
                <tr>
 +
                    <th> IDT <br> 2. Part sequence</th>
 +
                    <th> 593</th> 
 +
                </tr>
 +
          </table>
 +
      </td>
 +
      <td>
 +
          <table>
 +
                <tr>
 +
                    <th> beginning of the sequence</th>
 +
                    <th> end of the sequence</th> 
 +
                </tr>
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          </table>
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      </td>
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  </tr> 
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</table>
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</p>
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Revision as of 07:38, 11 October 2018

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Design

1st idea - Working with the whole sequence

The HMA3 Sequence is with 2619 bp quite big which make it a little bit difficult for us to actually isolate this sequence out of the A. halleri. That is the reason why we decided to get it synthesized by IDT.

First option was to use the whole sequence (2631 bp). A CaMV 35S (universal promoter, Cauliflower Mosaic Virus) is used as a promoter with a length of 345 bp.

For our Biobrick we decided to use the Vector called pSB1C3 with a chloramphenicol resistance. By including the sequence of the restriction sites of EcoRI at the beginning and PstI at the end of our sequence, which are the same restriction sites for the plasmide, we can insert our sequence into our targeted vector. In our specific case EcoRI would cut about two times into our Gene HMA3 for example. To make sure the restriction enzyme will not cut several times into our sequence we had to change some codons in the original sequence of our Gene HMA3 (you can see the changes of the codons below in red).

sequence length [bp] color
CaMV 35S promoter 245 letters
AhHMA3 cDNA 2.274 highlighted
restriction site of EcoRI (G/AATTC) 6 highlighted
restriction site of PstI (CTGCA/G) 6 highlighted
whole sequence 2.613

The whole promoter and gene sequence including the restriction sites to ligate into the vector pSB1C3

GAGTTC = changed codon to make sure that EcoRI will not cut several times

GAATTCTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCA TCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGC ACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGAATGGCGGAAGGTGAAGAGGCCAAGAAGAAGAATTTACAGACAAGTTACTTCGACGTCGTTGGAATCTGCTGTACATCG GAGGTTTCTATCGTCGGTGACGTTCTCCGTCCACTTGACGGCGTCAAAGAGTTCTCCGTTATCGTCCCTTCTAGAACCGTCATCGTTGTCCATGACACTTTCTTGATTTCTCCGCTTCAAATCGTCAAGGCTCTGAATCAAGC AAGACTAGAAGCAAGTGTGAGACCATACGGAGAAACAAGCTTGAAGAGTCAATGGCCAAGTCCTTTTGCAATACTTTCTGGGGTATTTCTTGCTCTCTCCTTCTTCAAATACTTTTATAGTCTGCTTGAATGGCTCGCTGTTG TTGCCGTGGTGGCCGGGATTTTCCCCATCCTTGCTAAAGCTGTTGCTTCGGTCACAAGGTTCAGACTTGATATCAACGCTCTCACTTTTATTGCTGTGATAGCAACACTATGTATGCAGAATTTCACAGAAGCTGCCACAATT GTGTTTCTATTCTCAGTTGCAGATTGGCTAGAGTCTAGTGCTGCTCATAAGGCAAGCACAGTAATGTCATCACTGATGAGCTTAGCGCCACGAAAGGCAGTGATAGCGGAAACTGGACACGAAGTCGATGTAGATGAGGTTAG GATCAACACAATTGTTTCAGTGAAAGCTGGAGAAAGTATACCGATTGATGGAGTTGTGGTGGATGGAAGCTGTGATGTGGATGAGAAAACATTGACAGGAGAGTCATTCCCTGTCTCCAAACAGAGAGATTCAACTGTTTTGG CTGCAACCATAAATCTTAATGGTTATATAAAGGTGAAAACTACAGCTCTAGCCCGGGACTGCGTAGTCGCGAAAATGACTAAGCTTGTAGAAGAAGCTCAAAAAAGCCAAACCAAAACTCAAAGGTTTATAGATAAATGTTCT CGCTACTACACTCCAGCTGTTGTCGTGTTAGCAGCATGTTTTGCGGTGATCCCGGTATTGTTAAAGCTTCAGGACCTTAGCCATTGGTTTCACTTAGCACTTGTAGTGTTAGTAAGTGGTTGTCCATGTGGTCTTATCTTATC CACACCTATTGCTACCTTTTGTGCTCTCACTAAGGCAGCCATGTCGGGGTTTCTGATCAAAACTGGTGATTGTCTAGAGACTCTTGCAAAGATCAAGATTGTTGCTTTTGACAAAACAGGAACTATTACAAAGGCTGAGTTCA TGGTCTCGGATTTTAGGTCTCTTTCTCACAATATCAATCTGCACAACTTGCTTTACTGGGTCTCGAGCATTGAGAGCAAGTCAAGTCATCCGATGGCAGCGGCGCTTATTGACTATGCAAGATCAGTTTCTGTTGAGCCTAAG CCTGATCTCGTTGAGAACTTTCAAAACTTTCCAGGAGAAGGAGTTTATGGGAGAATAGATGGTCAAGATATCTACATTGGAAACAAAAGAATTGCACAGAGAGCTGGATGCTTAACAGTTCCGGATATGGAAGCTAATATGAA GCGAGGTAAGACCATTGGTTACATATACATTGGAGCAAAACTGTCCGGAAGTTTCAACCTTATTGACAGTTGTCGATATGGGGTTGCTCAAGCTCTCAAGGAGCTCAAGTCTTTAGGAATCAAAACTGCAATGCTCACAGGAG ATAACCGAGACGCAGCCCTGTCTACTCAAGAACAGTTAGAGAATGCTTTGGATATTGTTCACTCTGAACTCCTTCCACAAGACAAAGCAAGAATCATCGATGAGTTCAAGATCCAAGGGCCTACAATGATGGTAGGAGACGGG CTTAACGATGCACCGGCTTTAGCGAAAGCAGACATTGGCCTTTCAATGGGGATCTCAGGGTCAGCACTTGCAACAGAGACAGGAGACATCATTCTTATGTCAAACGATATAAGGAAGATCCCGAAAGGGATGAGACTAGCGAA GAGAAGTCATAAGAAAGTGATTGAGAATGTTGTTTTGTCTGTGAGCATAAAAGGAGCAATCATGGTTCTTGCTTTTGTAGGTTACCCTCTGGTTTGGGCAGCTGTACTTGCAGATGCAGGAACTTGTTTGCTTGTGATACTCA ATAGTATGATGCTTCTACGCGATGAGCGTGAAGCCGTGTCTACATGTTACCGTGCTTCTTCTTCGCCGGTGAAACTTGAGGAGGATGAAGCAGAGGATCTAGAAGTTGGCTTGTTGCAGAAGAGTGAGGAGACAAATAAAAAG AGTTGTTGCTCTGGTTCTTGTAGTGGCCCTAAGGACAATCAACAAAAGTGACTGCAG

2nd idea - Separating the sequence into 4 parts

The second option is to separate our whole promoter and gene sequence in 4 fragment parts. You can see the parts in the table below.

Restriction site at the
Sequence Length [bp]
beginning of the sequence end of the sequence
IDT
1. Part sequence		 614
EcorRI: G/AATTC HindIII: A/AGCTT
IDT
2. Part sequence		 593
beginning of the sequence end of the sequence

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