Difference between revisions of "Team:HSHL/Design"

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<h1>Design</h1>
 
<h1>Design</h1>
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The HMA3 Sequence is with 2619 bp quite long, so we decided to get it synthesized by IDT.
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First option was to use the whole sequence (2631 bp). CAMV35S is used as promoter with 341 bp.  
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<div class="column two_thirds_size">
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<h3>1st idea <br>- Working with the whole sequence</h3>
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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 <a href="https://eu.idtdna.com/pages"  target="_blank">IDT</a>.
 
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<img src="https://static.igem.org/mediawiki/2018/c/c9/T--HSHL--design-biobrick.png">
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<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>
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 +
<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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    <img src="https://static.igem.org/mediawiki/2018/c/c9/T--HSHL--design-biobrick.png">
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    <p></p>
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    <a href="https://static.igem.org/mediawiki/2018/c/c9/T--HSHL--design-biobrick.png" target="_blank"><i class="fas fa-external-link-alt"></i></a>
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<div class="column two_thirds_size">
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<table>
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  <tr>
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      <th> Sequence </th>
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      <th> Length [bp] </th>
 +
      <th> Color</th> 
 +
  </tr>
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  <tr>
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      <td> CaMV 35S promoter</td>
 +
      <td> 345</td>
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      <td> letters</td>
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  </tr>
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  <tr>
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      <td> AhHMA3 cDNA</td>
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      <td> 2274</td>
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      <td> <span class="highlightGreen">highlighted</span></td>
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  </tr> 
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  <tr>
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      <td> Restriction site of EcoRI (G/AATTC)</td>
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      <td> 6 </td>
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      <td> <span class="highlightYellow">highlighted</span></td>
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  </tr> 
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  <tr>
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      <td> restriction site of PstI (CTGCA/G)</td>
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      <td> 6 </td>
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      <td> <span class="highlightLightBlue">highlighted</span></td>
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  </tr> 
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  <tr>
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      <td><b>whole sequence</b> </td>
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      <td><b>2613</b></td>
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      <td>  </td>
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  </tr>
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</table>
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<h3>Promoter and gene sequence</h3>
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<p> In the following the whole promoter and gene sequence including the restriction sites to ligate into the vector pSB1C3 is shown.
 +
<br><span class="highlightDarkBlue">GA<span class="highlightRed">G</span>TTC</span> = changed codon to make sure that EcoRI will not cut several times</p>
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<img src="https://static.igem.org/mediawiki/2018/1/15/T--HSHL--img_WholeGeneSequence.png">
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<h3>2nd idea <br>- Separating the sequence</h3>
 +
<p> The second option is to separate our whole promoter and gene sequence into 4 fragment parts. You can see the parts in the table to the right-hand side. </p>
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</div>
  
 
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<div class="column two_thirds_size">
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<h3>What should this page contain?</h3>
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<table>
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<ul>
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  <tr>
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<li>Explanation of the engineering principles your team used in your design</li>
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      <td colspan="2"></td>  
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<li>Discussion of the design iterations your team went through</li>
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      <td colspan="4"> <center> Restriction site at the </center></td> 
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<li>Experimental plan to test your designs</li>
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  </tr>
−
</ul>
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  <tr>
 +
      <th> Sequence </th>
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      <th> Length [bp]</th>
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      <th> beginning of the sequence</th>  
 +
      <th> end of the sequence</th>  
 +
  </tr>
 +
  <tr>
 +
      <td> IDT <br> 1. Part sequence</td>
 +
      <td> 614</td>
 +
      <td> EcorRI: G/AATTC</td>
 +
      <td> HindIII: A/AGCTT</td>
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  </tr>
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  <tr>
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      <td> IDT <br> 2. Part sequence</td>
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      <td> 593</td>
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      <td> HindIII: A/AGCTT</td>
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      <td> SmaI: CCC/GGG; blunt end</td>
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  </tr>
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  <tr>
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      <td> IDT <br> 3. Part sequence</td>
 +
      <td> 556</td>
 +
      <td> SmaI: CCC/GGG; blunt end</td>
 +
      <td> EcoRII: -/CCWGG; W=A/T</td>
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  </tr>
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  <tr>
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      <td> IDT <br> 4. Part sequence</td>
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      <td> 885</td>
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      <td> EcoRII: -/CCWGG; W=A/T</td>
 +
      <td> PstI: CTGCA/G</td>
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  </tr>
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  <tr>
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      <td> Total length of the synthesized fragments</td>
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      <td> 2648</td>
 +
      <td> </td>
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      <td> </td>
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  </tr>
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  <tr>
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      <td> Length of the sequence after the ligation</td>
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      <td> 2613 </td>
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      <td> </td>
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      <td> </td>
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  </tr>
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</table>
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</p>
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</div>
  
 +
<div class="clear"></div>
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<div class="column full_size" >
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<p>The following part shows the 4 fragments, which were synthesized by the IDT. The black letters indicates the promoter sequence and the green letters the HMA3 gene sequence. The restriction sites for each restriction enzyme are highlighted in different colors to clearly differentiate inbetween them. To ligate these fragments into the vector pSB1C3, the first fragment needs to have the restriction site of EcoRI at the beginning as well as the fourth fragment needs to end with the restriction site of PstI. For this sequence design, we also have to changed a few codons to make sure that EcoRI and HindIII will not cut several times in the sequence.
 +
All four fragments will bind together because of the different restriction sites. For example, the end of the first fragment and the beginning of the second fragment have the same restriction site. This means only one end fits the other end.
 +
</p>
 
</div>
 
</div>
  
 
<div class="column third_size">
 
<div class="column third_size">
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<div class="highlight decoration_A_full">
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<p><b> 1st fragment</b></p>
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<h3>Inspiration</h3>
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<p>Length: 614 bp</p>
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<ul>
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<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
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<p><b>Restriction enzymes</b><br>
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<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
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      EcoRI: <span class="highlightYellow">G/AATTC</span> <br>
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<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
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      HindIII: <span class="highlightRed">A/AGCTT </span> <br>
−
</ul>
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      <span class="highlightDarkBlue">GA<span class="highlightRed">G</span>TTC</span> = changed codon to make sure that EcoRI will not cut several times
 +
</p>
 
</div>
 
</div>
 +
 +
<div class="column two_thirds_size">
 +
<img src="https://static.igem.org/mediawiki/2018/6/6a/T--HSHL--fragment01.png">
 +
</div>
 +
<div class="clear"></div>
 +
<div class="column third_size">
 +
<p><b>2nd fragment</b></p>
 +
<p>Length: 593 bp</p>
 +
<p><b>Restriction enzymes</b><br>
 +
      HindIII: <span class="highlightRed">A/AGCTT </span> <br>
 +
      SmaI: <span class="highlightGreen">CCC/GGG</span>; blunt end
 +
</p>
 +
</div>
 +
 +
<div class="column two_thirds_size">
 +
<img src="https://static.igem.org/mediawiki/2018/a/a3/T--HSHL--fragment02.png">
 +
</div>
 +
 +
<div class="clear"></div>
 +
 +
<div class="column third_size">
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<p><b>3rd fragment</b></p>
 +
<p>Length: 556 bp</p>
 +
 +
<p><b>Restriction enzymes</b><br>
 +
      SmaI: <span class="highlightGreen">CCC/GGG</span>; blunt end <br>
 +
      EcoRII: <span class="highlightPurple">-/CCWGG</span>; W=A/T <br>
 +
      <span class="highlightGrey">AAGC</span><span class="highlightRed">T</span> = changed codon at the restriction site to make sure that HindIII will not cut several times
 +
</p>
 +
</div>
 +
 +
<div class="column two_thirds_size">
 +
<img src="https://static.igem.org/mediawiki/2018/4/49/T--HSHL--fragment03.png">
 +
</div>
 +
 +
<div class="clear"></div>
 +
 +
<div class="column third_size">
 +
<p><b>4th fragment</b></p>
 +
<p>Length: 885 bp</p>
 +
 +
<p><b>Restriction enzymes</b><br>
 +
      EcoRII: <span class="highlightPurple">-/CCWGG</span>; W=A/T <br>
 +
      PstI: <span class="highlightLightBlue">CTGCA/G </span>
 +
</p>
 +
</div>
 +
 +
<div class="column two_thirds_size">
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<img src="https://static.igem.org/mediawiki/2018/b/b5/T--HSHL--fragment04.png">
 
</div>
 
</div>
  

Latest revision as of 20:42, 17 October 2018

HOME
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JUDGING FORM ⇗

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 345 letters
AhHMA3 cDNA 2274 highlighted
Restriction site of EcoRI (G/AATTC) 6 highlighted
restriction site of PstI (CTGCA/G) 6 highlighted
whole sequence 2613

Promoter and gene sequence

In the following the whole promoter and gene sequence including the restriction sites to ligate into the vector pSB1C3 is shown.
GAGTTC = changed codon to make sure that EcoRI will not cut several times

2nd idea
- Separating the sequence

The second option is to separate our whole promoter and gene sequence into 4 fragment parts. You can see the parts in the table to the right-hand side.

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 HindIII: A/AGCTT SmaI: CCC/GGG; blunt end
IDT
3. Part sequence		 556 SmaI: CCC/GGG; blunt end EcoRII: -/CCWGG; W=A/T
IDT
4. Part sequence		 885 EcoRII: -/CCWGG; W=A/T PstI: CTGCA/G
Total length of the synthesized fragments 2648
Length of the sequence after the ligation 2613

The following part shows the 4 fragments, which were synthesized by the IDT. The black letters indicates the promoter sequence and the green letters the HMA3 gene sequence. The restriction sites for each restriction enzyme are highlighted in different colors to clearly differentiate inbetween them. To ligate these fragments into the vector pSB1C3, the first fragment needs to have the restriction site of EcoRI at the beginning as well as the fourth fragment needs to end with the restriction site of PstI. For this sequence design, we also have to changed a few codons to make sure that EcoRI and HindIII will not cut several times in the sequence. All four fragments will bind together because of the different restriction sites. For example, the end of the first fragment and the beginning of the second fragment have the same restriction site. This means only one end fits the other end.

1st fragment

Length: 614 bp

Restriction enzymes
EcoRI: G/AATTC
HindIII: A/AGCTT
GAGTTC = changed codon to make sure that EcoRI will not cut several times

2nd fragment

Length: 593 bp

Restriction enzymes
HindIII: A/AGCTT
SmaI: CCC/GGG; blunt end

3rd fragment

Length: 556 bp

Restriction enzymes
SmaI: CCC/GGG; blunt end
EcoRII: -/CCWGG; W=A/T
AAGCT = changed codon at the restriction site to make sure that HindIII will not cut several times

4th fragment

Length: 885 bp

Restriction enzymes
EcoRII: -/CCWGG; W=A/T
PstI: CTGCA/G

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