Difference between revisions of "Part:BBa K1640019"

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ChlH is the catalytic subunit of Magnesium chelatase. This oligomeric enzyme initiates the first committed step of the chlorophyll-a biosynthesis pathway via insertion of an Mg2+ ion into protoporphyrin IX to generate Mg-protoporphyrin IX. Specifically, ChlH is the subunit known to bind porphyrin, and potentially also the Mg2+ ion. During this process, ChlH interacts with two AAA ATPase-like subunits of Mg-chelatase (ChlI and ChlD) to catalyse the ATP-dependent insertion of Mg2+ into protoporphyrin IX (Adhikari <i>et al</i>., 2011).
 
ChlH is the catalytic subunit of Magnesium chelatase. This oligomeric enzyme initiates the first committed step of the chlorophyll-a biosynthesis pathway via insertion of an Mg2+ ion into protoporphyrin IX to generate Mg-protoporphyrin IX. Specifically, ChlH is the subunit known to bind porphyrin, and potentially also the Mg2+ ion. During this process, ChlH interacts with two AAA ATPase-like subunits of Mg-chelatase (ChlI and ChlD) to catalyse the ATP-dependent insertion of Mg2+ into protoporphyrin IX (Adhikari <i>et al</i>., 2011).
 
</p>
 
</p>
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<p>The 4166 bp ChlH gene was engineered synthetically by Integrated DNA Technologies (IDT) in 3 gene blocks (Table 1). The original gene sequence was taken from <i>Chlamydomonas Reinhardtii</i> and subsequently codon optimized for expression in <i>Escherichia coli.</i> Integrity of the protein sequence was closely maintained throughout this optimisation process, but translation of the original clone and the synthesised sequences has revealed one mutation (‘E’ → ‘D’; ‘GAG’ → ‘GAT’). </p>
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<h6><i>Table 1: Gene blocks</i></h6>
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<table border="1" style="width:40%">
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  <tr>
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    <td>1(G13)</td>
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    <td>1678 bp</td>
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      </tr>
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  <tr>
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    <td>2 (P2)</td>
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    <td>980 bp</td>
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  </tr>
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  <tr>
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    <td>3 (3-6)</td>
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    <td>1508 bp</td>
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  </tr>
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</table>
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<br>
 +
<!--- this is a table that shows the gene blocks and their lengths--->
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 +
<p>ChlH and the pSB1C3_001 KAN plasmid were successfully assembled in two parts.</p>
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<ul>
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<p>1. Assembled G13, the CAM vector and 3 - 6 via double restriction digest with EcoRI and EcoRI + PstI and ligation reaction</p>
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</ul>
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<p>2. Cloned P2 into the vector with the other parts via Gibson Assembly and then performed a restriction digest (EcoRI and EcoRI + PstI) on the assembly product to check for correct assembly (Figure 1).</p>
  
 
===Protein information===
 
===Protein information===

Revision as of 01:06, 19 September 2015

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 856
    Illegal BglII site found at 1606
    Illegal BglII site found at 2229
    Illegal BglII site found at 2308
    Illegal BglII site found at 3615
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1150
    Illegal AgeI site found at 35
    Illegal AgeI site found at 65
    Illegal AgeI site found at 959
    Illegal AgeI site found at 1127
    Illegal AgeI site found at 2645
    Illegal AgeI site found at 2699
    Illegal AgeI site found at 2918
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3044
    Illegal SapI.rc site found at 401
    Illegal SapI.rc site found at 2974


Overview

Biology & Literature

ChlH is the catalytic subunit of Magnesium chelatase. This oligomeric enzyme initiates the first committed step of the chlorophyll-a biosynthesis pathway via insertion of an Mg2+ ion into protoporphyrin IX to generate Mg-protoporphyrin IX. Specifically, ChlH is the subunit known to bind porphyrin, and potentially also the Mg2+ ion. During this process, ChlH interacts with two AAA ATPase-like subunits of Mg-chelatase (ChlI and ChlD) to catalyse the ATP-dependent insertion of Mg2+ into protoporphyrin IX (Adhikari et al., 2011).

The 4166 bp ChlH gene was engineered synthetically by Integrated DNA Technologies (IDT) in 3 gene blocks (Table 1). The original gene sequence was taken from Chlamydomonas Reinhardtii and subsequently codon optimized for expression in Escherichia coli. Integrity of the protein sequence was closely maintained throughout this optimisation process, but translation of the original clone and the synthesised sequences has revealed one mutation (‘E’ → ‘D’; ‘GAG’ → ‘GAT’).

Table 1: Gene blocks
1(G13) 1678 bp
2 (P2) 980 bp
3 (3-6) 1508 bp


ChlH and the pSB1C3_001 KAN plasmid were successfully assembled in two parts.

    1. Assembled G13, the CAM vector and 3 - 6 via double restriction digest with EcoRI and EcoRI + PstI and ligation reaction

2. Cloned P2 into the vector with the other parts via Gibson Assembly and then performed a restriction digest (EcoRI and EcoRI + PstI) on the assembly product to check for correct assembly (Figure 1).

Protein information

References