Difference between revisions of "Part:BBa K2664005"

 
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<partinfo>BBa_K2664005 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2664005 SequenceAndFeatures</partinfo>
  
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===Overview===
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<html><center><img src=https://static.igem.org/mediawiki/parts/6/6a/CHLH.PNG></center></html>
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<center>Figure:1  The protein structure of ChlH.
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<br>Source: Chen et al., 2015. </center>
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When induced, the synthetic operon of which this ChlH part is a component will enable ''Escherichia coli'' to generate Mg-protoporphyrin IX as a first step of the chlorophyll a biosynthesis pathway.
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This part comprises a subunit of the enzyme Magnesium chelatase, catalysing the first step of the chlorophyll-a biosynthesis pathway. After combining this composite part (ChlH + GUN4) with three additional synthetically engineered operons [(2 with Chli, ChlD), (3 with ChlM,  CTH1, plasto, and YCF54), and (4 with POR, ChlP, DVR1, and ChlG)] and transfecting into ''E. coli'', the bacterial cells should express the functional ''C. reinhardtii''-derived chlorophyll synthesis complex.
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===Biology & Literature===
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<p>
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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).
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</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>
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<!--- 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>
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(added trc)
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 03:35, 14 October 2018


trc-ChlH

trc-ChlH

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 1
    Illegal BglII site found at 2001
    Illegal BglII site found at 2307
    Illegal BglII site found at 3693
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 559
    Illegal NgoMIV site found at 2225
    Illegal AgeI site found at 1205
    Illegal AgeI site found at 2723
    Illegal AgeI site found at 2777
    Illegal AgeI site found at 2996
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2327
    Illegal BsaI.rc site found at 3122
    Illegal SapI.rc site found at 3052

Overview

Figure:1 The protein structure of ChlH.
Source: Chen et al., 2015.

When induced, the synthetic operon of which this ChlH part is a component will enable Escherichia coli to generate Mg-protoporphyrin IX as a first step of the chlorophyll a biosynthesis pathway.

This part comprises a subunit of the enzyme Magnesium chelatase, catalysing the first step of the chlorophyll-a biosynthesis pathway. After combining this composite part (ChlH + GUN4) with three additional synthetically engineered operons [(2 with Chli, ChlD), (3 with ChlM, CTH1, plasto, and YCF54), and (4 with POR, ChlP, DVR1, and ChlG)] and transfecting into E. coli, the bacterial cells should express the functional C. reinhardtii-derived chlorophyll synthesis complex.

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).

(added trc)