Difference between revisions of "Part:BBa K2664005"
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<partinfo>BBa_K2664005 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2664005 SequenceAndFeatures</partinfo> | ||
+ | ===Overview=== | ||
+ | <html><center><img src=https://static.igem.org/mediawiki/parts/6/6a/CHLH.PNG></center></html> | ||
+ | <center>Figure:1 The protein structure of ChlH. | ||
+ | <br>Source: Chen et al., 2015. </center> | ||
+ | |||
+ | 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=== | ||
+ | <p> | ||
+ | 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>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> | ||
+ | |||
+ | <h6><i>Table 1: Gene blocks</i></h6> | ||
+ | <table border="1" style="width:40%"> | ||
+ | <tr> | ||
+ | <td>1(G13)</td> | ||
+ | <td>1678 bp</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2 (P2)</td> | ||
+ | <td>980 bp</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3 (3-6)</td> | ||
+ | <td>1508 bp</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | <!--- this is a table that shows the gene blocks and their lengths---> | ||
+ | |||
+ | <p>ChlH and the pSB1C3_001 KAN plasmid were successfully assembled in two parts.</p> | ||
+ | <ul> | ||
+ | <p>1. Assembled G13, the CAM vector and 3 - 6 via double restriction digest with EcoRI and EcoRI + PstI and ligation reaction</p> | ||
+ | </ul> | ||
+ | |||
+ | <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> | ||
+ | (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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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
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)