Part:BBa_K2664005
trc-ChlH
ChlH with high expression Trc promoter.
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.
Protein Expression - Increasing transcription of the ChlH gene would improve the efficiency of chlorophyll production. As such in 2018, we decided not to use many of the parts assembled by previous Macquarie teams. We created the second iteration of our design, in which a trc protomoter was placed before the genes. Modelling this promoter swap based on gene expression data obtained by Tegel, Ottosson, and Hober (2011), we were confident our improved plasmid would be more efficient at producing chlorophyll.
Part Verification
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).
Biobrick Design:
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.
In 2018 team Macquarie changed the lac promoter of the part to a higher expression Trc promoter. The promoter was changed to Trc from lac using standard assembly from another part containing the promoter.
Protein information
Magnesium chelatase sits at the branch point of the common tetrapyrrole pathway and inserts Mg2+ into Proto to produce Mg-Proto, the first unique intermediate of the chlorophyll biosynthetic pathway. It is known that the BchH/ChlH subunit binds the substrate and, for this reason, is thought to be the catalytic component of the enzyme. The ChlH subunit makes conformational changes upon binding its porphyrin substrate.
A study done on Rhodobacter capsulatus has demonstrated the apo structure to contain three major lobe-shaped domains connected at a single point, with additional densities at the tip of two lobes termed the “thumb” and “finger” (figure: 1). This independent reconstruction of a substrate-bound ChlH complex permitted insight into substrate-induced conformational changes (Sirijovski et al., 2008).
Number of amino acids: 1378
Molecular weight: 152265.2
References
Adhikari, N.D., Froehlich, J.E., Strand, D.D., Buck, S.M., Kramer, D.M., Larkin, R.M. (2011) GUN4-Porphyrin Complexes Bind the ChlH/GUN5 Subunit of Mg-Chelatase and Promote Chlorophyll Biosynthesis in Arabidopsis. Plant Cell 23: 1449-1467.
Chen, X., Pu, H., Fang, Y., Wang, X., Zhao, S., Lin, Y., Zhang, M., Dai, H-E., Gong, W., Liu, L. (2015). Crystal Structure of the catalytic subunit of magnesium chelatase. Nature Plants, 1, doi:10.1038/nplants.2015.125.
Sirijovski, N., Lunqvist, J., Rosenback, M., Elmlund, H., Al-Karadaghi, S., Willows, R.D., Hansson, M. (2008). Substrate-binding Model of the Chlorophyll Biosynthetic Magnesium Chelatase BchH Subunit. Journal of Biological Chemistry, 283, 11652-11660.
Tegel, H., Ottosson, J. and Hober, S., 2011. Enhancing the protein production levels in Escherichia coli with a strong promoter. The FEBS journal, 278(5), pp.729-739.
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