Difference between revisions of "Part:BBa K1640019"

Latest revision as of 03:35, 21 October 2016

ChlH

Sequence and Features

Overview

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

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

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: 2). 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

Visualisation
The following gel image shows the expected ChlH banding in lanes 2 and 3, comprising of a EcoRI digest and EcoRI + PstI double digest respectively.

Part update by 2016 Macquarie Australia iGem team

The 2016 Macquarie Australia iGem team added a promoter sequence for the ChlH gene. Previous parts containing the ChlH gene have used the Tac promoter (pTac promoter). Instead, by adding the lactose promoter (pLac promoter), we are able to control the expression of the ChlH gene through the presence of IPTG, a lactose analog, in the growth media. This has allowed us to transform this regulatory enzyme, involved in cell signalling, into E.Coli to express protoporphyrin IX, an intermediate of the chlorophyll biosynthesis pathway (pathway shown below). We have used this part to build our part to build another part this year (https://parts.igem.org/Part:BBa_K1998000) and have been able to prove and characterise it's function in our experiments (http://2016.igem.org/Team:Macquarie_Australia/Proof). Future applications of this part include the expression of the chlorophyll biosynthesis pathway in non-photosynthetic organisms but also as a key regulatory part for photosynthetic energy projects.

Part confirmation
The following gel image shows a ladder in lane 1, the ChlH gene by itself in lane 2 and the plac-ChlH sequence in lanes 3-10. Lane 3 shows a slightly smaller band then the others and was excluded from sequencing results. Lanes 4-10 were confirmed as correct through sequencing. The lac promoter has a 200 base pair sequence which was added to the start of the gene and taken from iGem part BBa_R0010.

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.