CRISPR and Magnesium Chelatase plasmid
In the chloroplast of photosynthetic organisms, the enzyme magnesium chelatase catalyses the conversion of protoporphyrin IX (PPIX) to Magnesium protoporphyrin IX – a precursor of chlorophyll a. The enzyme is a hexameric motor complex made up of 3 different proteins – ChlI1, ChlD and ChlI2. ChlH and GUN4 proteins assist the binding of PPIX to magnesium chelatase and in the presence of Mg2+ and ATP, Mg-PPIX is formed.
The assembly of our magnesium chelatase plasmid [BBa_K1998000] allows E. coli to produce the hexameric complex along with its two substrates GUN4 and ChlH. Since E. coli produces PPIX (as precursor of heme), it is theoretically possible to engineer E. coli to produce Mg-PPIX and subsequently induce the production of chlorophyll a with the MgPPIX - chlorophyll a plasmid [BBa_K1998013]. However, Mg-PPIX production would be limited in E. coli as it competes with the biosynthesis of heme. To overcome this, we have integrated CRISPR into our project to produce hemH deficient mutants of E. coli. By disrupting the hemH gene, there is subsequently more PPIX available for the magnesium chelatase plasmid to produce Mg-PPIX. The hemeH mutants would need to be grown in media containing glucose for survival.
Our team have successfully constructed the Magnesium chelatase plasmid and have also produced functional in vivo assays of the plasmid. Efforts to develop CRISPR-Cas9 Δheme mutants were successful. According to the literature, a fluorescences excitation and emission spectra should peaks at 418nm and 592nm respectively.
The assembly of this complex requires millimolar concentrations of Mg2+ and ATP to assemble. The substrate complex also requires micromolar concentrations of Protoporphyrin IX and ChlH for optimal activity.
Plasmid Confirmation and Functional Assay
1. The digests of Mg-chelatase plasmid reveal the correct band sizes of the insert (approx 11 kbp). ChlH protein (approx. 150kDa) is also highly expressed and visible on SDS-PAGE.
2. Mass spectrometry confirms the presence of magnesium chelatase subunits
Fig 1. Protein expression of the Mg-chelatase plasmid induced with IPTG. Lane 2 is the uninduced culture. Highly expressed band at approximately 144 kDa represents the ChlH protein. The MW of other proteins of interest include ChlI1 (40 kDa), ChlD (63 kDa), Gun4 (24 kDa), ChlI2 (40 kDa) and CTH1 (43 kDa).
Fig 2. Protein expression of the Mg-chelatase plasmid induced with IPTG. Lane 2 is the uninduced culture. Highly expressed band at approximately 144 kDa represents the ChlH protein. The MW of other proteins of interest include ChlI1 (40 kDa), ChlD (63 kDa), Gun4 (24 kDa), ChlI2 (40 kDa) and CTH1 (43 kDa).
Fig 3. All bands the size of the proteins of interest expressed from the induced magnesium chelatase plasmid were extracted from the SDS PAGE. MALDI TOF analysis of these proteins revealed that ChlH and ChlI2 were expressed successfully according to GPM.
Fig 4. We observe the increase in MgPPIX concentration at day 3 in the figure above. According to graphs produced on the modelling page, that is when PPIX is starting to be generated. We therefore conclude that we only get accumulation of MgPPIX when PPIX increases within the cell and when we induce with IPTG.
3. Mg-protoporphyrin is not produced in DH5-alpha E.coli containing this plasmid even when induced with IPTG. However, the deltahemH strain created via CRISPR accumulates protoporphyrin IX within the cell.
4. Transformed Mg chelatase plasmid into ΔhemH mutants and it produced an increasing amount of Mg-PPIX demonstrating that the metabolic engineering of E. coli is required to generate sufficient PPIX substrate for the assembled magnesium chelatase complex to function
Fig 5. Fluorescence excitation and emission spectra demonstrates the production of Mg-PPIX upon the addition of Mg-chelatase plasmid to ΔhemeH mutants. Mg-PPIX has an emission and excitation spectra of 418nm and 592nm respectively.
Fig 6. Emission and excitation spectra of the in vitro assay of Mg-chelatase from the Mg-P plasmid. Zero time control is the initial emission/excitation spectra when Mg-chelatase is added to 10mM PPIX, 10mM ATP and 15mM MgCl2. After incubating the assay under constant reagents as the control at room temperature, Mg-PPIX production increases. When additional 50mM MgCl2 is added to the assay in comparison to the control, Mg-PPIX production increases further. However, upon addition of 50mM MgCl2 including 20nM of ChlID complex, the production of Mg-PPIX remains the same. Since adding additional ChlID enzyme to the last assay does not change Mg-PPIX production, the ChlID complex is not limiting.