Difference between revisions of "Team:Macquarie Australia/Results"

Project Results

On this page we describe the results of our project and our future plans.

Summary of achievements

• Successful completion of the chlorophyll-a synthesis pathway
• all 13 genes required for chlorophyll-a synthesis in E. coli successfully placed into BioBricks
• Functional characterisation of the ChlM enzyme
• Functionality demonstrated in converting Magnesium-Protoporphyrin IX (Mg-PPIX) to Magnesium-Protoporphyrin IX-Monomethyl Ester (Mg-PPIX-ME)
• Presence of ChlM further confirmed by running of extracted protein on an SDS-PAGE gel and mass spectrometry analysis
• Successfully demonstrated protein expression of GUN4 and Plasto genes
• through SDS-PAGE gel and mass spectrometry analysis
• Successfully constructed BioBricks containing 14 of the 17 genes required for biosynthesis of Photosystem II
• Modelling of pathway from 5-aminolevulinic acid to PPIX
• Determined what concentration of 5-aminolevulinic acid (5-ALA) resulted in optimum yield of Protoporphyrin IX (PPIX)

We have successfully constructed BioBricks containing all 13 of the required genes for the synthesis of chlorophyll-a from protoporphyrin IX. This was achieved through the design and construction of four operons containing these 13 genes in BioBricks. The four operons were constructed by assembling our 13 BioBricks into composite parts using a restriction digestion and ligation protocol. All of our assembled parts were sequenced for further confirmation of our success.

Figure 2. Single (EcoRI) and double (EcoRI + PstI) restriction digests of four operon constructs containing 13 genes required for the chlorophyll-a synthesis pathway on a 1% agarose gel. A 1kb DNA ladder (NEB) is shown in the far left lane running alongside digest products.

ChlH

We successfully constructed the ChlH(BBa_K1080001)(4166kb) BioBrick. ChlH is part of operon 1 constituting the chlorophyll-a biosynthesis pathway in E. coli.

The ChlH construct’s purpose is to generate Mg-protoporphyrin IX from Protoporphyrin IX, as the very first step towards synthesising chlorophyll-a from Protoporphyrin IX⁽¹⁾. The role of ChlH is to chelate Mg²⁺ to Protoporphyrin IX, thereby synthesising MG-protoporphyrin IX ⁽¹⁾.

This part was constructed in two steps, first via restriction digest and ligation Gibson assembly⁽²⁾, whereby three Gblocks were combined into the vector.

Figure 3. Image of ChlH digested with EcoRI and EcoRI + PstI, showing bands at ~6kb, showing the complete assembly of all the three parts (~4kb) + KAN vector (~2kb) for single digestion with EcoRI and at ~4kb (ChlH) and ~2kb (KAN vector) for double digest with EcoRI and PstI.

Protein expression

We were successful in inducing protein expression of the genes ChlM, GUN4, and Plasto in E. coli. This was confirmed by running of protein extracts on an SDS-PAGE gel, which produced distinct gel bands corresponding to the theoretical sizes of these three proteins (figure 4).

Figure 4. SDS-PAGE protein expression analysis of transformed E. coli cells each expressing ChlM, Plastocyanin, and GUN4. Protein extracts were loaded onto 4-12% Bis-Tris gel. A unique band is present at the molecular weight of approximately 30kDa (theoretical mass of ChlM) signifying expression of S-Adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase. Overexpression of plastocyanin and GUN4 was also observed in the gel (as highlighted). These are further confirmed by mass spectrometry/

Functional assay of submitted parts

We have demonstrated the functionality of ChlM enzyme encoding S-Adenosyl-L-methionine:magnesium protoporphyrin IX methyltransferase (SAM:MgPMT) (lac+ChlM: BBa_K1640018), an enzyme within the third operon in the chlorophyll-a biosynthesis pathway. SAM catalyses the methylation of a carboxyl group in magnesium-protoporphyrin IX, yielding magnesium-protophorphyrin IX mono-methyl ester(3);

S-adenosyl-L-methionine + magnesium protoporphyrin IX ? S-adenosyl-L-homocysteine + magnesium protoporphyrin IX 13-methyl ester.

Figure 5. Catalysis of Mg-protoporphyrin IX to Mg-protoporphyrin IX monomethylester by ChlM(4).

To demonstrate functionality of ChlM, we added a lac promoter creating a lac + ChlM composite part (Figure 4). This was transformed into E. coli strains DH5a, K12 and XL-blue. Protein was induced using auto-induction media ZYM5052 (5).

Functional assay of ChlM

Cell lysate containing over expressed ChlM was added with substrates and products were separated by FPLC (Figure 3).

Figure 6. FPLC chromatogram of the products of ChlM activity alongside positive and negative controls. The substrate MG-PPIX elutes at 3.04 mins, while the product Mg-PPIX mono-ester elutes at 4.22 mins.

The FPLC chromatogram data enabled determination of ChlM enzyme activity (Figure 3). This data enabled further determination of levels of Mg-Protoporphyrin IX converted to Mg-Protoporphyrin IX-ME by ChlM. In this instance, 26% of the precursor molecule Mg-Protoporphyrin IX had been catalysed by the enzyme ChlM into Mg-Protoporphyrin IX-ME.

When compared to ChlM pET (positive control) expression, which successfully converted 12% of the precursor molecule, it is apparent that our expression of ChlM was very successful.

Mass Spectrometry Analysis of ChlM

Upon expression of ChlM (see Figure 4) mass spectrometry analysis of excised and trypsin digested gel band at 30kDa with ESI found peptides matching.

Photosystem II

We designed 5 operons consisting of a total of 17 genes from C. reinhardtii which encoded for essential proteins for Photosystem II (see table 1). We divided these 17 genes arbitrarily into 10 BioBricks.

Our team successfully constructed 8 of the 10 BioBricks designed for Photosystem II. These were confirmed by sequencing.

Table 2. The 10 BioBricks we attempted to create, whether we were able to detect a constituent gene fragment in a gel-electrophoresis and whether confirmed an accurate sequence from an extracted plasmid.

Biobrick	Operon	Constructed
PsbE-L-J	2	Y
PsbQ-R	5	Y
PsbM-Z-H	4	Y
PsbA	1	Y
PsbW-K	4	Y
PsbP	5	Y
PsbO	5	Y
PsbC	1	Y
PsbT-B	3	N
PsbD	1	N

Figure 8. Single (EcoRI) and double (EcoRI + PstI) restriction digests of eight BioBricks containing 14 genes required for the construction of PSII on a 1% agarose gel. A 1kb DNA ladder (NEB) is shown in the far left lane running alongside digest products.

If we achieve this we can then transform E. coli which we have engineered to develop chlorophyll and hydrogenase, this will allow the E. coli to produce a functioning PSII and allow the production of H₂.

Modelling

ALA is the first precursor on the pathway to biosynthesis of both chlorophyll-a and heme (6). As an organism which produces its own heme, E. coli itself produces 5-ALA and converts it to PPIX (the point of divergence between the heme and chlorophyll-a synthesis pathways) through a multi-step pathway for which it naturally possesses the necessary genes (6).

We successfully modelled the production of PPIX from 5-ALA in non-genetically transformed E. coli, comparing how different concentrations of the precursor 5-ALA affected the production of PPIX (figure 9).

Figure 9. Concentration of PPIX produced from various initial concentrations of 5-ALA for E. coli cell culture concentrations of 0.2mM, with MgCl added at a concentration of 2mM.

Figure 9 shows that the greatest PPIX concentration was achieved in the presence of a 5mM concentration of 5-ALA. Higher initial concentrations of 5-ALA did not result in a higher production of PPIX due to substrate inhibition, whereby Protoporphyrinogen IX inhibits conversion of ALA to porphobilinogen via porphobilinogen synthase (7) (figure 10).

Figure 10. A schematic representation of Protoporphyrinogen IX causing substrate inhibition of 5-ALA’S conversion to porphobilinogen via porphobilinogen synthase.

Future Direction of our Project

• Demonstrate functionality of our constructed parts for chlorophyll-a synthesis
• Successfully induce protein expression of all 13 genes
• Functionally characterise all 13 genes by successfully catalysing each step in the chlorophyll-a synthesis pathway
• Finish the construction of Photosystem II
• Construction BioBricks for the remaining components (PsbD and PsbT-B)
• Building of composite parts by joining the constructed BioBricks
• Development of a BioBrick containing all 5 operons
• Biosynthesis of a hydrogenase enzyme
• Allowing for the combination of electrons and protons for the synthesis of hydrogen gas
• Building of a functional prototype

References

1. Peter, E., Wallner, T., Wilde, A. & Grimm, B. (2011). Comparative functional analysis of two hypothetical chloroplast open reading frames (ycf) involved in chlorophyll biosynthesis from Synechocystis sp. PCC6803 and plants. Journal of Plant Physiology, 168(12), 1380-1386. doi:10.1016/j.jplph.2011.01.014
2. Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A. & Smith, H.O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345. doi:10.1038/nmeth.1318.
3. Sawicki, A., & Willows, R. D. (2007). S-Adenosyl-L-methionine:magnesium-protoporphyrin IX O-methyltransferase from Rhodobacter capsulatus: mechanistic insights and stimulation with phospholipids. The Biochemical Journal, 406(3), 469–478. doi:10.1042/BJ20070284
4. Shepherd, M., Reid, J. D., & Hunter, C. N. (2003). Purification and kinetic characterization of the magnesium protoporphyrin IX methyltransferase from Synechocystis PCC6803. Biochemical Journal, 371(2), 351–360. doi:10.1042/BJ20021394
5. Studier, F.W. (2014). Stable expression clones and auto-induction for protein production in E. coli. Methods in Molecular Biology, 1091, 17-32. doi: 10.1007/978-1-62703-691-7_2.
6. Ilag, L.L., Kumar, A.M. & Soll, D. (1994). Light regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis. The Plant Cell, 6(2), 265-275. doi10.2307/3869644
7. Patrick-Stanford, N.P.J, Capretta, A. & Battersby, A.R. (1995). Expression, purification, and characterisation of the product from the Bacillus subtilis hemD gene, uroporphyrinogen III synthase. European Journal of Biochemistry, 231(1), 236-241. doi:10.1111/j.1432-1033.1995.0236f.x