Team:Macquarie Australia/Project/Results

We have demonstrated the functionality of the first step within the biosynthetic pathway of chlorophyll a: Magnesium chelatase (lac+Chli1+ChlD:BBa_K1326004). This was performed initially through the construction of each operon through our composite part assembly method (Ligation Protocol) and subsequent protein expression of each gene product by SDS-PAGE and MALDI mass spectrometry. The functionalities of our two operons was then demonstrated by in vitro spectrophotometric assay of the E.coli cell lysates. The cell lysates from part of Operon 1 were able to convert their expected biochemical substrates into the expected product for their respective step in the biosynthetic pathway. Prior to biochemical pathway modeling it was expected that expressed GUN4 protein product would sequester protoporphyrin IX from the heme biosynthesis pathway. However HPLC analysis of expressed GUN4 lysate showed insignificant levels of protoporphyrin IX. The theoretical modeling of this step in the pathway was demonstrated to be consistent with our experimental observations.

Construction of operons

The engineering of the chlorophyll a biosynthetic pathway was constructed through the design and building of three operons holding 11 of the 12 essential gene BioBricks required for the biosynthesis of chlorophyll pathway. Assembly of the composite parts to construct these operons was performed using a restriction digestion and ligation protocol. Assembly was performed in a stepwise sequential manner to generate each of the three operons for subsequent functional analysis. Composite parts were constructed by sequentially adding one BioBrick gene at a time. Each gene part added was excised from sequence-confirmed BioBricks. All assembled constructs were DNA sequenced for a greater validation of the correct assembly of the genes for each operon.

Figure 1. Single (EcoRI) and double (EcoRI + PstI) restriction digests of the three operon constructs. All the double digests show that the three operons contain a single band that correspond to the combined molecular weight of each expected composite gene construct. A 1 kB DNA ladder (NEB) is run alongside digest products.

Our project was also successful in repairing a 50 bp deletion that we identified in the Macquarie_University 2013 ChlD BioBrick. The missing 50bp fragment in the 2013 ChlD construct was for a poly-Proline rich region essential for functionality. This was achieved by excising a fragment, using ApaI and MluI to digest, from a pET vector containing the correct sequence of ChlD (kindly provided by R. Willows at Macquarie University).The correct sequence was then inserted back into the 2013 ChlD BioBrick. Successful insertion of the additional 50bp was visualized by a SacI and MluI double digest, where the repaired BioBrick would have a fragment of 750bp (figure 2). This part was resent to the registry and re-characterised in the database for its corrected sequence. The functionality of the repaired ChlD BioBrick was also confirmed from the functional assays of Operon 1.

Figure 2. Image of the ChlD “Repair” for the 50 bp deletion on the 2013 ChlD BioBrick part BBa_k1080002. The 50bp difference between the 2013 ChlD BioBrick and our 2014 ChlD repaired BioBrick is clearly visualised when digested with SacI and MluI to isolate the gene fragment (comparison of Lanes 6/7 boxed in green with Lane 8). The repaired ChlD BioBrick was submitted to the registry. The missing 50bp fragment in the 2013 ChlD construct was for a poly-Proline rich region essential for functionality.

Expression of Protein Products for the three Operons

The protein expression for each gene within our assembled operons was analysed after IPTG induction of the lac promoter. The E.coli cells were lysed using a French Press and centrifuged to collect the protein extract supernatant. The supernatant fractions were then run on an SDS-PAGE gel and protein bands corresponding to the sizes expected for the gene products were cut out for trypsin digestion and subsequent MALDI TOF/TOF mass spectrometry. The MS/MS data was searched against a Chlamydomonas reinhardtii through the Global Proteome Machine (GPM) database to identify our proteins of interest (Fig. 5).

Figure 3. Protein expression analysis of SDS-PAGE gel electrophoresis of protein extracts from transformants of E. coli with Operon 1 (Mg-chelatase) and Operon 3 (chlorophyll a). Locations of each gel piece that was excised based on the theoretical molecular weight of the expressed proteins: ChlD (77 kDa), Chli1 (40 kDa) and Gun4 (24 kDa) for Operon 1; POR (41 kDa), DVR1 (45 kDa), ChlP (47 kDa) and ChlG (37 kDa) for operon 2.

Figure 4. Protein expression analysis through SDS-PAGE gel electrophoresis of protein extracts from transformant of E. coli with Operon 2 (proto-chlorophyllide). Protein extracts were collected after ultracentrifugation to obtain the membrane fraction. The pellet membrane fraction (P) and the soluble fraction or supernatant (S) were both run on an SDS-PAGE. Locations for each gel excision based on the theoretical molecular weight of the expressed proteins are shown; CTH1 (44 kDa), ChlM (30 kDa), YCF54 (17 kDa) and Plastocyanin (10 kDa). However, plastocyanin was not able to be visualised for excision as it has ran off the gel.

Figure 5. Identification of ChlI1.

The inability to identify all of our designed genes products in the E. coli extracts is likely due to the crude protein separation on a SDS-PAGE; the high level of E.coli background proteins; and the low coverage of a MALDI TOF/TOF proteomics analysis. Future replications could utilise shotgun identification methods such as LC-MS/MS to provide a more comprehensive coverage of peptides of the expressed proteins, or employ a crude protein purification step prior to the SDS-PAGE.

Functional assays of submitted parts

An assay of ChlI1 + ChlD, two of three genes in Operon 1, was performed to analyse the the functionality of Mg-Chelatase in the biosynthetic pathway of chlorophyll a. Furthermore, establishing functionality, as predicted, further confirmed the correct assembly of gene products for the construct.

Operon 1 - Magnesium chelatase (lac+Chli1+ChlD)
The functionality of operon 1(BBa_K1326004) was demonstrated through the photo-spectral measurements which detected the appearance of Mg-protoporphyrin IX (Fig. 6 & 7) [1]. This supports the conversion step of protoporphyrin IX by the Mg-chelatase enzyme (Operon 1) which is the first required step in the chlorophyll a biosynthetic pathway.

Figure 6. Increase in fluorescence intensity due to formation of Mg-Protoporphyrin by Mg-Chelatase (Operon 1). The sample was excited at 420nm and fluorescence emission monitored at 595nm over 50 minutes. A distinct increase in emission intensity is observable for the cell lysate containing the protein products from (BBa_K1326004).

Figure 7. Fluorescence spectra at 420nm excitation of induced Chl1+ChlD BBa_K1326004 extract in the presence of purified ChlH, GUN4 and Protoporphyrin following a 1hr incubation (as shown in Figure 6). Peak observed at 595nm is consistent with Mg-Protoporphyrin IX production, demonstrating clear activity of the submitted BioBrick part.

The functionality of BBa_K1326004 in converting Protophoryrin IX to Mg-Protophoryrin IX was confirmed by the photoscopic assay. The biochemical pathway due to the action of Mg-chelatase produced by Operon 1 was then modelled.

Experimental Validation of Our Model

Understanding the functional role of GUN4
GUN4 expressed cells (BBa_K108003 were lysed and the protein precipitated by methanol. The supernatant was then loaded onto a UHPLC column (reverse phase) to separate organic compounds. The target compound protoporphyrin-IX is expected to elute at 11.1 mins, based on a commercial protoporphyrin-IX standard (figure 9).

Figure 8. UHPLC trace of methanol solvent only. No peak at 11.1 mins observed.

Figure 9. UHPLC trace of Protoporphyrin IX standard. Note peak intensity at 11.1 mins consistent with Protoporphyrin IX.

Figure 10. UHPLC trace of organic compounds from GUN4 expressed cell lysate. Protoporphyrin-IX peak is at 11.1 mins, with a peak area of 29.

Figure 11. HPLC trace for YCF54 expressed cells (control) . Protoporphyrin IX peak is at 11.1 mins, with a peak area of 158.

Peak area of both traces at 11.1 minutes for cells expressing either GUN4 or YCF54 were significantly different, such that E.coli cells expressing GUN4 show significantly less levels of Protoporphyrin IX.. It is suggested in the literature that the expressed GUN4 may sequester protoporphyrin-IX. This would then result in an increase in the amount of protoporphyrin-IX in the cell lysate. However, we did not see an increase in the presence of GUN4, but rather the converse. Our modelling also examined the effect of the binding of GUN4 to protoporphyrin IX in the heme-chlorophyll biosynthetic pathway. Our modelling showed that an increase would be expected. This is an interesting and novel result because this was opposite to the observations in our expressed construct. This complex system requires further investigation to fully characterise the sequestering activity of GUN4 within the chlorophyll pathway.

References

• [1] Zhou, S., Sawicki, A., Willows, R. D., & Luo, M. (2012). C-terminal residues of Oryza sativa GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis. FEBS letters, 586(3), 205-210.