Team:Macquarie Australia/NPSSummary

For the photosystem II subproject, the aim was to successfully join and assemble the operons of the photosystem II protein complex using 3A assembly. Within the subproject, we successfully assembled psbDCA( [BBa_K1998014]), psbTB [BBa_K1998002], psbMZHWK [BBa_K1998004] and psbOPQR [BBa_K1998006]. psbELJ was assembled by the 2015 Macquarie Australia iGEM team. Additionally, we joined operons 2 and 3 together to form psbELJ-TB and operons 4 and 5 to form psbMZHWK-OPQR. These formed larger composite parts which have been uploaded to the Parts Registry for future users.
gel of bio-bricks.
Fig 1. Gel electrophoresis of the operons and single parts constituting the Photosystem II pathway implemented in this project. Samples psbDCA to psbOPQR are the five operons designed. Lanes D and CA are individual samples submitted which make up operon psbDCA. The final two lanes; psbELJ-TB, and psbMZHWK-OPQR; are the final composite operons from the Photosystem II design. All parts shown have been sequenced and submitted. This gel validates all Biobrick parts and backbones to the designed constructs.
BioBrick Operon Constructed (Y/N) Size (bp)
psbDCA 1 Y 3501
psbELJ 2 Y 495
psbTB 3 Y 1623
psbMZHWK 4 Y 885
psbOPQR 5 Y 2331
psbELJ-TB 2 and 3 Y 2118
psbMZHWK-OPQR 4 and 5 Y 3216
psbD 1 Y 1059
psbCA 1 Y 2442

MZHWK Protein Results

Fig 2.Protein Gel of fractionated MZHWK expressing E. coli. Lane 2 soluble protein fraction, lane 3 urea washing of pellet and lane 4 insoluble pellet) over expressed. MZHWK is shown here to be the correct molecular size (27kD). Following SDS treatment and boiling this single band remains in high yields suggesting it has an extremely hydrophobic nature.

This Biobrick construct is a composite part of 5 individual genes. Each gene encodes a protein which range in size from 4 kDa for the smallest to 9 kDa as the largest. When expressed these proteins combined size is 27 kDa, which is exactly the size of the protein produced in E. coli containing this plasmid induced with IPTG as it appeared on our SDS-PAGE protein gel (Fig 1). We have a single band, expressing at a very high yield, representing a homogenous complex of the 5 expressed protein.

The complex is present as both a soluble protein and as an insoluble aggregate. The complex is also extremely stable. We cannot dissociate the individual proteins from this complex in any extreme condition tested (boiling with 50% SDS and 500mM DTT). We believe this extremely hydrophobic and stable complex could be membrane associated. In addition, digestion with Trypsin and MS proteomic analysis does not produce any identifiable peptides. This is consistent with the difficulty in ionisation of hydrophobic peptides by MS. In addition, there are very few Lys or Arg residues producing Tryptic peptides within the size range 700-4000 Da predicted to be produced by any of these proteins.

Trying to understand more about this we spoke to Professor Min Chen, a leading expert in the biochemical mechanisms of photo-regulated processes in photosynthetic organisms, at The University of Sydney. Prof Chen was very excited to see our results showing the expression and assembly of this complex. The function of this group of proteins is unclear, but it is suspected that they are important in assembly of Photosystem II, possibly in the delivery of chlorophyll. Prof Min Chen commented, that the complex, and in particular psbM, is ‘one of the most hydrophobic proteins known’. Prof Chen also said that the function of the complex may be to bind chlorophyll.

Test tubes
Fig 2.Binding of chlorophyll to MZHWK complex as shown by green pellet on left indicting chlorophyll has associated with insoluble pellet fraction (Tube 1, from left). Corresponding soluble fraction is shown in Tube 2. In contrast, chlorophyll does not associate with the Cas9 control cells with the majority of the chlorophyll remaining in the soluble fraction following centrifugation (Tube 4, far right) and is not found associated with insoluble pellet fraction (Tube 3).

To investigate the function of this complex, as suggested by Prof Willows and Prof Chen, we performed a chlorophyll binding experiment on MZHWK fractions (used in Fig 1 SDS-PAGE). The majority of the complex was found in the insoluble pellet fraction (Fig 1. lane 4). We tested chlorophyll binding by mixing the insoluble membrane pellet fraction with purified chlorophyll, and we observed that the pellet turns green, while an unrelated inclusion body remains white; the supernatant of the unrelated protein control remains green, while the MZHWK supernatant turns clear. This provides evidence that the MZHWK protein complex binds chlorophyll.