Our contribution: 6GIX

A novel water-soluble chlorophyll binding protein

6GIX is a 180-amino acid water-soluble chlorophyll binding protein (WSCP) which is hypothesized to play a role as a transient chlorophyll shuttle or to be involved in anti-photobleaching responses in Lepidium virginicum (Takahashi et al., 2013). 6GIX is capable of binding chlorophyll a and b, but it has been shown to have higher affinity for chlorophyll b (Bednarczyk, Takahashi, Satoh, & Noy, 2015; Palm et al., 2018).

6GIX exists as a homotetramer that is capable of binding four chlorophyll molecules (Bednarczyk, Takahashi, Satoh, & Noy, 2015). Chlorophyll is a hydrophobic pigment and is therefore soluble only in organic solvents. This part can be used for aqueous phase capture of chlorophyll using emulsions. iGEM Calgary successfully created seven inducible genetic circuits for high-level production of 6GIX using various parts from our collection

DsbA signal peptide 6GIX circuit (BBa_K3114016)
MalE signal peptide 6GIX circuit (BBa_K3114017)
OmpA signal peptide 6GIX circuit(BBa_K3114018)
PhoA signal peptide 6GIX circuit (BBa_K3114019)
YcbK signal peptide 6GIX circuit (BBa_K3114020)
TorA signal peptide 6GIX circuit (BBa_K3114021)
6GIX circuit with no signal peptide (BBa_K311402)

Part Design

Maximizing expression, minimizing aggregation, optimizing purification

When designing this part and the rest of our collection, we were interested in creating parts that could be used in Golden Gate assembly right out of the distribution kit without the need to first domesticate them in a Golden Gate entry vector. As such, these parts are not compatible with the iGEM Type IIS RFC[1000] assembly standard because we included the BsaI restriction site and MoClo standard fusion site in the part’s sequence.

As per the MoClo standard, the 5’ cds fusion sequence included in this part is AGGT, and the 3’ cds fusion sequence is GCTT (Weber et al., 2011).

Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).

This part does not contain a start codon, as it was designed to be used with one of the signal peptides in the collection. The native L. virginicum signal peptide was excluded from this sequence. A double stop codon was introduced to the sequence.

A 6xHistidine affinity chromatography tag was added to the N-terminus of this sequence for purification. Our modelling informed us that this tag would likely not interfere with 6GIX’s folding or function. Regardless, we added a thrombin proteolytic site between the tag and the 6GIX sequence in case it needed to be removed following purification.

The sequence has been codon optimized for high expression in E. coli.

Characterization

Validation of 6GIX’s design and function

We were able to purify 6GIX produced by this genetic construct using the 6xHis tag and Ni-NTA column chromatography. The SDS-PAGE gel below shows the protein in the whole cell lysate (WCL) and in different elution fractions following purification. Purification was conducted as per our protocol. The second elution fraction shows the strongest band. The empty destination vector (EVC) was used as a control.

Figure 2. SDS-PAGE gel showing whole cell lysate and Ni-NTA purification fractions for 6GIX without a signal peptide and an empty vector control. The marker used is the NEB colour protein standard. The arrow denotes correct band size of 21 kDa for the 6GIX protein.

We also conducted Western blotting as per our protocol to verify the identity of the bands seen in SDS-PAGE.

Figure 3. Western blot of whole cell lysate and Ni-NTA purification fractions for 6GIX without a signal peptide and an empty vector control. A His-tagged positive control was also included. The marker used is the NEB colour protein standard. The primary antibody used was anti-His MAb from mouse, and the secondary antibody was anti-mouse IgG conjugated with HRP.

We used 6GIX protein that we purified in our emulsion experiments in order to demonstrate its ability to capture chlorophyll from green seed canola oil in an emulsion. In the image below, emulsions were created for 6GIX, BSA positive control (used due to its slight nonspecific chlorophyll-binding property), and no protein buffer negative control. For the 6GIX emulsion, the lower aqueous phase is visibly more green in colour and the upper oil phase is slightly lighter in colour than the controls. This indicates that 6GIX is capturing chlorophyll from the green oil more efficiently than BSA. The industry standard of acid-activated clay treatment was also included for reference.

Figure 4. Acid-activated clay, BSA positive control, 6GIX, and buffer negative control emulsions. BSA and 6GIX were emulsified at equal concentrations. The upper phase is a pure oil phase and the lower phase is an oil in water microemulsion. Processed and unprocessed canola oil samples were also imaged for reference.

The results from these assays demonstrate that we can successfully produce and purify 6GIX using our genetic constructs. The experiments also show that the purifed 6GIX functions within our emulsion system as expected.

References

Bednarczyk, D., Takahashi, S., Satoh, H., & Noy, D. (2015). Assembly of water-soluble chlorophyll-binding proteins with native hydrophobic chlorophylls in water-in-oil emulsions. Biochimica et Biophysica Acta - Bioenergetics, 1847(3), 307–313. https://doi.org/10.1016/j.bbabio.2014.12.003

Palm, D. M., Agostini, A., Averesch, V., Girr, P., Werwie, M., Takahashi, S., … Paulsen, H. (2018). Chlorophyll a/b binding-specificity in water-soluble chlorophyll protein. Nature Plants, 4(11), 920–929. https://doi.org/10.1038/s41477-018-0273-z

Takahashi, S., Yanai, H., Oka-Takayama, Y., Zanma-Sohtome, A., Fujiyama, K., Uchida, A., … Satoh, H. (2013). Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chlorophyll-binding protein (WSCP) from Virginia pepperweed (Lepidium virginicum), a unique WSCP that preferentially binds chlorophyll b in vitro. Planta, 238(6), 1065–1080. https://doi.org/10.1007/s00425-013-1952-7

Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0016765

Team:Calgary/Validate