Difference between revisions of "Part:BBa K3114007"

Latest revision as of 02:25, 22 October 2019

Modified water-soluble chlorophyll binding protein (ModGIX) with 6xHis tag

Usage and Biology

ModGIX, short for modified 6GIX, was engineered in silico for greater stability in emulsions. It is based on 6GIX (BBa_K3114006), a 180-amino acid water-soluble chlorophyll binding protein (WSCP) which can bind chlorophyll a and b (Bednarczyk, Takahashi, Satoh, & Noy, 2015; Palm et al., 2018).

6GIX has 12 amino acids that contribute to variance in its crystalline structure when functioning in an emulsion system. At the water-oil interface, these amino acids cause 6GIX to denature faster. To address this issue, iGEM Calgary designed and used a genetic algorithm called iGAM. This algorithm allowed us to modify the 12 amino acids with the aim of increasing the binding affinity and stability of ModGIX.

ModGIX exists as a homotetramer that is capable of binding four chlorophyll molecules. 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.

An inducible genetic circuit for high-level production of ModGIX (BBa_K3114023) was created using various parts from our collection.

Design

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 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 6X Histidine 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 ModGIX’s folding or function. Regardless, we added a thrombin proteolytic site between the tag and the ModGIX sequence in case it needed to be removed following purification.

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

Characterization

We characterized this part in silico through the use of RMSF curves. As seen in Figure 2 below, the ModGIX protein RMSF values all measured less than 0.5nm, which shows that this protein is extremely stable.

Please see our modelling page for more information.

Experimental characterization in the laboratory is ongoing.

Sequence and Features

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

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