Difference between revisions of "Part:BBa K3114022"

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6xHis-tagged water-soluble chlorophyll binding protein (6GIX) circuit with no signal peptide

Usage and Biology

This part can be used for IPTG-inducible expression and secretion of the water-soluble chlorophyll binding protein 6GIX, which contains a 6xHis tag for purification. This circuit does not contain a signal peptide.

Figure 1. iGEM Calgary's genetic construct scheme. Each construct consists of a T7 inducible promoter, a strong RBS, one of six signal peptides or no signal peptide, the water-soluble chlorophyll binding protein 6GIX with a 6X His tag, and a bidirectional terminator.

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_K3114022)

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.

Design

When designing this circuit 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, the basic 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.

Figure 2. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).

This part was constructed using component parts listed below. It is iGEM Type IIS RFC[1000] compatible.

• T7 Promoter and strong RBS (BBa_K3114012)
• 6xHis-tagged 6GIX (BBa_K3114006)
• Double terminator (BBa_K3114013)

The circuit was designed for inducible, high-level expression and has been codon optimized for E. coli. A 6X Histidine affinity chromatography tag was added to the N-terminus of the 6GIX coding sequence for purification.

Characterization

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 3. 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.

For experimental characterization of 6GIX’s function, please see (BBa_K3114006).

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

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