Composite

Part:BBa_K3114020

Designed by: Cassandra Sillner, Sara Far, Sravya Kakumanu, Nimaya De Silva, Andrew Symes   Group: iGEM19_Calgary   (2019-10-08)


6xHis-tagged water-soluble chlorophyll binding protein (6GIX) circuit with YcbK 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 contains the YcbK 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, the water-soluble chlorophyll binding protein 6GIX with a 6xHis 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.

T--Calgary--6GIXgifRegistry.gif

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.

The YcbK signal peptide is a 30-amino acid sequence which targets fused proteins to the Tat secretion pathway in E. coli (Tullman-Ercek et al., 2007). In this pathway, proteins are fully or partially folded before secretion to the periplasm. The signal peptide is cleavage site has not been experimentally determined.


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 Golden Gate assembly and parts listed below. It is iGEM Type IIS RFC[1000] compatible because the BsaI sites are removed during assembly.

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 secrete 6GIX with the YcbK signal peptide using this genetic construct. The SDS-PAGE gel below shows the protein recovered from the periplasm for each of our genetic constructs as per our periplasmic protein isolation protocol. The construct with the MalE signal peptide showed the strongest band of the correct size, but bands are also visible for the DsbA and PhoA signal peptide samples.

Figure 3. SDS-PAGE gel showing periplasmic purification of 6GIX with six different signal peptides, no 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 734
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 259
    Illegal AgeI site found at 202
    Illegal AgeI site found at 518
  • 1000
    COMPATIBLE WITH RFC[1000]

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

Tullman-Ercek, D., Delisa, M. P., Kawarasaki, Y., Iranpour, P., Ribnicky, B., Palmer, T., & Georgiou, G. (2007). Export Pathway Selectivity of Escherichia coli Twin Arginine Translocation Signal Peptides. https://doi.org/10.1074/jbc.M610507200

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


[edit]
Categories
//cds
//chassis/prokaryote/ecoli
Parameters
None