Coding
ModGIX

Part:BBa_K3114007

Designed by: Andrew Symes, Cassandra Sillner, Sara Far, Sravya Kakumanu, Nimaya De Silva   Group: iGEM19_Calgary   (2019-10-08)
Revision as of 09:25, 21 October 2019 by Cassandrasillner (Talk | contribs) (Characterization)


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

Usage and Biology

F

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

Please see our modelling page for more information.

Experimental characterization in the laboratory is ongoing.

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 594
    Illegal XhoI site found at 4
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 119
    Illegal AgeI site found at 62
    Illegal AgeI site found at 378
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 605

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


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Categories
//cds
//chassis/prokaryote/ecoli
Parameters
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