Difference between revisions of "Part:BBa K3114023"
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===Usage and Biology===
 
===Usage and Biology===
  
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[[Image:T--Calgary--ModGIXConstruct.png|500px|thumb|right|Figure 1. iGEM Calgary's genetic construct design for ModGIX. Each construct consists of a T7 inducible promoter, a strong RBS, the water-soluble chlorophyll binding protein ModGIX with a 6X His tag, and a bidirectional terminator.]]
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[[Image:T--Calgary--ModGIXConstruct.png|400px|thumb|right|Figure 1. iGEM Calgary's genetic construct design for ModGIX. Each construct consists of a T7 inducible promoter, a strong RBS, the water-soluble chlorophyll binding protein ModGIX with a 6X His tag, and a bidirectional terminator.]]
 
This part can be used for IPTG-inducible expression of the modified water-soluble chlorophyll binding protein ModGIX, which contains a 6xHis tag for purification. This circuit does not contain a signal peptide.  
 
This part can be used for IPTG-inducible expression of the modified water-soluble chlorophyll binding protein ModGIX, which contains a 6xHis tag for purification. This circuit does not contain a signal peptide.  
  
  
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[[Image:T--Calgary--ModGIXRegistry.png|500px|frameless|right|F]]
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[[Image:T--Calgary--ModGIXRegistry.png|400px|frameless|right|F]]
 
ModGIX, short for modified 6GIX, was engineered <i>in silico</i> for greater stability in emulsions. It is based on 6GIX [https://parts.igem.org/Part:BBa_K3114006 (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).
 
ModGIX, short for modified 6GIX, was engineered <i>in silico</i> for greater stability in emulsions. It is based on 6GIX [https://parts.igem.org/Part:BBa_K3114006 (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).
  
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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 _____. This algorithm allowed us to modify the 12 amino acids with the aim of increasing the stability of ModGIX
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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 [https://2019.igem.org/Team:Calgary/iGAM iGAM]. This algorithm informed us to modify the 12 amino acids with the aim of increasing the 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.
 
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.
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===Design===
 
===Design===

Latest revision as of 05:16, 21 October 2019


6xHis-tagged modified water-soluble chlorophyll binding protein (ModGIX) circuit

Usage and Biology

Figure 1. iGEM Calgary's genetic construct design for ModGIX. Each construct consists of a T7 inducible promoter, a strong RBS, the water-soluble chlorophyll binding protein ModGIX with a 6X His tag, and a bidirectional terminator.

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


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 informed us to modify the 12 amino acids with the aim of increasing the 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.

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 and BioBrick RFC[10] compatible.

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 ModGIX coding sequence for purification.


Characterization

For modelling characterization of ModGIX, please see (BBa_K3114007).

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 654
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 179
    Illegal AgeI site found at 122
    Illegal AgeI site found at 438
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 765

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