Difference between revisions of "Part:BBa K3114010"
 
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===Design===
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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.
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As per the MoClo standard, the 5’ signal peptide fusion sequence included in this part is AATG, and the 5’ signal peptide fusion sequence is AGGT.
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[[Image:T--Calgary--MoClo1.png|900px|thumb|center|Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).]]
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This part includes a start codon. A Gly-Gly spacer sequence was added to the end of the signal peptide sequence in order to ensure that the fused protein of interest will be in-frame following Golden Gate assembly.
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This sequence has been codon optimized for high expression in <i>E. coli</i>.
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Latest revision as of 23:40, 18 October 2019


Magnesium Dechelatase Stay Green (SGR)

The conversion of chlorophyll a to pheophytin a is the first step in the natural chlorophyll degradation pathway. The enzyme Magnesium Dechelatase, encoded by the SGR gene, catalyzes this reaction by extracting magnesium (Mg2+) from chlorophyll, along with cofactors 2H+ (Shimoda et al. 2016)

Usage and Biology

The conversion of chlorophyll a to pheophytin a is the first step in the natural chlorophyll degradation pathway. The enzyme Magnesium Dechelatase, encoded by the SGR gene, catalyzes this reaction by extracting magnesium (Mg2+) from chlorophyll, along with cofactors 2H+ (Shimoda et al. 2016).

BBa_K3114010 is a golden gate compatible part which allows insertion of the SGR gene into genetic circuits to express the enzyme Magnesium Dechelatase. In creating this part, iGEM Calgary wished to allow the golden gate cloning of this plant enzyme into E.coli for other teams to use in projects involving degradation of chlorophyll. This part was designed to be golden-gate compatible based on the Mo-Clo standard (Weber et al., 2011). It is also compatible with the BioBrick RFC[10] standard.

This part is compatible with other golden gate parts created by iGEM Calgary including the following:


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’ signal peptide fusion sequence included in this part is AATG, and the 5’ signal peptide fusion sequence is AGGT.

Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).

This part includes a start codon. A Gly-Gly spacer sequence was added to the end of the signal peptide sequence in order to ensure that the fused protein of interest will be in-frame following Golden Gate assembly.

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



Sequences and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 4
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1508
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 1883

References

Shimoda, Y., Ito, H., & Tanaka, A. (2016). Arabidopsis STAY-GREEN, Mendel’s green cotyledon gene, encodes magnesium-dechelatase. The Plant Cell, 28(9), 2147-2160.