Difference between revisions of "Part:BBa K3114009"

 
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7-Hydroxymethyl chlorophyll a reductase (7-HCAR) is a naturally occurring plant enzyme prominent in the chlorophyll degradation pathway. 7-HCAR and cofactor Ferredoxin<sub>reduced</sub> catalyze the conversion of 7-Hydroxymethyl Chlorophyll a to Chlorophyll a (Meguro et al. 2011).
  
 
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===Usage and Biology===
 
===Usage and Biology===
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BBa_K3114009 is a golden gate compatible part which allows insertion of the gene encoding for 7-HCAR into genetic circuits to express the enzyme 7-Hydroxymethyl chlorophyll a reductase.
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==His Tag Purification==
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Meguro et al. 2011 purified 7-HCAR using a his-tag/nickel column, but the recombinant protein did not exhibit enzymatic activity. The authors conjectured that the "His tag at the N terminus disturbed the HCAR activity" (Meguro et al. 2011).
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The 2019 Calgary iGEM team set out to get around this problem. The team modelled the electrostatic interactions of 7-HCAR and discovered that 7-HCAR has a highly electronegative core, which is likely interfering with the positively charged His tag. iGEM Calgary developed a universal spacer sequence (https://parts.igem.org/Part:BBa_K3114014) which permitted successful His tag purification.
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===Collection of Parts===
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This part was designed to be Golden Gate compatible with certain Golden Gate parts in iGEM Calgary's 2019 part collection including the following:
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1. DsbA signal peptide - Bba_K3114000 https://parts.igem.org/Part:BBa_K3114000
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2. MalE signal peptide - Bba_K3114001 https://parts.igem.org/Part:BBa_K3114001
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3. OmpA signal peptide - Bba_K3114002 https://parts.igem.org/Part:BBa_K3114002
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4. PhoA signal peptide - Bba_K3114003 https://parts.igem.org/Part:BBa_K3114003
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5. Tat signal peptide - Bba_K3114004 https://parts.igem.org/Part:BBa_K3114004
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6. TorA signal peptide - Bba_K3114005 https://parts.igem.org/Part:BBa_K3114005
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7. Double Terminator - Bba_K3114013 https://parts.igem.org/Part:BBa_K3114013
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===References===
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Meguro, M., Ito, H., Takabayashi, A., Tanaka, R., & Tanaka, A. (2011). Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis. The Plant Cell, 23(9), 3442-3453.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3114009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3114009 SequenceAndFeatures</partinfo>
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Revision as of 04:44, 17 October 2019


7-Hydroxymethyl Chlorophyll A Reductase (7-HCAR)

7-Hydroxymethyl chlorophyll a reductase (7-HCAR) is a naturally occurring plant enzyme prominent in the chlorophyll degradation pathway. 7-HCAR and cofactor Ferredoxinreduced catalyze the conversion of 7-Hydroxymethyl Chlorophyll a to Chlorophyll a (Meguro et al. 2011).

Sequence 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 NgoMIV site found at 869
    Illegal AgeI site found at 293
    Illegal AgeI site found at 386
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 1403