Part:BBa_K3114009

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 Ferredoxin_reduced catalyze the conversion of 7-Hydroxymethyl Chlorophyll a to Chlorophyll a (Meguro et al. 2011).

Usage and Biology

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.

His Tag Purification

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).

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.

Collection of Parts

This part was designed to be Golden Gate compatible with certain Golden Gate parts in iGEM Calgary's 2019 part collection including the following: 1. DsbA signal peptide - Bba_K3114000 https://parts.igem.org/Part:BBa_K3114000
2. MalE signal peptide - Bba_K3114001 https://parts.igem.org/Part:BBa_K3114001
3. OmpA signal peptide - Bba_K3114002 https://parts.igem.org/Part:BBa_K3114002
4. PhoA signal peptide - Bba_K3114003 https://parts.igem.org/Part:BBa_K3114003
5. Tat signal peptide - Bba_K3114004 https://parts.igem.org/Part:BBa_K3114004
6. TorA signal peptide - Bba_K3114005 https://parts.igem.org/Part:BBa_K3114005
7. Double Terminator - Bba_K3114013 https://parts.igem.org/Part:BBa_K3114013

References

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.

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