Part:BBa_K3114011

Pheophytinase (PPH)

Usage and Biology

Pheophytinase is a chloroplast-located hydrolase which dephytylates the pigment pheophytin during the chlorophyll degradation pathway in plants and algae (Schelbert et al., 2009). Pheophytinase catalyzes the reaction removing the phytyl chain from pheophytin resulting in the formation of pheophorbide a. In several plant species, it has been shown that absence of pheophytinase results in a stay-green phenotype (Guyer et al., 2018)

BBa_K3114009 is a golden gate compatible part which allows insertion of the gene encoding 7-HCAR into genetic circuits to express the enzyme. 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:

• DsbA signal peptide (BBa_K3114000)
• MalE signal peptide (BBa_K3114001)
• OmpA signal peptide (BBa_K3114002)
• PhoA signal peptide (BBa_K3114003)
• TorA signal peptide (BBa_K3114005)
• YcbK signal peptide (BBa_K3114004)
• Double Terminator (BBa_K3114013)
• Universal Spacer with 6XHis tag and Double Terminator (BBa_k3114014)

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 NgoMIV site found at 218
  Illegal AgeI site found at 509
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI.rc site found at 1328
  

References

Luzia Guyer, Kathrin Salinger, Undine Krügel, Stefan Hörtensteiner, Catalytic and structural properties of pheophytinase, the phytol esterase involved in chlorophyll breakdown, Journal of Experimental Botany, Volume 69, Issue 4, 6 February 2018, Pages 879–889, https://doi.org/10.1093/jxb/erx326

Schelbert, S., Aubry, S., Burla, B., Agne, B., Kessler, F., Krupinska, K., & Hörtensteiner, S. (2009). Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. The Plant Cell, 21(3), 767-785.

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